USOO8852916B2
(12) United States Patent (10) Patent No.: US 8,852,916 B2 Hyde et al. (45) Date of Patent: Oct. 7, 2014
(54) COMPOSITIONS AND METHODS FOR g: R 7.58 SanrOWn et al.al THERAPEUTIC DELVERY WITH 6,605,286 B2 8, 2003 Steidler et al. MCROORGANISMS 6,610,529 B1 8/2003 Curtiss, III et al. (75) Inventors: Roderick A. Hyde, Redmond, WA (US); 6,670,4276,652,849 B1B2 12/200311/2003 UlbrichtBrown et et al. al. Edward K. Y. Jung, Bellevue, WA (US); 6,797.522 B1 9/2004 Still et al. Royce A. Levien, Lexington, MA (US); 6,852,511 B2 2/2005 Romano et al. Robert W. Lord, Seattle, WA (US); 6,875,356 B2 4/2005 Perriello Mark A. Malamud, Seattle, WA (US); 3.R. 338; Ea John D. Rinaldo, Jr., Bellevue, WA 7341,860 B3 32008 Curtiss, Iliet al. (US); Lowell L. Wood, Jr., Bellevue, 7,344,710 B2 3/2008 Dang et al. WA (US) 7.447,595 B1 1 1/2008 Pohlschroder et al. 7.462,708 B2 12/2008 Singh et al. (73) Assignee: The Invention Science Fund I, LLC, 7,510,852 B2 3/2009 Royer et al. Bellevue, WA (US) 7.550,558 B2 6/2009 Leite et al. 7,780,961 B2 8, 2010 Steidler (*) Notice: Subject to any disclaimer, the term of this 38.8.28 A. ck 9.38 Eas al ...... 435/252.3 patent is extended or adjusted under 35 2003/0064074 A1 4/2003 Changa etC al.a. U.S.C. 154(b) by 377 days. 2004/0018508 A1 1/2004 Friedman 2004/0043003 A1 3/2004 Chen et al...... 424.93.2 (21) Appl. No.: 12/6.57,607 2004/0241849 A1 12/2004 Kapat 2005, OO31643 A1 2/2005 Szalay et al. 1-1. 2005/0101005 A1* 5, 2005 Steidler ...... 435/252.3 (22) Filed: Jan. 22, 2010 2005, 0112133 A1 5, 2005 Druhe (65) Prior Publication Data 28393. A ck 39 Star 435/723 2005/0276788 A1 12/2005 Steidler et al. US 2011 FO183347 A1 Jul. 28, 2011 2006, O121054 A1 6/2006 Sun et al. 2007/01 10723 A1 5/2007 Hans et al. (51) Eao (2006.01) 2007/O122427 A1 5, 2007 Hans et al. s 2007,0213659 A1 9, 2007 Trovato et al.
CI2N I/00 (2006.01) 3888. A 658. SEtedler et al. 3. 2. R 2008/0254014 A1 10, 2008 Rottiers et al. CI2N L/36 3:08: 2009/0115603 A1* 5/2009 Tabe ...... 340,540 A6 IK38/6 (2006.01) (Continued) A6 IK35/74 (2006.01) A6 IK36/064 (2006.01) FOREIGN PATENT DOCUMENTS CI2N 15/63 (2006.01) WO WO96,40947 * 12, 1996 ...... C12N 15,64 (52) U.S. Cl. WO WOO1 (24690 A2 4/2001 CPC ...... CI2N 15/635 (2013.01); A61 K38/2066 WO WO 2005/041848 A2 5, 2005 (2013.01); A61K 38/1841 (2013.01): CI2N OTHER PUBLICATIONS I/36 (2013.01); A61 K38/208 (2013.01); A61 K Qiu et al. Environment-sensitive hydrogels for drug delivery. 2001. 38/2013 (2013.01); A61 K38/162 (2013.01): Advanced Drug Delivery Reviews. vol. 53, pp. 321-329.* A61K 35/74 (2013.01); A61 K35/747 Narvaez-Vasquez et al. Chapter 15 Systemins and AtPeps: Defense (2013.01); A61 K36/064 (2013.01) Related Peptide Signals. 2008. Induced Plant Resistance to USPC ...... 435/252.3; 435/243 Herbivory. pp. 313-328.* (58) Field of Classification Search Syto et al. Structural and biological stability of human interleukin 10 None glimer Dec. 1998. Biochemistry, vol. 37, No. 48, pp. 16943 See application file for complete search history. Ui et al. The production of D-acetoin by a transgenic Escherichia coli. Letters in Applied Microbiology. 1998, vol. 26, pp. 275-278.* (56) References Cited Nichol et al.; “Effectiveness of Live, Attenuated Intranasal Influenza Virus Vaccine in Healthy, Working Adults'; JAMA: Jul. 14, 1999: pp. U.S. PATENT DOCUMENTS 137-144; vol. 281, No. 2: American Medical Association. 4,837,151 A 6, 1989 Stocker (Continued) 5,017,373 A 5, 1991 Herrnstadt et al. 5,316,940 A 5/1994 Georgiou et al. Primary Examiner — Anne Gussow 5,643,771 A 7, 1997 Stocker Assistant Examiner — Channing S Mahatan 5,663,063 A 9/1997 Peoples et al. 5,710,027 A 1/1998 Hauptmann et al. (57) ABSTRACT 5,804,563 A 9, 1998 Still et al. 5,823,993 A 10, 1998 Lemelson Certain embodiments disclosed relate to compositions, 5,831,012 A 11/1998 Nilsson et al. including therapeutic compositions, methods, devices, and 5,888,396 A 3, 1999 Perriello systems that include modified microorganisms including at 5,928,635 A 7, 1999 Schmidt least one genetic element encoding at least one therapeutic 6,066,343 A 5/2000 Megeed et al. 6,100,388 A 8/2000 Casas et al. agent or environmental treatment agent. 6.242,194 B1 6, 2001 Kullen et al. 34 Claims, 44 Drawing Sheets US 8,852,916 B2 Page 2
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U.S. Patent Oct. 7, 2014 Sheet 43 of 44 US 8,852,916 B2
U.S. Patent US 8,852,916 B2
US 8,852,916 B2 1. 2 COMPOSITIONS AND METHODS FOR K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Mala THERAPEUTIC DELVERY WITH mud, John D. Rinaldo, Jr., and Lowell L. Wood, Jr. as inven MCROORGANISMS tors, filed 22 Jan. 2010, which is currently co-pending, or is an application of which a currently co-pending application is CROSS-REFERENCE TO RELATED entitled to the benefit of the filing date. APPLICATIONS For purposes of the USPTO extra-statutory requirements, the present application constitutes a continuation-in-part of The present application is related to and claims the benefit U.S. patent application Ser. No. 127657,605, Docket No. of the earliest available effective filing date(s) from the fol 0905-002-030E-000000, entitled COMPOSITIONS AND lowing listed application(s) (the “Related Applications') 10 METHODS FOR THERAPEUTIC DELIVERY WITH (e.g., claims earliest available priority dates for other than MICROORGANISMS, naming Roderick A. Hyde, Edward provisional patent applications or claims benefits under 35 K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Mala USC S 119(e) for provisional patent applications, for any and mud, John D. Rinaldo, Jr., and Lowell L. Wood, Jr. as inven all parent, grandparent, great-grandparent, etc. applications tors, filed 22 Jan. 2010, which is currently co-pending, or is an of the Related Application(s)). All subject matter of the 15 application of which a currently co-pending application is Related Applications and of any and all parent, grandparent, entitled to the benefit of the filing date. great-grandparent, etc. applications of the Related Applica tions is incorporated herein by reference to the extent such SUMMARY Subject matter is not inconsistent herewith. Particular embodiments disclosed herein relate to various RELATED APPLICATIONS compositions, methods of administering, computer systems, computer-implemented methods, and associated products For purposes of the USPTO extra-statutory requirements, related to compositions including modified microorganisms the present application is related to U.S. patent application modified to produce particular agents for at least one Sub Ser. No. 127657,606, entitled COMPOSITIONS AND 25 Strate. METHODS FOR THERAPEUTIC DELIVERY WITH In an embodiment, a composition comprises: at least one MICROORGANISMS, naming Roderick A. Hyde, Edward auxotrophic microorganism including at least one pH induc K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Mala ible promoter operably coupled to at least one heterologous mud, John D. Rinaldo, Jr., and Lowell L. Wood, Jr. as inven genetic element encoding at least one therapeutic agent for at tors, filed 22 Jan. 2010, which is currently co-pending, or is an 30 least one biological tissue, and including at least one genetic application of which a currently co-pending application is element inducible to initiate death of the at least one aux entitled to the benefit of the filing date. otrophic microorganism; and wherein the at least one com For purposes of the USPTO extra-statutory requirements, position includes at least one metabolite required by the at the present application is related to U.S. patent application least one auxotrophic microorganism. Ser. No. 127657,608, entitled COMPOSITIONS AND 35 In an embodiment, a composition comprises: at least one METHODS FOR THERAPEUTIC DELIVERY WITH auxotrophic microorganism including at least one tempera MICROORGANISMS, naming Roderick A. Hyde, Edward ture inducible promoter operably coupled to at least one het K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Mala erologous genetic element encoding at least one therapeutic mud, John D. Rinaldo, Jr., and Lowell L. Wood, Jr. as inven agent for at least one biological tissue, and including at least tors, filed 22 Jan. 2010, which is currently co-pending, or is an 40 one genetic element inducible to initiate death of the at least application of which a currently co-pending application is one auxotrophic microorganism; and wherein the at least one entitled to the benefit of the filing date. composition includes at least one metabolite required by the For purposes of the USPTO extra-statutory requirements, at least one auxotrophic microorganism. the present application is related to U.S. patent application In an embodiment, a composition comprises: at least one Ser. No. 127657,604, entitled COMPOSITIONS AND 45 auxotrophic microorganism including at least one tempera METHODS FOR THERAPEUTIC DELIVERY WITH ture inducible repressor operably coupled to at least one het MICROORGANISMS, naming Roderick A. Hyde, Edward erologous genetic element encoding at least one therapeutic K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Mala agent for at least one biological tissue, and including at least mud, John D. Rinaldo, Jr., and Lowell L. Wood, Jr. as inven one genetic element inducible to initiate death of the at least tors, filed 22 Jan. 2010, which is currently co-pending, or is an 50 one auxotrophic microorganism; and wherein the at least one application of which a currently co-pending application is composition includes at least one metabolite required by the entitled to the benefit of the filing date. at least one auxotrophic microorganism. For purposes of the USPTO extra-statutory requirements, In an embodiment, a composition comprises: at least one the present application is related to U.S. patent application auxotrophic microorganism including at least one pH induc Ser. No. 127657,609, entitled COMPOSITIONS AND 55 ible repressor operably coupled to at least one heterologous METHODS FOR THERAPEUTIC DELIVERY WITH genetic element encoding at least one therapeutic agent for at MICROORGANISMS, naming Roderick A. Hyde, Edward least one biological tissue, and including at least one genetic K.Y. Jung, Royce A. Levien, Robert W. Lord, Mark A. Mala element inducible to initiate death of the at least one aux mud, John D. Rinaldo, Jr., and Lowell L. Wood, Jr. as inven otrophic microorganism; and wherein the at least one com tors, filed 22 Jan. 2010, which is currently co-pending, or is an 60 position includes at least one metabolite required by the at application of which a currently co-pending application is least one auxotrophic microorganism. entitled to the benefit of the filing date. In an embodiment, a method of administering at least one For purposes of the USPTO extra-statutory requirements, therapeutic agent to at least one biological tissue comprises: the present application is related to U.S. patent application providing a composition to at least one biological tissue; Ser. No. 127657,605, entitled COMPOSITIONS AND 65 wherein the composition includes at least one auxotrophic METHODS FOR THERAPEUTIC DELIVERY WITH microorganism including at least one pH inducible heterolo MICROORGANISMS, naming Roderick A. Hyde, Edward gous genetic element encoding at least one therapeutic agent US 8,852,916 B2 3 4 formulated for at least one biological tissue, and including at one biological tissue, and including at least one genetic ele least one genetic element inducible to initiate death of the at ment inducible to initiate death of the at least one auxotrophic least one auxotrophic microorganism; and the composition microorganism; and at least one metabolite required by the at further including at least one metabolite required by the at least one auxotrophic microorganism. least one auxotrophic microorganism. In an embodiment, a computer program product com In an embodiment, a composition comprises: at least one prises: a recordable medium bearing one or more instructions modified microorganism including at least one heterologous for regulating dispensing of at least one delivery device, genetic element encoding at least one environmental medium wherein the delivery device includes at least one composition, treatment agent; and at least one genetic element inducible to wherein the at least one composition includes at least one initiate death of the at least one modified microorganism. 10 auxotrophic microorganism including at least one pH induc In an embodiment, a method of administering at least one environmental medium treatment agent to at least one envi ible heterologous genetic element encoding at least one thera ronmental medium comprises: providing at least one compo peutic agent formulated for at least one biological tissue, and sition to at least one environmental medium; wherein the at including at least one genetic element inducible to initiate least one composition includes at least one modified micro 15 death of the at least one auxotrophic microorganism; and at organism including at least one heterologous genetic element least one metabolite required by the at least one auxotrophic encoding at least one environmental medium treatmentagent, microorganism. and at least one genetic element inducible to initiate death of In an embodiment, a system comprises: at least one com the at least one modified microorganism. puting device; at least one delivery device configured to retain In an embodiment, a delivery device comprises: a housing and dispense at least one composition to at least one environ including at least one reservoir containing at least one com mental medium; and a recordable medium including one or position, the at least one composition including at least one more instructions that when executed on a computing device first constituent including at least one auxotrophic microor cause the computing device to regulate dispensing of at least ganism including at least one pH inducible promoteroperably a portion of the at least one composition, wherein the at least coupled to at least one heterologous genetic element encod 25 one composition includes at least one modified microorgan ing at least one therapeutic agent, and at least one genetic ism including at least one heterologous genetic element element inducible to initiate death of the at least one aux encoding at least one environmental medium treatment agent; otrophic microorganism; and at least one second constituent and at least one genetic element inducible to initiate death of including at least one metabolite required by the at least one the at least one modified microorganism. auxotrophic microorganism; the reservoir configured to 30 In an embodiment, a computer-implemented method com receive, retain, and dispense the at least one composition; and prises: one or more instructions for regulating dispensing of at at least one constituent configured to administer the at least least one composition from at least one delivery device to at one composition to at least one biological tissue. least one environmental medium, the at least one composition In an embodiment, a delivery device comprises: a housing including at least one modified microorganism including at including at least one reservoir containing at least one com 35 least one heterologous genetic element encoding at least one position, the at least one composition including at least one environmental medium treatment agent; and at least one modified microorganism including at least one heterologous genetic element inducible to initiate death of the at least one genetic element encoding at least one environmental medium modified microorganism. treatment agent, and at least one genetic element inducible to In an embodiment, a computer program product com initiate death of the at least one modified microorganism, the 40 prises: a recordable medium bearing one or more instructions reservoir configured to receive, retain, and dispense at least a for regulating dispensing of at least one delivery device, portion of the at least one composition; and at least one wherein the delivery device includes at least one composition, component configured to administer the at least one compo wherein the at least one composition includes at least one sition to at least one environmental medium. modified microorganism including at least one heterologous In an embodiment, a system comprises: at least one com 45 genetic element encoding at least one environmental medium puting device; at least one delivery device configured to retain treatment agent; and at least one genetic element inducible to and dispense at least one composition to at least one biologi initiate death of the at least one modified microorganism. cal tissue; and a recordable medium including one or more In an embodiment, a computer-implemented method, sys instructions that when executed on the computing device tem, or computer program product thereof, relates to making cause the computing device to regulate dispensing of at least 50 or administering the compositions described. a portion of the at least one composition, wherein the at least one composition includes at least one first constituent includ BRIEF DESCRIPTION OF THE FIGURES ing at least one auxotrophic microorganism including at least one pH inducible promoter operably coupled to at least one FIG. 1 illustrates a partial view of various embodiments heterologous genetic element encoding at least one therapeu 55 disclosed herein. tic agent, and including at least one genetic element inducible FIG. 2 illustrates a partial view of various embodiments to initiate death of the at least one auxotrophic microorgan disclosed herein. ism; and at least one second constituent including at least one FIG. 3 illustrates a partial view of particular genetic ele metabolite required by the at least one auxotrophic microor ments utilized in various embodiments disclosed herein. ganism. 60 FIG. 4 illustrates a partial view of particular genetic ele In an embodiment, a computer-implemented method com ments utilized in various embodiments disclosed herein. prises: one or more instructions for regulating dispensing at FIG. 5 illustrates a partial view of a particular embodiment least one composition from at least one delivery device to at of a delivery device disclosed herein. least one biological tissue, the at least one composition FIG. 6 illustrates a partial view of various embodiments of including at least one auxotrophic microorganism including 65 the device of FIG. 5. at least one pH inducible heterologous genetic element FIG. 7 illustrates a partial view of various embodiments of encoding at least one therapeutic agent formulated for at least the device of FIG. 5. US 8,852,916 B2 5 6 FIG. 8 illustrates a partial view of various embodiments of FIG. 41 illustrates apartial view of various embodiments of the device of FIG. 5. the computer-implemented method of FIG. 38. FIG. 9 illustrates a partial view of various embodiments of FIG. 42 illustrates a partial view of a particular embodi the device of FIG. 5. ment of a computer program product disclosed herein. FIG.10 illustrates apartial view of various embodiments of FIG. 43 illustrates apartial view of various embodiments of the device of FIG. 5. the computer program product of FIG. 42. FIG.11 illustrates apartial view of various embodiments of FIG. 44 illustrates apartial view of various embodiments of the device of FIG. 5. a delivery device including a composition disclosed herein. FIG. 12 illustrates apartial view of various embodiments of the device of FIG. 5. 10 DETAILED DESCRIPTION FIG. 13 illustrates apartial view of various embodiments of In the following detailed description, reference is made to the device of FIG. 5. the accompanying drawings, which form a parthereof. In the FIG. 14 illustrates a partial view of a particular embodi drawings, similar symbols typically identify similar compo ment of a delivery device disclosed herein. 15 nents, unless context dictates otherwise. The illustrative FIG. 15 illustrates apartial view of various embodiments of embodiments described in the detailed description, drawings, the device of FIG. 14. and claims are not meant to be limiting. Other embodiments FIG.16 illustrates apartial view of various embodiments of may be utilized, and other changes may be made, without the device of FIG. 14. departing from the spirit or scope of the Subject matter pre FIG.17 illustrates apartial view of various embodiments of sented here. the device of FIG. 14. The present application uses formal outline headings for FIG. 18 illustrates apartial view of various embodiments of clarity of presentation. However, it is to be understood that the the device of FIG. 14. outline headings are for presentation purposes, and that dif FIG. 19 illustrates apartial view of various embodiments of ferent types of Subject matter may be discussed throughout the device of FIG. 14. 25 the application (e.g., method(s) may be described under com FIG.20 illustrates apartial view of various embodiments of position heading(s) and/or kit headings; and/or descriptions the device of FIG. 14. of single topics may span two or more topic headings). Hence, FIG. 21 illustrates a partial view of a particular embodi the use of the formal outline headings is not intended to be in ment of a system disclosed herein. any way limiting. FIG.22 illustrates apartial view of various embodiments of 30 Modified Microorganisms the system of FIG. 21. The compositions, methods, devices, and systems FIG.23 illustrates apartial view of various embodiments of described herein relate to at least one modified microorgan the system of FIG. 21. ism (e.g., an auxotrophic microorganism) that is configured FIG.24 illustrates apartial view of various embodiments of or modified to produce or deliver at least one agent (e.g., a the system of FIG. 21. 35 therapeutic agent, environmental medium treatment agent, FIG.25 illustrates apartial view of various embodiments of etc.) to at least one Substrate (e.g., biological tissue, environ the system of FIG. 21. mental medium, etc.). FIG. 26 illustrates apartial view of various embodiments of For example, in an embodiment, a composition comprises the system of FIG. 21. at least one auxotrophic microorganism including at least one FIG. 27 illustrates a partial view of a particular embodi 40 pH inducible promoter operably coupled to at least one het ment of a computer-implemented method disclosed herein. erologous genetic element encoding at least one therapeutic FIG.28 illustrates apartial view of various embodiments of agent for at least one biological tissue, and including at least the computer-implemented method of FIG. 27. one genetic element inducible to initiate death of the at least FIG.29 illustrates apartial view of various embodiments of one auxotrophic microorganism; and wherein the at least one the computer-implemented method of FIG. 27. 45 composition includes at least one metabolite required by the FIG.30 illustrates apartial view of various embodiments of at least one auxotrophic microorganism. In an embodiment, a the computer-implemented method of FIG. 27. composition comprises at least one auxotrophic microorgan FIG.31 illustrates apartial view of various embodiments of ism including at least one temperature inducible promoter the computer-implemented method of FIG. 27. operably coupled to at least one heterologous genetic element FIG. 32 illustrates a partial view of a particular embodi 50 encoding at least one therapeutic agent for at least one bio ment of a computer program product disclosed herein. logical tissue, and including at least one genetic element FIG.33 illustrates apartial view of various embodiments of inducible to initiate death of the at least one auxotrophic the computer program product of FIG. 32. microorganism; and wherein the at least one composition FIG. 34 illustrates a partial view of a particular embodi includes at least one metabolite required by the at least one ment of a system disclosed herein. 55 auxotrophic microorganism. FIG.35 illustrates apartial view of various embodiments of In an embodiment, a composition comprises at least one the system of FIG. 33. auxotrophic microorganism including at least one tempera FIG.36 illustrates apartial view of various embodiments of ture inducible repressor operably coupled to at least one het the system of FIG. 33. erologous genetic element encoding at least one therapeutic FIG.37 illustrates apartial view of various embodiments of 60 agent for at least one biological tissue, and including at least the system of FIG. 33. one genetic element inducible to initiate death of the at least FIG. 38 illustrates a partial view of a particular embodi one auxotrophic microorganism; and wherein the at least one ment of a computer-implemented method disclosed herein. composition includes at least one metabolite required by the FIG. 39 illustrates apartial view of various embodiments of at least one auxotrophic microorganism. the computer-implemented method of FIG. 38. 65 In an embodiment, a composition comprises at least one FIG. 40 illustrates apartial view of various embodiments of auxotrophic microorganism including at least one pH induc the computer-implemented method of FIG. 38. ible repressor operably coupled to at least one heterologous US 8,852,916 B2 7 8 genetic element encoding at least one therapeutic agent for at example, by allowing selective growth of the microorganism, least one biological tissue, and including at least one genetic or selective agent production (e.g., lactic acid suppressor element inducible to initiate death of the at least one aux gene), the microorganism can be directed or controlled. In otrophic microorganism; and wherein the at least one com another example, the modified microorganism includes a position includes at least one metabolite required by the at recombinant microorganism with a mutation that renders the least one auxotrophic microorganism. microorganism dependent on an external factor for Survival In an embodiment, a composition comprises at least one (e.g., thymidylate synthase gene). In another example, the modified microorganism including at least one heterologous modified microorganism contains an essential gene and a genetic element encoding at least one environmental medium control sequence that regulates expression of the lethal gene treatment agent; and at least one genetic element inducible to 10 Such that an essential gene is expressed in a permissive envi initiate death of the at least one modified microorganism. ronment and not expressed in a nonpermissive environment. In an embodiment, the modified microorganism includes at In another example, the modified microorganism contains a least one genetic element (e.g., heterologous genetic element) lethal gene and a control sequence that regulates expression encoding the at least one agent. In an embodiment, the com Such that the lethal gene is expressed in a nonpermissive position further comprises at least one metabolite utilizable or 15 environment, and not expressed when in a permissive envi required by the at least one modified microorganism. In an ronment. See, for example, U.S. Patent Application Publica embodiment, the at least one modified microorganism tion No. 20080253990, and U.S. Patent Application Publica includes at least one inducible genetic element configured to tion No. 20080254014, each of which is incorporated herein initiate death of the modified microorganism. In an embodi by reference. ment, the metabolite includes the at least one therapeutic In an embodiment, the microorganism includes at least one agent or environmental treatment agent produced by the genetic element inducible to initiate death in the at least one modified microorganism. In an embodiment, the metabolite microorganism. In this manner, the microorganism includes is provided by the at least one biological tissue or environ Suicide genetic elements that are inducible to initiate death in mental medium. the microorganism upon encountering at least one inducer In an embodiment, a modified microorganism includes a 25 that regulates the genetic elements. In an embodiment, the microorganism that has been chemically, physically, or genetic elements inducible to initiate death in the microor genetically modified from a naturally occurring microorgan ganism include at least one of an inducible promoter, induc ism, or a microorganism that has been artificially synthesized. ible enhancer, or inducible repressor, any of which is operably In an embodiment, the modified microorganism includes an coupled to the genetic element that encodes the particular auxotrophic microorganism. For example, in an embodiment, 30 Suicide gene or other death-initiating gene. In an embodi an auxotrophic microorganism includes at least one modifi ment, the genetic elements that regulate the induction of death cation that renders it dependent on at least one metabolite for of the microorganism are incorporated as part of the microo maintenance of physiological processes (e.g., metabolism, ganisms own chromosomal or genetic constitution. In an reproduction, sporulation, etc.). In an embodiment, the modi embodiment, the genetic elements that regulate the induction fied microorganism is unable to manufacture the metabolite 35 of death of the microorganism are included as part of a vector itself, and instead must receive the metabolite from the envi (e.g., plasmid, cosmid, etc.). See, for example, U.S. Patent ronment. In particular instances, the metabolite acts as at least Application No. 20050276788, which is incorporated herein one of a repressor or inducer of the at least one genetic by reference. element inducible to initiate death of the at least one modified In an embodiment, at least one of the promoter, enhancer, microorganism. Examples of particular metabolites are 40 or repressor operably coupled to the heterologous genetic described herein, and include but are not limited to, energy element encoding the at least one therapeutic agent or the at Sources or nutrients for metabolic pathways. least one environmental medium treatment agent is an epige In an embodiment, the metabolite is utilizable but not netic element. required by the modified microorganism. For example, in an In an embodiment, at least one of the promoter, enhancer, embodiment an auxotrophic microorganism may require one 45 or repressor operably coupled to the at least one heterologous metabolite but also be able to utilize other metabolites genetic element encoding the at least one therapeutic agent or (whether or not such metabolites can replace the required the at least one environmental medium treatment agent is an metabolite). epigenetic element. In an embodiment, the at least one metabolite includes at Many standard lethal genes or lethal gene systems are least one promoter operably coupled to at least one heterolo 50 known. For example, in an embodiment, a conditional lethal gous genetic element encoding the at least one therapeutic system for eukaryotic cells (e.g., fungal cells such as yeast, or agent or at least one environmental medium treatment agent. other eukaryotic cells) is utilized by providing intracellular In an embodiment, the at least one metabolite includes a production of the Serratia marcescens nuclease in the cell, time-release formulation. In an embodiment, the wherein the which destroys the genetic material in the cell. See, for metabolite is formulated for pH-dependent release or activa 55 example, Balan and Schenberg, Yeast, Vol. 22, pp. 203-212 tion. (2005), which is incorporated herein by reference. As In an embodiment, the pH level of the release or activation reported, under normal conditions, the nuclease, encoded by of the at least one pH dependent metabolite is different than the nucA gene, is secreted into the extracellular medium. the pH level of the pH inducible promoteroperably coupled to Cloning it without the signal sequence, however, results in the at least one heterologous genetic element encoding at 60 killing the yeast cell upon glucose depletion from the least one therapeutic agent or at least one environmental medium. Id. The conditional lethal system also disfavors medium treatment agent. horizontal gene transfer from recombinant yeast cells to other In an embodiment, the administered microorganism microorganisms found in the environment. Id. remains relatively localized. For example, by utilizing a In an embodiment, a lac-hok cassette is utilized for induc microorganism including at least one genetic element that 65 ing death, wherein the hok gene from plasmid R1 belongs to allows for controlled growth, the microorganism can be a family of genes encoding Small polypeptides (about 50 directed toward or away from a particular location. In one amino acids) which, when overexpressed, collapse the mem US 8,852,916 B2 10 brane potential and lead to cell death. See, for example, Con sensing peptide, Extracellular Death Factor, is a signal mol treras, et al., App. Env. Microbiol. Vol. 57, no. 5, pp. 1504 ecule required for mazEF-mediated cell death. Id. 1508 (1991), which is incorporated herein by reference. In an In an embodiment, the at least one genetic element induc embodiment, a gef system, a chromosomally encoded E. coli ible to initiate death of the modified microorganism includes gene highly homologous to hok, is utilized for inducing death at least one genetic element inducible to interfere with the in the modified microorganism. For example, the modified utilization of the at least one metabolite (e.g., by encoding a microorganism Survives only in the presence of effectors of peptide or protein that interferes with the microorganism’s the meta-cleavage pathway encoded by the TOL plasmid of P metabolism, etc.). putida. Id. For example, in an embodiment, microorganisms In an embodiment, the at least one inducible genetic ele modified to degrade substituted benzoates utilize a LacI pro 10 tein (Lac repressor) expressed from a Pm::lacI fusion ment configured to initiate death of the modified microorgan represses transcription from a Ptac::gef cassette in the pres ism includes at least one of extracellular death factor, maZF, ence of XylS effectors (coding for the regulator necessary to or maZEF. As illustrated in FIG.3, an example of an inducible activate transcription from Pm, in the presence of an effector genetic element, including an inducible promoter 300 is Such as 3-methylbenzoate, in this example), whereas in the 15 capable of regulating expression of at least one gene 310. In absence of XylS effectors, expression of the gef gene is no the absence of an inducer 320, the gene 310 is not transcribed longer repressed, leading to cell killing. Id. Substitution of (as indicated by the “X”). However, in the presence of the XylS for another protein expands the range of response. Id. inducer 320, the promoter 300 directs transcription of the Thus, in an embodiment, a similar construct is developed for gene 310, resulting in production of at least one transcript regulation of production of a particular therapeutic or envi 330. Likewise, in the presence of a repressor 340, the pro ronmental medium treatment agent in a modified microor moter 300 does not support gene transcription of the gene 310 ganism, as described herein. (as indicated by the “X”). In an embodiment, the at least one In an embodiment, the at least one modified microorgan genetic element inducible to initiate death of the at least one ism includes at least one genetic element inducible to initiate modified microorganism includes at least one genetic ele death of the at least one microorganism. In an embodiment, 25 ment inducible to initiate autophagocytosis of the at least one the inducible genetic element includes at least one secretory modified microorganism. signal sequence. Various mechanisms can be employed to As described herein, in an embodiment the at least one initiate death of the microorganism. For example, the genetic composition includes at least one metabolite that is required element inducible to initiate death can include a genetic ele by the at least one modified microorganism. In an embodi ment inducible to initiate at least one of programmed cell 30 ment, the modified microorganism has a strict requirement death, to initiate autophagocytosis of the at least one micro for at least one metabolite that is not present or is present at organism, to lyse the at least one microorganism, or by other low concentrations in the external environment (e.g., aux CaS. otrophic). For example, the at least one metabolite includes, In an embodiment, the at least one genetic element induc but is not limited to, at least one of an organic or inorganic ible to initiate death of the at least one modified microorgan 35 Small molecule, nucleic acid, amino acid, peptide, polypep ism includes at least one genetic element inducible to initiate tide, protein, glycopeptide, glycoprotein, glycolipid, programmed cell death. In an embodiment, the genetic ele lipopolysaccharide, peptidoglycan, proteoglycan, lipid, met ment inducible to initiate death of the at least one modified alloprotein, metal, liposome, carbohydrate, or radiation. In an microorganism includes at least one of programmed cell embodiment, the at least one metabolite includes at least one death 1 gene (PDCD1), programmed cell death 2 gene 40 of arabinose, lactose, maltose. Sucrose, glucose, Xylose, (PDCD2), programmed cell death 3 gene (PDCD3), pro galactose, rhamnose, fructose, melibiose, starch, inunlin, grammed cell death 4 gene (PDCD4), programmed cell death lipopolysaccharide, arsenic, cadmium, hydrocarbon, chro 5 gene (PDCD5), programmed cell death 6 gene (PDCD6), mium, ultra-violet radiation, infrared radiation, electromag programmed cell death 7 gene (PDCD7), programmed cell netic radiation, visible radiation, antibiotic, oxygen, carbon death 8 gene (PDCD8), programmed cell death 9 gene 45 dioxide, nitrogen, Xylan, or nisin. Other non-limiting (PDCD9), programmed cell death 10 gene (PDCD10), pro examples of metabolites include at least one of L-arabinose, grammed cell death 11 gene (PDCD11), programmed cell allolactose, D-glucose, D-xylose, D-galactose, amplicillin, death 12 gene (PDCD12), caspase gene, rel gene, hok gene, tetracycline, penicillin, pristinamycin, retinoic acid, or inter Sok gene, diaminopimelate gene, nuclease gene, methylase feron. In an embodiment, the at least one metabolite includes gene, DNA ligase gene, DNA gyrase gene, toxin-antitoxin 50 the at least one therapeutic or environmental medium treat module, relF gene, triclosan, lysine, or lysine-holin. In an ment agent, or at least one component thereof. In certain embodiment, the toxin-antitoxin module includes at least one instances, the at least one metabolite is provided or produced of masEF, chpBIK, relBE, yefM-yoeB, dinJ-yafl, or ecna by the at least one biological tissue or environmental medium. ecnB. In an embodiment, the at least one inducible genetic In an embodiment, the at least one metabolite utilizable by the element configured to lyse the at least one auxotrophic micro 55 modified microorganism is the same as at least one inducer organism includes at least one of a nuclease gene, or lysis capable of inducing at least one inducible genetic element of gene E. the modified microorganism. In an embodiment, the at least one inducible genetic ele In an embodiment, the at least one metabolite includes at ment configured to initiate death of the modified microorgan least one repressor of the at least one inducible genetic ele ism includes at least one toxin-antitoxin. Toxin-antitoxin 60 ment configured to initiate death of the at least one aux modules are generally gene pairs specifying for a toxin and its otrophic or modified microorganism. In an embodiment, the antitoxin, and are found on the chromosomes of many bacte at least one inducible genetic element configured to initiate ria. For example, in E. coli, maZF encodes a stable toxin death of the at least one auxotrophic or modified microorgan (MaZF), and mazE encodes a labile antitoxin (mazE) which ism includes at least one inducible promoter. In an embodi prevents the lethal effect of MazF. See, for example, J. Bac 65 ment, the at least one inducible genetic element configured to teriol., vol. 186, no. 24, pp. 8295-8300, (2004), which is initiate death of the at least one auxotrophic or modified incorporated herein by reference. As published, the quorum microorganism includes at least one regulatory sequence. In US 8,852,916 B2 11 12 an embodiment, the at least one regulatory sequence includes Communication between the two populations directs the at least one terminator fragment, enhancer sequence, or “prey’ to rescue the predator, but once the “predator recov marker sequence. ers to a sufficiently high density, it begins to kill the “prey. Id. In an embodiment, the at least one metabolite is provided In an embodiment, a cooperative relationship includes, but by at least one modified biological cell. The modified biologi is not limited to biofilm formation, colonization, virulence, cal cell can include, for example, at least one of bacteria, proliferation, communication, and other activities. See, for protozoa, rotifers, algae, archaeon, or fungi. In an embodi example, Brenner, et al., Trends Biotech. Vol. 26, no. 9, pp. ment, the modified biological cell includes at least one 483-489 (2008), which is incorporated herein by reference. prokaryote cell or eukaryote cell. In an embodiment, the For example, the at least one modified microorganism can modified biological cell includes at least one modified micro 10 participate in at least one microbial consensus consortium for organism (e.g., an auxotrophic microorganism). In an cross-talk and cell-cell signaling. See, for example, Brenner, embodiment, the modified biological cell includes at least one et al., PNAS, vol. 104, no. 44, pp. 17300-17304 (2007), which blood cell, muscle cell, nerve cell, fibroblast, adipose cell, is incorporated herein by reference. stem cell, pluripotent cell, epithelial cell, skin cell, neoplastic Published studies describe synthetic ecosystems of at least cell, or other biological tissue or organ cell. In an embodi 15 one microbial population capable of communicating bi-direc ment, the modified biological cell includes at least one autolo tionally through quorum sensing and regulating gene expres gous cell or modified autologous cell. In an embodiment, the sion and Survival of other population members, by way of modified biological cell is at least part of at least one cell engineered gene circuits. See, for example, Balagadde, et al., mass. In an embodiment, the at least one cell mass includes at Mol. Sys. Biol. vol. 4, no. 187, pp. 1-8 (2008), which is least one tumor, scar, pore, pit, eschar, granuloma, keloid, incorporated herein by reference. As discussed herein, in an artheromatous plaque, abscess, pustule, Scaling (e.g., psoria embodiment, the “predator cells' kill the “prey' by inducing sis or eczema), infected tissue, hair follicle, necrotic tissue, expression of a killer protein in the “prey, while the “prey' stratum corneum, wrinkle, wound, tumor, skin structure, rescues the “predators' by eliciting expression of an antidote nevus, cyst, lesion, callus, neoplastic tissue, gangrenous tis protein in the “predator'. Thus, extinction, coexistence and Sue, fetal tissue, placental tissue, or cellular deposit. 25 oscillatory dynamics of the “predator” and “prey” popula In an embodiment, the modified biological cell is posi tions are possible depending on the operating conditions, tioned for at least one of a commensal or cooperative rela which can be determined mathematically. Id. In an embodi tionship with the at least one modified microorganism. In an ment, the “predator” includes the at least one modified bio embodiment, the modified biological cell is positioned for logical cell, and the “prey” includes the at least one modified obligatory cooperation with the at least one modified micro 30 microorganism. In an embodiment, the "predator” includes organism. the at least one modified microorganism, and the “prey' Developing a modified biological cell that is formulated to includes the at least one modified biological cell. coexist in a cooperative relationship with another cell is con In an embodiment, the at least one modified microorgan ducted using routine laboratory procedures. For example, ism is in a syntrophic relationship with at least one other populations of Saccharomyces cerevisiae have been modified 35 modified microorganism. See, for example, Marx, Science to create obligatory cooperation by mutating each strain in vol. 324, pp. 1150-1151 (2009), which is incorporated herein Such a manner as to render each strain nutritionally deficient by reference. For example, one modified microorganism may without the other. See, for example, Shou, et al. PNAS, Vol. convert the primary resource to an intermediate that can be 104, no. 6, pp. 1877-1882 (2007), which is incorporated used by another modified microorganism. In an embodiment, herein by reference. For example, a first S. cerevisiae strain 40 one modified microorganism provides motility for another was modified in order to require adenine to grow and over modified microorganism, which may in turn provide a nutri produce lysine, while a second S. cerevisiae Strain was modi ent source. Id. fied to require lysine to grow and overproduce adenine. Id. In an embodiment, the modified microorganism includes at These modified, nonmating yeast strains compose a synthetic least one nucleic acid construct, including a heterologous obligatory cooperative system, termed COSMO (cooperation 45 genetic element that includes at least one artificial operon. In that is synthetic and mutually obligatory) by providing an an embodiment, the at least one artificial operon encodes at essential metabolite to the other strain. Id. As published, least one polycistronic mRNA transcript. persistent cooperation can be established with the modified In an embodiment, the modified microorganism includes at yeast strains, and is mathematically and experimentally least one heterologous genetic element encoding at least one shown to be viable over a wide range of initial conditions, 50 agent. In an embodiment, the heterologous genetic element with oscillating population ratio settling to a value predicted includes at least one of an inducible promoter, enhancer, or by nutrient Supply and consumption. Id. repressor operably coupled to the heterologous gene. In an Furthermore, even in the absence of explicitly engineered embodiment, the at least one inducible promoter includes at mechanisms to stabilize cooperation, the system can consis least one neutral, base or acid inducible promoter. In an tently develop increased ability to Survive reductions in popu 55 embodiment, the genetic element inducible to initiate death of lation density. Id. For example, members of a microorganism the at least one modified microorganism includes at least one consortium can exert both positive and negative control over of an inducible promoter, enhancer, or repressor operably one another's activities by exchanging metabolic intermedi coupled to the Suicide or other death-inducing gene. In an ates that either assist or compromise the growth of their embodiment, at least one inducer for one or more various neighbors. In one example, engineered acyl-HSL communi 60 inducible promoters, enhancers, or repressors is located in the cation has been used in biological "circuits’ that coordinate biological tissue or environmental medium to which the com population-wide behaviors ranging from population density position is administered or is to be administered. dependent fluorescence, cell Suicide, and invasion of cancer In an embodiment, at least one of the inducible promoter, cells, to pattern formation. Id. In one example, upon induction inducible enhancer, or inducible repressor includes at least of the biological circuit that encodes the communication and 65 one pH or temperature inducible promoter, enhancer, or the programmed cellular response, one population (preda repressor. In an embodiment, the inducible promoter, tor') dies out in the absence of the other (“prey') population. enhancer, or repressor includes an acid inducible promoter, US 8,852,916 B2 13 14 enhancer, or repressor. In an embodiment, the inducible pro medium treatment agent, or therapeutic agent, or the genetic moter, enhancer, or repressor includes a base inducible pro element inducible to initiate death in the modified microor moter, enhancer, or repressor. In an embodiment, the induc ganism. ible promoter, enhancer, or repressor includes a neutral As described herein for various inducible genetic elements, inducible promoter, enhancer, or repressor. the inducible genetic elements can include at least one induc Examples of acid inducible promoters include, but are not ible promoter, inducible repressor, or inducible enhancer. limited to HVA1 promoter (plant cells), P170, P1, or P3 Examples of inducers include, but are not limited to, at least (Lactococcus), baiA1, baiA3 (Eubacteria), lipF promoter one of radiation, temperature change, alcohol, antibiotic, Ste (Mycobacteria), FF-ATPase promoter (Lactobacillus, roid, metal, Salicylic acid, ethylene, benzothiadiazole, or 10 other compound. In an embodiment, the at least one inducer Streptococcus, or Enterococcus), gadC, gad D (Lactococcus, includes at least one of arabinose, lactose, maltose. Sucrose, Shignella), glutamate decarboxylase promoter (Mycobacte glucose, Xylose, galactose, rhamnose, fructose, melibiose, ria, Clostridium, Listeria, Lactobacillus), or similar operons. starch, inunlin, lipopolysaccharide, arsenic, cadmium, chro See, for example, Cotter and Hill, Microbiol. and Mol. Biol. mium, temperature, light, antibiotic, oxygen level, Xylan, Rev. Vol. 67, no. 3, pp. 429–453 (2003); Hagenbeek, et al., 15 nisin, L-arabinose, allolactose, D-glucose, D-xylose, D-ga Plant Phys., vol. 123, pp. 1553-1560 (2000); Madsen, et al., lactose, amplicillin, tetracycline, penicillin, pristinamycin, Abstract, Mol. Microbiol. vol. 56, no. 3, pp. 735-746 (2005): retinoic acid, or interferon. Other examples of inducers U.S. Pat. No. 6.242, 194; Richter, et al., Abstract, Gene, Vol. include, but are not limited to, at least a portion of one of an 395, no. 1-2, pp. 22-28 (2007), Mallonee, et al., J. Bacteriol. organic or inorganic Small molecule, clathrate or caged com vol. 172, no. 12, pp. 701 1-7019 (1990); each of which is pound, protocell, coacervate, microsphere, Janus particle, incorporated herein by reference. Examples of base inducible proteinoid, laminate, helical rod, liposome, macroscopic promoters include, but are not limited to, alkaline phos tube, niosome, Sphingosome, vesicular tube, Vesicle, Small phatase promoters. unilamellar vesicle, large unilamellar vesicle, large multila In an embodiment, the acid inducible promoter, enhancer, mellar vesicle, multivesicular vesicle, lipid layer, lipid or repressor is inducible at a pH of approximately 0.0, 25 bilayer, micelle, organelle, nucleic acid, peptide, polypeptide, approximately 0.5, approximately 1.0, approximately 1.5. protein, glycopeptide, glycolipid, lipoprotein, lipopolysac approximately 2.0, approximately 2.5, approximately 3.0, charide, Sphingolipid, glycosphingolipid, glycoprotein, pep approximately 3.5, approximately 4.0, approximately 4.5, tidoglycan, lipid, carbohydrate, metalloprotein, proteogly approximately 5.0, approximately 5.5, approximately 6.0, can, chromosome, nucleus, acid, buffer, protic solvent, approximately 6.5, approximately 6.6, approximately 6.7, 30 aprotic solvent, nitric oxide, vitamin, mineral, nitrous oxide, nitric oxide synthase, amino acid, micelle, polymer, copoly approximately 6.8, approximately 6.9, or any value therebe mer, monomer, prepolymer, cell receptor, adhesion molecule, tween or less. cytokine, chemokine, immunoglobulin, antibody, antigen, In an embodiment, the base inducible promoter, enhancer, extracellular matrix, cell ligand, Zwitterionic material, cat or repressor is inducible at a pH of approximately 7.1, 35 ionic material, oligonucleotide, nanotube, piloxymer, trans approximately 7.5, approximately 8.0, approximately 8.5. fersome, gas, element, contaminant, radioactive particle, approximately 9.0, approximately 9.5, approximately 10.0, radiation, hormone, virus, quantum dot, temperature change, approximately 10.5, approximately 11.0, approximately thermal energy, or contrast agent. See, for example, Theys, et 11.5, approximately 12.0, approximately 12.5, approxi al., Abstract, Curr. Gene Ther. Vol. 3, no. 3 pp. 207-221 mately 13.0, approximately 13.5, approximately 14.0, or any 40 (2003), which is incorporated herein by reference. In an value therebetween or greater. embodiment, the at least one inducer is produced by at least In an embodiment, the neutral inducible promoter, one microorganism. enhancer, or repressor is inducible at a pH of approximately In an embodiment, the at least one inducible genetic ele 7.0. ment is temperature inducible. For example, various heat The pH level of a particular biological tissue can affect the 45 shock protein promoters have been isolated from microorgan inducibility of the pH inducible promoter. For example, the isms, animal and plant cells (including maize and Soybeans), gastric pH of humans is between 1.5 (fasting state), and and other temperature inducible promoters have been isolated 3.0-5.0 (following feeding). Id. Likewise, human urine has a from various organisms and microorganisms. Some non-lim pH of approximately 6.0, human skin has a pH of approxi iting examples of promoters induced by a change in tempera mately 5.5, human ear canal has a pH of approximately 4.5, 50 ture include hsp70 (e.g., animal cells, maize), Gimhsp17.5-E while blood and cerebrospinal fluid tend to have a pH of (e.g., soybeans), Gimhsp17.6-L, HS6871 (e.g., soybeans), approximately 7.3-7.4. See, for example, Boron, et al., Medi HSP18.2 (Arabidopsis), HSP18.1 (Arabidopsis), Hsp70B, P2 cal Physiology: A Cellular and Molecular Approach. (Bacillus), P7 (Bacillus), PhS (E. coli), Tetrahymena thero Elsevier/Saunders. (2004), ISBN 1-4160-2328-3, which is phila promoter (protozoan), Hansenula polymorpha pro incorporated herein by reference. Furthermore, dental caries 55 moter (yeast), Schizosaccharomyces pombe promoters and tooth demineralization is initiated at a pH of approxi (yeast), or P, (lambda phage). See, for example, Taylor, et al. mately 5.2. Cotter and Hill, Ibid. Thus, a composition that is Cell, Abstract, vol. 38, no. 2, pp. 371-381 (1984); U.S. Pat. activated at various pH levels is useful for particular embodi No. 6,852,511, Wang, et al., Biochem. and Biophys. Res. ments. In an embodiment, the pH inducible promoter is Commun. Abstract, Vol. 358, no. 4, pp. 1148-1153 (2007), induced at a particular pH (e.g., chewing gum containing the 60 U.S. Pat. No. 7.462,708, each of which is incorporated herein composition described herein which is inducible at low pH in by reference. the mouth). The pH level of plant leaves is approximately 4.2. In an embodiment, the at least one modified microorgan See, for example, Sargent and Blackman, Abstract, J. Exp. ism includes at least one of a prokaryote or a eukaryote. In an Botany, Vol. 21, no. 1, pp. 219-227 (2005), which is incorpo embodiment, the at least one modified microorganism rated herein by reference. In an embodiment, the at least one 65 includes at least one of bacteria, protozoa, rotifers, algae, inducible promoter or inducible enhancer facilitates tran archaeon, or fungi. In an embodiment, the at least one modi Scription of the genetic element encoding the environmental fied microorganism includes at least one of a non-pathogenic US 8,852,916 B2 15 16 Strain, transgenic microorganism, magnetotactic microorgan bacillus minutus, Lactobacillus mucosae, Lactobacillus ism, anaerobic or aerobic microorganism, food grade strain, murinus, Lactobacillus nagelii, Lactobacillus Oris, Lactoba obligate microorganism, attenuated microorganism strain, cillus panis, Lactobacillus parabuchneri, Lactobacillus facultative anaerobe, non-invasive strain, probiotic, coloniz paracasei, Lactobacillus paracasei Subsp. paracasei, Lacto ing microorganism, element-modifying microorganism, or 5 bacillus paracasei Subsp. tolerans, Lactobacillus parakefiri, photosynthetic microorganism. In an embodiment, the at Lactobacillus paralimentarius, Lactobacillus paraplan least one element-modifying microorganism includes at least tarum, Lactobacillus pentosus, Lactobacillus perolens, Lac one of a nitrogen-fixing microorganism, nitrifying microor tobacillus piscicola, Lactobacillus plantarum, Lactobacillus ganism, denitrifying microorganism, hydrocarbon-utilizing pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lac microorganism, dechlorinating microorganism, or a Sulfate 10 tobacillus rimae, Lactobacillus rogosae, Lactobacillus rumi reducing microorganism. nis, Lactobacillus sakei, Lactobacillus sakei Subsp. carnosus, In an embodiment, the at least one auxotrophic or modified Lactobacillus sakei Subsp. sakei, Lactobacillus salivarius, microorganism includes at least one of Bifidobacterium, Lac Lactobacillus salivarius Subsp. salicinius, Lactobacillus Sali tococcus, Lactobacillus, Salmonella, Clostridium, Escheri varius Subsp. salivarius, Lactobacillus Sanfranciscensis, chia, Listeria, Streptococcus, Staphlococcus, Bacillus, 15 Lactobacillus sharpeae, Lactobacillus Suebicus, Lactobacil Marinobacter, Micrococcus, Dietzia, Oceanobacillus, Cit lus trichodes, Lactobacillus uli, Lactobacillus vaccinoster riococcus, Georgenia, Microbacterium, Stappia, Isopteri cus, Lactobacillus vaginalis, Lactobacillus viridescens, Lac colla, Cellulomonas, Rhizobia, Frankia, Klebsiella, Nocar tobacillus vitulinus, Lactobacillus xylosus, Lactobacillus dioform Actinomycetes, Cytophagacia, Corynebacterium, yamanashiensis, Lactobacillus vananashiensis Subsp. mall, Vibrionacia, Cyanobacteria, Pseudomonas, Rhastonia, Lactobacillus vananashiensis Subsp. Yamanashiensis, Lac Sphaerotilus, Shewanella, Wolbachia, or Azotobacter, tobacillus zeae, Clostridium novyi, Clostridium sordellii, AzOspirillum. In an embodiment, the at least one auxotrophic Bifidobacterium longum, Escherichia coli, Salmonella typh microorganism includes at least one of Saccharomyces, Can imurium, Salmonella paratyphi, Salmonella pneumoniae, dida, Brettanomyces, Zygosaccharomyces, Yarrowia, Salmonella enterica, Streptococcus pneumoniae, Streptococ Schizosaccharomyces, Torulaspora, Neotyphodium, or Cryp 25 cus pyogenes, Streptococcus group B, Streptococcus mutans, to COCC tiS. Streptococcus sobrinus, Streptococcus equi, Staphylococcus Some non-limiting examples of microorganisms that can ssp., Erysipelothrix rhusiopathiae, Bacillus anthracis, List be modified according to various embodiments described eria monocytogenes, Mycobacterium leprae, Mycobacte herein include: Lactococcus garvieae, Lactococcus lactis, rium tuberculosis, Clostridium tetani, Clostridium botuli Lactococcus lactis Subsp. Cremoris, Lactococcus lactis 30 num, Clostridium difficile, Clostridium perfingens, Subsp. hordniae, Lactococcus lactis, Lactococcus lactis Corynebacterium diphtheriae, Mycoplasma ssp., Rhodococ Subsp. Lactis, Lactococcus piscium, Lactococcus plantarum, cus, Nocardia, Gordona, Jensenia, Shewanella, Shewandella Lactococcus raffinolactis, Lactobacillus acetotolerans, Lac Oneidensis, Dehalococcoides, Burkholderia Zenovorans, tobacillus acidophilus, Lactobacillus agilis, Lactobacillus Comamonas, Cupriavidus, Sphingomonas, Acidovorax, Des algidus, Lactobacillus alimentarius, Lactobacillus amy 35 ulfo vibrio, Anabaena cylindrica, Plectonema, Nostoc com lolyticus, Lactobacillus amylophilus, Lactobacillus amylo mune, Rhodobacter sphaeroides, Rhodopseudomonas palus vorus, Lactobacillus animalis, Lactobacillus aviarius, Lac tris, or Rhodobacter capsulatus, Escherichia coli Nissle tobacillus aviarius Subsp. araffinosus, Lactobacillus aviarius 1917, Salmonella typhimurium 14028, Salmonella typhimu Subsp. aviarius, Lactobacillus bavaricus, Lactobacillus rium SL1344, Salmonella typhi, Salmonella abortus-ovi, Sal bifermentans, Lactobacillus brevis, Lactobacillus buchneri, 40 monella abortus-equi, Salmonella Dublin, Salmonella galli Lactobacillus bulgaricus, Lactobacillus carnis, Lactobacil narum, Salmonella pullorum, Shigella fexneri, Shigella lus casei, Lactobacillus casei Subsp. alactosus, Lactobacillus sonnei, Haemophilus influenzae, Bordetella pertussis, Nis casei Subsp. casei, Lactobacillus casei Subsp. pseudoplan seria meningitides, Nisseria gonorrohia, Pasteuralla multo tarum, Lactobacillus casei Subsp. rhamnosus, Lactobacillus cida, Yersinia pestis, Escherichia coli 4608–58, Salmonella casei Subsp. tolerans, Lactobacillus catenaformis, Lactoba 45 flexneri 2a SC602, Escherichia coli CFTO73, Escherichia cillus cellobiosus, Lactobacillus collinoides, Lactobacillus coli Top10, Escherichia coli MC1000, Escherichia coli confusus, Lactobacillus coryniformis, Lactobacillus coryni NF1815, Escherichia coli NF1830, Escherichia coli HB101, formis Subsp. coryniformis, Lactobacillus coryniformis Escherichia coli BD3364, Escherichia coli BD3364, Subsp. torquens, Lactobacillus Crispatus, Lactobacillus cur Escherichia coli HfrC, Escherichia coli BD3342, Escheri vatus, Lactobacillus curvatus Subsp. curvatus, Lactobacillus 50 chia coli BD3346, Escherichia coli XAC, Escherichia coli curvatus Subsp. melibiosus, Lactobacillus delbrueckii, Lac BD71, Escherichia coli BD76, Escherichia coli S17.1, Lac tobacillus delbrueckii Subsp. bulgaricus, Lactobacillus del tococcus lactis MG 1363, Lactococcus lactis-Thy 12, brueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. Clostridium novyi-NT, Lactobacillus plantarum lactis, Lactobacillus divergens, Lactobacillus farcininis, NCIMB8826, Lactobacillus plantarum NCIMB8826Intl. Lactobacillus fermentum, Lactobacillus fomicalis, Lactoba 55 Lactobacillus fermentum KLD, Lactobacillus plantarum cillus fructivorans, Lactobacillus fructosus, Lactobacillus MD007, Lactobacillus plantarum MD007Intó, Lactococcus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, lactis NZ3900, MC-1 magnetotactic bacteria, Lactococcus Lactobacillus halotolerans, Lactobacillus hamsteri, Lacto lactis PH3960, Streptococcus gordonii, Lactobacillus zeae, bacillus helveticus, Lactobacillus heterohiochii, Lactobacil Streptococcus mutans, Bacteroides ovatus, Bacteroides fra lus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, 60 gilis, PrevOtella, Saccharomyces boulardii, Firmicutes, Giam Lactobacillus intestinalis, Lactobacillus jensenii, Lactoba maproteobacteria, PrevOtellaceae, Archaea, Listeria cillus johnsonii, Lactobacillus kandleri, Lactobacillus kefiri, innocua, Staphylococcus xylosus, Staphylococcus Carnosus, Lactobacillus kefiranofaciens, Lactobacillus kefirgranum, Listeria monocytogenes, Klebsiella pneumoniae, Azoto Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus bacter vinlandii, Anabaena cylindrica, Plectonema, Nostoc leichmannii, Lactobacillus lindneri, Lactobacillus malefer 65 commune, Rhodobacter sphaeroides, Rhodopseudomonas mentans, Lactobacillus mali, Lactobacillus maltaronicus, palustris, Rhodobacter capsulatus, Clavibacter; Alcaligenes, Lactobacillus manihotivorans, Lactobacillus minor, Lacto Sphingobacterium, Phyllobacterium, Aeromonas, Stenotro US 8,852,916 B2 17 18 phomonas, Acidovorax, Comamonas, Desulfovibrio, Steintro incorporated herein by reference. Microorganism popula phomonas, Serratia, Pseudomonas aeruginosa, Steintroph tions vary according to age, disease state or health State, and Omonas maltophilia, Serratia marsescens, Variovorax, diet. Id. For example, Lactobacili are dominant among Chryseobacterium, Comamonas, Acidovorax, Stenotroph microflora associated with the urogenital tract of healthy Omonas, Sphingobacterium, Xanthomonas, Frateuria, Zoog women but are almost completely absent in patients who loea, Alcaligenes, Flavobacterium, Derxia, Lampropedia, develop most forms of urinary tract infections. See, for Brucella, Xanthobacter. Thermus, Thermonicrobium, example, Hanniffy, et al., Adv. Applied Microbiol. Vol. 56, Halomonas, Alteromonas, Serpens, Janthinobacterium, Bor pp. 1-64 (2004), which is incorporated herein by reference. detella, Paracoccus, Beijerinckia, Francisella, Eubacteria, Published reports indicate that the human gut microbiota Actinomycetes, Nocardia, Rhodococcus, Gordona, Nocar 10 dioides, Saccharopolyspora, Micropolyspora, Promi affects nutrition, development, metabolism, pathogen resis cromonospora, Intrasporangium, Pseudonocardia, Oerisk tance, and regulation of immune responses; and the micro Ovia, Stomatococcus, Planococcus, Aerococcus, biota community also changes with antibiotic use. See, Deth Peptococcus, Peptostreptococcus, Coprococcus, Gemella, lefsen, et al., PLOS Biol., vol. 6, no. 11, pp. 2383-2400 Pediococcus, Leuconostoc, Ruminococcus, Sarcina, Aeromo 15 (2008), which is incorporated herein by reference. For nas, Photobacterium, Vibrio, Plesiomonas, Zymomonas, example, as published, approximately 3300-5700 bacterial Chronobacterium, Cardiobacterium, Calymmatobacterium, taxa were identified as accounting for over 99% of the vari Streptobacillus, Eikenella, Gardnerella, Phyllobacterium, able region sequence tags obtained. Id. Also as published, Rhizobium, Bradyrhizobium, Agrobacterium, Cytophaga, antibiotic use of ciprofloxacin influences about one third of Flexibacter, Saprospira, Flexithrix, Herpetosiphon, Capno the bacterial taxa in the gut. Id. cytophaga, Sporocytophaga, Aureobacterium, Agromyces, Other published studies have found that Lactococcus lactis Arachnia, Rothia, Acetobacterium, Actinomyces, Arthro is well adapted to deliver medical proteins to the mucosal bactera, Arcanobacterium, Lachnospira, Propionibacte immune system, and is capable of delivery proteins that elicit rium, Eubacterium, Butyrivibria, Brevibacterium, Bifidobac both systemic and mucosal immune responses. For example, terium, Microbacterium, Caseobacter; Of 25 L. lactis has been used as a live vector for in situ delivery of Thermoanaerobacter, Enterobacter sp. 638, or Burkholderia biologically active IL-12. See, for example, Bermudez-Hu cepacia BU72, or other strain. maran, et al., J. Mol. Microbiol. Biotechnol. Abstract. Vol 14 In an embodiment, the at least one modified microorgan (1-3), pp. 80-89 (2008), which is incorporated herein by ism includes MC-1 magnetotactic bacteria. For example, reference. magnetotactic bacteria have flagella that provide for mobili 30 Further published reports indicate high diversity of micro Zation, and can be combined with nanoparticles of magnetite organisms located in the human mouth, with a small majority or magnetosome chains embedded in the bacteria for direct in common amongst any particular geographical region ing the microbes. See, for example, Felfoul, et al., IEEE group. See, for example, Myles, et al., Abstract, BMC Med. Xplore Abstract, issue 22-26, pp. 1463-1466 (2007), which is Gen. Vol. 2, no. 45 (2009), which is incorporated herein by incorporated herein by reference. 35 reference. For example, the populations of individual groups In an embodiment, the modified microorganism includes at of bacteria located in the saliva of one person tested were not least one microorganism with the designation of 'generally largely common with other people located in a similar geo regarded as safe” (GRAS) status in the food industry. graphical region. Id. Many microorganism strains are registered with the Ameri In an embodiment, at least one microorganism (or a popu can Type Culture Collection (ATCC, Rockville, Md., USA), 40 lation thereof) is obtained from at least one subject, modified, and can be adapted for use with various embodiments and returned to the at least one Subject, and possibly others described herein. (e.g., members of a household). In an embodiment, the at least In an embodiment, any microorganism for which at least a one modified microorganism (or a population thereof) is portion of the genome has been sequenced can be utilized in returned to approximately the same location of the Subject compositions described herein. For example, by sequencing 45 from which it was extracted. In an embodiment, the at least the genome of a particular microorganism, regulatory ele one modified microorganism (or a population thereof) is ments and sites for chromosomal insertion can be identified. returned to a different location in the subject source. For Furthermore, bioinformatics and promoter-trapping strate example, at least one microorganism can be extracted from gies can assist in locating endogenous promoters to further the intestine of a subject, modified, and returned to the oral regulate production of the at least one therapeutic or environ 50 cavity, otic cavity, Stomach, or intestine of that same Subject. mental medium treatment agent. In an embodiment, at least one microorganism is extracted, In an embodiment, at least one modified microorganism modified, and returned to the Subject at approximately the includes a microorganism that is isolated from at least one same location from where it was extracted, and the modified biological tissue, environmental medium, a Subject, or other microorganism is able to translocate to another location Substrate. The microorganism is modified, and placed into at 55 within the Subject or to another Subject (e.g., maternal trans least one biological tissue, environmental medium, Subject, fer, peristalysis through the digestive tract, etc.). In an or other substrate (which may be the same or different sub embodiment, at least Some genetic sequence information strate as the source of the microorganism). from the microorganism is obtained prior to or Subsequent to Selection of the particular microorganism can be based on modifying the microorganism. In an embodiment, the at least calculations of particular Sub-populations in a larger popula 60 one modified microorganism is amplified prior to reinstating tion, a particular Subject, a particular state of health of the it in the at least one biological tissue, or Subject. Subject, a particular organ, a particular biological tissue, or a Published studies indicate that the human gut contains particular biological cell type. For example, the intestinal distinct differences in the microflora of obese subjects, nor microflora can contribute to pathogenesis in Susceptible Sub mal weight Subjects, and Subjects who have undergone gastric jects, or it can contribute to the metabolism and overall health 65 bypass Surgery. See, for example, Zhang, et al., PNAS, pp. of the subject. See, for example, O'Hara and Shanahan, 1-6; available online at 10.10973/pnas.0812600106 (printed EMBO reports, vol. 7, no. 7, pp. 688-693 (2006), which is 2009, available 2008), which is incorporated herein by refer US 8,852,916 B2 19 20 ence. Further, these studies indicate a shift in microbiota includes at least one of an inducible promoter, inducible populations following gastric bypass Surgery. Id. enhancer, or inducible repressor. In an embodiment, a microorganism is selected from at In an embodiment, the at least one modified microorgan least one subject based on a desired state of health, modified ism produces at least one therapeutic agent or environmental to produce at least one therapeutic agent, and placed in the 5 medium treatment agent. In an embodiment, the at least one same or different subject. Selection of a particular microor therapeutic agent or environmental medium treatment agent ganism can be conducted utilizing conventional techniques, includes at least a portion of one of an organic or inorganic including but not limited to barcoded 16S pyrosequencing. Small molecule, proteinoid, nucleic acid, peptide, polypep See, for example, Andersson, et al., PLOS One vol. 3, no. 7, tide, protein, glycopeptide, glycolipid, lipoprotein, pp. 1-8 (2008), which is incorporated herein by reference. 10 lipopolysaccharide, Sphingolipid, glycosphingolipid, glyco The relatively small genome size of microorganisms and the availability of high-throughput sequencing facilities has protein, peptidoglycan, lipid, carbohydrate, metalloprotein, allowed for the production of sequence information of more proteoglycan, Vitamin, mineral, amino acid, polymer, copoly than 90 bacterial genomes for the public domain. Id. mer, monomer, prepolymer, cell receptor, adhesion molecule, In an embodiment, the at least one modified microorgan 15 cytokine, chemokine, immunoglobulin, antibody, antigen, ism includes at least one vector including at least one genetic extracellular matrix component, cell ligand, oligonucleotide, element for producing an agent, or initiating death in the element, hormone, or contrast agent. microorganism. Various vectors can be utilized, as described In an embodiment, the polymer or co-polymer includes at herein, including at least one of a plasmid, bacteriophage, least one of polyester, polylactic acid, polyglycolic acid, cel cosmid, artificial chromosome, or other vector. In an embodi lulose, nitrocellulose, urea, urethane, or other polymer. For ment, the modified microorganism includes multiple vectors, example, in an embodiment, at least one microorganism that some of which can be the same or different from the other is capable of synthesizing at least one thermoplastic polymer vectors. In an embodiment, the modified microorganism (e.g., polyester) is utilized. See, for example, U.S. Pat. No. includes at least one vector that is able to regulate initiation of 5,663,063, which is incorporated herein by reference. For death of the at least one modified microorganism, and also 25 example, as described in U.S. Pat. No. 5,663,063, E. coli generate the at least one therapeutic agent or environmental strains can be modified to produce polyhyroxybutyrate and medium treatment agent. polyhydroxyalkanoate polyesters. Id. Vectors suitable for microbiological applications are well In an embodiment, at least one microorganism that is known in the art, and are routinely designed and developed capable of degrading at least one polymer (e.g., aliphatic for particular purposes. Some non-limiting published 30 polyester) is utilized. See, for example, Suyama, et al., App. examples of vectors that have been used to transform bacterial Env. Microbiol. vol. 64, no. 12, pp.5008-5011 (1998), which strains include the following plasmids: pMW211, pBAD is incorporated herein by reference. For example, in an DEST49, plDONRP4-P1R, pFNTR-P pENTR-DUAL, embodiment, the at least one microorganism is capable of pENTR-term, pBR322, pIDESTR4-R3, pBGS18-N9uc8, degrading at least one of poly(beta-hydroxyalkanoate), poly pBS24Ub, pUbNuc, pIXY 154, pBR322DEST, 35 (epsilon-caprolactone), or poly(hexamethylene carbonate). pBR322DEST-P-DUAL-term, p.JIM2093, pTG2247, Id. pMEC10, pMEC46, pMEC127, pTX, pSK360, p.ACYC184, In an embodiment, the polymer or co-polymer includes at pBOE93, pBR327, pIDW205, pKCL11, pKK2247, pMR60, least one of polyester, polylactic acid, polyglycolic acid, cel pOU82, pR2172, pSK330, pSK342, pSK355, puHE21-2, lulose, nitrocellulose, urea, urethane, or other polymer, as pEHLYA2-SD, See, for example, Stritzker, et al. Intl. J. Med. 40 described herein. Microbiol. Vol. 297, pp. 151-162 (2007); Grangette et al., In an embodiment, the therapeutic agent or environmental Infect. Immun. Vol. 72, pp. 2731-2737 (2004), Knudsen and medium treatment agent includes at least one of calcium, Karlstrom, App. and EnV. Microbiol. pp. 85-92, Vol. 57, no. 1 carbon, nitrogen, Sulfur, nitrate, nitrite, copper, magnesium, (1991), Rao et al., PNAS pp. 11193-11998, vol. 102, no. 34 Selenium, boron, Sodium, aluminum, phosphorus, potassium, (2005), each of which is incorporated herein by reference. 45 titanium, chromium, manganese, iron, nickel, Zinc, silver, Therapeutic Agents and Environmental Medium Treatment barium, lead, Vanadium, tin, strontium, or molybdenum. Agents In an embodiment, the at least one therapeutic agent or Examples of the at least one therapeutic agent or environ environmental medium treatment agent includes at least one mental medium treatment agent produced by the at least one enzyme able to convert at least one prodrug or precursor modified microorganism are described herein. As indicated, 50 compound into an active state. In an embodiment, the at least the at least one therapeutic agent is produced for at least one one enzyme includes at least one of beta glucuronidase, biological tissue, while the at least one environmental cytosine deaminase, or nitroreductase. In an embodiment, the medium treatment agent is produced for at least one environ at least one therapeutic agent or environmental medium treat mental medium. Thus, in certain instances, the at least one ment agent includes at least one nutraceutical. In an embodi therapeutic agent or at least one environmental medium treat 55 ment, the at least one therapeutic agent or environmental ment agent are similar (e.g., mineral), and in other instances, medium treatment agent excludes nutraceuticals. they are different and correspond to the particular biological In an embodiment, the at least one therapeutic agent or tissue, or environmental medium, respectively. environmental medium treatment agent is pH dependent. In an embodiment, the at least one therapeutic agent or Thus, in certain embodiments, the agent can be administered environmental treatment agent is encoded by at least one 60 to one particular location and remain inactive until conditions vector. In an embodiment, the vector includes at least one of change that result in alteration of the pH of the location (or the a plasmid, bacteriophage, cosmid, artificial chromosome, or modified microorganism travels to another location with a other vector. In an embodiment, the vector encodes two or different pH), and the agent becomes active. The activity of more therapeutic agents or environmental treatment agents. the agent can be gradual (along a gradation), or immediate. As In an embodiment, the two or more therapeutic agents or 65 described herein, various formulations for the compositions environmental treatment agents are linked to different pro disclosed can be provided, depending on the location and the moters. In an embodiment, as described herein, the vector therapeutic agent. US 8,852,916 B2 21 22 In an embodiment, the at least one therapeutic agent is In an embodiment, the at least one therapeutic agent capable of modulating at least one immune response. In an includes at least one vaccine. In an embodiment, the at least embodiment, wherein the at least one immune response one vaccine includes at least one of an antigenic peptide, includes at least one allergic or autoimmune response. In an antigenic protein, or antigenic carbohydrate. In an embodi embodiment, the at least one therapeutic agent is capable of 5 ment, the at least one vaccine includes at least one of an inducing apoptosis in one or more cells of the at least one envelope protein, capsid protein, Surface protein, toxin, biological tissue. In an embodiment, the at least one admin polysaccharide, oligosaccharide, or enzyme needed to make istered therapeutic agent modulates the viability, prolifera at least one thereof. In an embodiment, the vaccine compo tion, or metastasis of at least one tumor cell in the at least one sition further comprises at least one adjuvant. In an embodi 10 ment, the at least one therapeutic agent includes at least one biological tissue. anti-inflammatory cytokine. In an embodiment, the at least one therapeutic agent In an embodiment, at least one therapeutic agent includes includes at least one of insulin, clacitonin, lutenizing hor at one of Interleukin-1, Interleukin-2, Interleukin-3, Interleu mone, parathyroid hormone, Somatostatin, thyroid stimulat kin-4, Interleukin-5, Interleukin-6, Interleukin-7. Interleu ing hormone, vasoactive intestinal polypeptide, tumor necro 15 kin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleu sis metabolite, endostatin, angiostatin, anti-angiogenic kin-12, Interleukin-13, Interleukin-14, Interleukin-15, antithrombin II, fibronectin, prolactin, thrombospondin I, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin laminin, procollagen, collagen, integrin, steroid, corticoster 19, Interleukin-20, Interleukin-21, Interleukin-22, Interleu oid, virus antigen, microorganism antigen, trefoil protein, or kin-23, Interleukin-24, Interleukin-25, Interleukin-26, Inter lipase. leukin-27, Interleukin-28, Interleukin-29, Interleukin-30, The trefoil peptide can include, but not be limited to, TFF1 Interleukin-31, Interleukin-32, Interleukin-33, Interleukin (also known as pS2, or breast cancer estrogen inducible 34, Interleukin-35, Interleukin-36, Interleukin-37, Interleu gene), TFF2 (also known as SP, or spasmolytic peptide), or kin-38, Interleukin-39, Interleukin-40, Interleukin-41, Inter TFF3 (also known as ITF, or intestinal trefoil factor). See, for leukin-42, Interferon-Y, Interferon-C. Interferon-?3. example, U.S. Patent Application Publication Nos. 25 Transforming Growth factor, Granulocyte Macrophage 200701 10723, and 20070122427; each of which is incorpo Colony Stimulating Metabolite, Macrophage-Colony Stimu rated herein by reference. See also, U.S. Pat. No. 7,220,418, lating Metabolite, Scarecrow, Erythropoietin, Granulocyte which is incorporated herein by reference. The vaccine can Colony Stimulating Metabolite, Leukemia Inhibitory include but not be limited to antigenic peptides, proteins, or Metabolite. Oncostatin M, Ciliary Neurotrophic Metabolite, carbohydrates. For example, the vaccine can include but not 30 Growth Hormone, Prolactin, Fibroblast Growth factor, Nerve be limited to envelope proteins, capsid proteins, Surface pro Growth factor, Platelet Derived Growth factor, Epidermal teins, toxins, polysaccharides, oligosaccharides, or enzymes Growth factor, Fas, Fas ligand, CD40, CD27, CD4, CD8, needed to make at least one thereof. CD2, CD3, Tumor Necrosis Metabolite-C, or Tumor Necro In an embodiment, the virus antigen includes at least one sis Metabolite-B. antigen from one or more of a double-stranded DNA virus, 35 Chemokines are biochemical signaling molecules that act single-stranded DNA virus, double-stranded RNA virus, (+) to attract other particular molecules, including but not limited single-strand RNA virus, (-) single-strand RNA virus, single to cells, to a specific site. In at least one embodiment, the strand RNA-Reverse Transcriptase virus, or double-stranded therapeutic agent includes one or more chemokines. In at DNA-Reverse Transcriptase virus. In an embodiment, the least one embodiment, the one or more chemokines include at virus antigen includes at least one antigen of a virus from one 40 least one of a CC chemokine, CXC chemokine, C chemokine, or more of the family of Adenoviridae, Arenaviridae, Bun or CX3C chemokine. In an embodiment, the at least one yaviridae, Calciviridae, Circoviridae, Coronaviridae, Filov therapeutic agent includes at least one of CCL1, CCL2. iridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orth CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, omyxoviridae, Papovaviridae, Papillomaviridae, CCL11, CCL12, CCL13, CCL14. CCL15, CCL16, CCL17, Polyomaviridae, Paramyxoviridae, Parvoviridae, Picor 45 CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, naviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviri CCL25, CCL26, CCL27, CCL28, CCL29, CXCL1, CXCL2, dae, Pseudoviridae, or Togaviridae. In an embodiment, the CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, virus antigen includes at least one antigen from one or more of CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14. human immunodeficiency virus (HIV) type I, HIV-type 2, CXCL15, CXCL16, CXCL17, CXCL18, CXCL19, simian immunodeficiency virus (SIV), or feline leukemia 50 CXCL20, CXCL21, CXCL22, XCL1, XCL2, XCL3, XCL4, virus. In an embodiment, the virus antigen includes at least XCL5, CX3CL1, CX3CL2, or CX3CL3. one antigen from one or more of respiratory syncytial virus In an embodiment, administration of the at least one thera (RSV), influenza (flu), adenovirus, rhinovirus, enterovirus, peutic agent results in at least one of nucleic acid transcrip poliovirus, rubella virus, paramyxovirus, herpes simplex tion, protein translation, cell division, apoptosis, necrosis, virus type I (HSV-1), Herpes simplex virus 2 (HSV-2), rotavi 55 cytoskeletal rearrangement, cell differentiation, or secretion rus, neurotropic virus, coxsackie virus, hepatitis virus type A, in the at least one biological tissue. hepatitis virus type B, hepatitis virus type C, or oncovirus. In In an embodiment, the at least one therapeutic agent an embodiment, the at least one antigen includes at least one includes at least one prodrug or precursor compound. For antigen from one or more of Brucella, Chlamydia, Citro example, the at least one prodrug or precursor compound bacter, Coxiella brunetii, Escherichia coli, Francisella tulla 60 includes at least one glucuronide prodrug, Such as at least one rensis, Haemophilius, Legionella, mycobacterium, Neisseria, glucuronide of epirubicin, 5-fluorouracil, 4-hydroxycyclo Pseudomonas, Rickettsia, Salmonella, Shigella, Staphyllo phosphamide, or 5-fluorocytosine. In another example, the at coccus, Streptococcus, Candida, Cryptococcus, Aspergillus, least one prodrug or precursor compound includes 5-(aziri Blastomyces, Histoplasma, Paracoccidioides, Nosema, din-1-yl)-2,4-dinitrobenzamide. In an embodiment, the at Encephalitozoon, Torulopsis, Pneumocystis, Trypanosoma, 65 least one therapeutic agent includes at least one prodrug. In an Leishmania, Theileria, Plasmodium, Cryptosporidium, or embodiment, at least one modified microorganism delivers at Toxoplasma. least one prodrug, while at least one other modified microor US 8,852,916 B2 23 24 ganism delivers at least one enzyme capable of converting the In an embodiment, the at least one modified microorgan at least one prodrug into active form. For example, published ism includes at least one synthetic protein scaffold positioned studies demonstrating strains of recombinant Saccharomyces to modulate the stoichiometry of the synthesis of the at least cerevisiae expressing plant P450 73A1 show that the enzyme one therapeutic agent or environmental medium treatment is active and able to convert trans-cinnamic acid into p-cou agent. For example, in an embodiment, the at least one syn maric acid in vivo. See, for example, Garrait, et al., Applied thetic protein scaffold is positioned to spatially or temporally Env. Microbiol. vol. 73, no. 11, pp. 3566-3574 (2007), and recruit at least one protein or fragment thereof. For example, Blanquet, et al., Applied EnV. Microbiol. Vol. 69, no. 5, pp. synthetic protein scaffolds that spatially recruit metabolic 2884-2892 (2003), each of which is incorporated herein by enzymes can be designed and modulate the stochiometry of a reference. In an embodiment, the at least one therapeutic 10 biosynthetic pathway. See, for example, Dueber et al., Nat. agent or environmental medium treatment agent includes at Biotech. vol. 27, no. 8, pp. 753-759 (2009), which is incor least one converting enzyme responsive to the at least one porated herein by reference. In one example, at least one flux prodrug or precursor compound. In an embodiment, the at of an enzymatic reaction of a particular biosynthetic pathway least one therapeutic agent or environmental medium treat can be balanced to, for example, limit the accumulation of ment agent includes at least one time-release formulation. In 15 intermediates, optimize production levels, prevent loss of an embodiment, the at least one therapeutic agent or environ intermediates, protect unstable intermediates from degrada mental medium treatment agent is pH dependent or tempera tion, circumvent unfavorable equilibria and kinetics imposed ture dependent. by bulk-phase metabolite concentration, etc. Id. In an In an embodiment, the at least one modified microorgan embodiment, Scaffolds provide a means for controlling ism delivers at least two different therapeutic agents. In cer design by physically separating catalytic activities from bind tain cases, the at least two different therapeutic agents are ing element, which allows modification (i.e., single interac regulated by at least one different promoter. In an embodi tion to each enzyme) to the enzyme needed for attaining ment, at least one therapeutic agent includes at least one modular control over complex formation. Id. Designing Such antigen, and at least one cytokine. In an embodiment, the at scaffolds can be performed by tethering a short peptide ligand least one antigen includes tetanus toxin fragment. In an 25 to the enzyme, for example an SH2 domain, SH3 domain, embodiment, the at least one cytokine includes at least one of GTPase binding domain, etc. In one particular embodiment, a IL-2 or IL-6. In an embodiment, the antigen is contained synthetic complex is designed by attaching a varying number within the at least one modified microorganism, while the of SH3 interaction ligands to the C terminus of one enzyme cytokine is secreted. See, for example, Steidler, et al. Infect. (e.g., synthase), and attaching an SH3 domain to the N ter Immun. pp. 3183-3189, vol. 66, no. 7 (1998), and U.S. Pat. 30 minus of another enzyme (e.g., reductase). Id. No. 6,605.286; each of which is incorporated herein by ref In an embodiment, the at least one modified microorgan erence. In another example, published studies show secretion ism includes at least one riboregulated transcriptional cas of human myelin basic protein (hMBP) or hMBP as a fusion cade counter. See, for example, Friedland, et al., Science, Vol. protein with beta-glucuronidase from E. coli. The heterolo 324, pp. 1199-1202, (2009), which is incorporated herein by gous products are produced by L. casei. See, for example, 35 reference. In an embodiment, the at least one riboregulated Maassen, et al., Vaccine, Abstract, Vol. 17, no. 17, pp. 2117 transcriptional cascade counter is positioned to produce at 2128 (1999), which is incorporated herein by reference. least one product upon exposure to at least one inducer. The at least one modified microorganism includes at least Various inducers are described herein. Several non-limit one heterologous genetic element encoding at least one thera ing examples include physical or chemical inducers, such as peutic agent or environmental medium treatment agent by at 40 radiation, temperature, carbohydrate, peptide, protein, alco least one of mode, including but not limited to synthesizing hol, antibiotic, steroid, metal, salicylic acid, ethylene, ben the at least one agent, expressing the at least one agent on its Zothiadiazole, or other compound. In an embodiment, the at Surface, or extracellular secretion of the at least one therapeu least one antibiotic includes at least one of amplicillin, tetra tic agent or environmental medium treatment agent. cycline, penicillin, pristinamycin, or other antibiotic. In an In an embodiment, the at least one modified microorgan 45 embodiment, multiple different inducers may be required for ism includes at least one heterologous genetic element encod the various inducible genetic elements. ing at least one therapeutic agent and expresses at least a In an embodiment, the at least one riboregulated transcrip portion of the agent (e.g. antibody) on its Surface. In an tional cascade counter is positioned to produce at least one embodiment, the at least one modified microorganism sequence of products upon exposure to at least one sequence expresses at least a portion of a carbohydrate-binding motif 50 of inducers. In an embodiment, the at least one riboregulated on its Surface. In one example, the at least one modified transcriptional cascade counter is positioned to produce at microorganism expresses at least one of a heparin or mannose least one product that is different than at least one other binding motif its surface. consecutive product. In an embodiment, the at least one ribo For example, as illustrated in FIG.4, in an embodiment, a regulated transcriptional cascade counter is positioned to pro vector 400 including at least one heterologous genetic ele 55 duce at least one first product upon exposure to at least one ment 410 is placed into at least one microorganism 420 by first inducer. In an embodiment, the at least one riboregulated methods known in the art (e.g., electroporation, transforma transcriptional cascade is inducible to produce at least one tion, etc.). Once incorporated into the microorganism 420, the second product upon exposure to at least one second inducer. heterologous genetic element 410 is transcribed, resulting in In an embodiment, the at least one riboregulated transcrip production of at least one transcript 430, which is converted 60 tional cascade is inducible to produce at least one third prod intracellularly into at least one protein 440. In an embodi uct upon exposure to at least one third inducer, etc. See ment, the protein can remain intracellularly 440 or be secreted Friedland, Ibid. In an embodiment, one or more of the first into a periplasmic space (not shown). Thus, the protein is product, the second product, or the third product includes the obtained through lysis of the microorganism 420. In an at least one therapeutic agent or environmental medium treat embodiment, the protein is expressed on the surface 450 of 65 ment agent. the microorganism 420. In an embodiment, the protein is In an embodiment, the at least one modified microorgan secreted extracellularly 460. ism is inducible to produce the at least one agent by at least US 8,852,916 B2 25 26 one twin-arginine translocation system. See, for example, tively to a location (e.g., by utilizing particular cell ligand Widdick, et al., PNAS, vol. 103, no. 47, pp. 17927-17932 receptor interactions, oxygen or other gas levels, magnetic (2006), which is incorporated herein by reference. For particles tagged to the microorganism, or other mechanism example, in most bacteria, the general Secretory pathway for directing the microorganism, etc.). (Sec) is the predominant route for protein export. Id. Proteins Likewise, in an embodiment, the at least one environmen exported via Sec are translocated across the membrane in an tal medium treatment agent is able to translocate to at least unfolded State through a membrane-embedded translocon, to one other location in the environmental medium. For which they are targeted by cleavable N-terminal signal pep example, in an embodiment, the at least one modified micro tides. Id. A second export pathway designated Tat (for twin organism delivers at least one environmental medium treat arginine translocation), transports prefolded protein Sub 10 ment agent to the environmental medium and the environ strates. Id. Proteins are targeted to the Tat pathway by mental medium treatment agent spreads by osmosis, water tripartite N-terminal signal peptides, which contain a con flow, etc. to at least one other location in the environmental served twin-arginine motif in the N region of Tat signal pep medium. In an embodiment, the translocation is directed tides. Id. The motif is described as R-R-X-db-db, where did (e.g., by water flow direction, etc.). represents a hydrophobic amino acid. The consecutive argi 15 In an embodiment, the modified microorganism produces nine residues are almost invariant and believed to be impor at least one environmental medium treatment agent or thera tant for transport by this pathway. Id. The Tat system is peutic agent that catalyzes the conversion of a prodrug to its capable of producing large proteins, and proteins with lipid active state. For instance, in an embodiment, at least one anchors, which are sometimes difficult to produce by way of anaerobic microorganism is administered to a tumor that has the Sec system. Id. According to published studies, the twin a largely anaerobic environment. The anaerobic microorgan arginine translocation system has been demonstrated to be ism is allowed to proliferate and produce an enzyme or other usable in various microorganisms. See, for example, Mail factor. Prior to, during, or after administration of the at least lard, et al., PNAS, vol. 104, no. 40, pp. 15641-15646; U.S. one anaerobic microorganism, at least one prodrug is admin Pat. No. 7,447,595; each of which is incorporated herein by istered to the tumor (e.g., by way of a microorganism or other reference. 25 form of administration). The enzyme or other factor secreted In an embodiment, the at least one modified microorgan by the at least one anaerobic microorganism converts the at ism includes a microorganism having one or more non-revert least one prodrug to an active state (e.g., to a cytotoxic agent). ing mutations. For example, a non-reverting mutation can For example, the at least one enzyme includes at least one of involve a polynucleotide of greater than about 1 nucleotide, beta glucuronidase or cytosine deaminase. In an embodiment, greater than about 2 nucleotides, greater than about 3 nucle 30 the at least one prodrug includes at least one glucuronide otides, greater than about 4 nucleotides, greater than about 5 prodrug. In an embodiment, the at least one glucuronide nucleotides, greater than about 10 nucleotides, greater than prodrug includes at least one glucuronide of epirubicin, about 15 nucleotides, greater than about 20 nucleotides, or 5-fluorouracil, 4-hydroxycyclophosphamide, or 5-fluorocy any value therebetween. tosine. See, for example, U.S. Pat. No. 6,652,849, which is In an embodiment, the non-reverting mutation blocks at 35 incorporated herein by reference. least one biosynthetic pathway of the microorganism. For In another example, the enzyme includes nitroreductase, example, the non-reverting mutation can include one or more and the prodrug includes 5-(aziridin-1-yl)-2,4-dinitrobenza of a deletion, insertion, inversion, or any combination of mide (CB1954). See, for example, U.S. Pat. No. 6,416,754, these. Developing modified microorganisms containing non which is incorporated herein by reference. reverting mutations is a routine practice, and is based on 40 In an embodiment, the composition is formulated for several non-limiting factors, including the ability to mutate a administration to at least one biological tissue by at least one particular gene without destroying the viability of the micro route including peroral, topical, transdermal, epidermal, organism, the nature of the at least one therapeutic agent that intravenous, intraocular, tracheal, transmucosal, intracavity, is to be delivered by the modified microorganism, the nature Subcutaneous, intramuscular, inhalation, fetal, intrauterine, of the microorganism, and in some cases, the nature of the 45 intragastric, placental, intranasal, interdermal, intradermal, subject that will host the modified microorganism. For enteral, parenteral, Surgical, or injection. In an embodiment, example, in an embodiment the mutated gene prevents pro the intracavity route includes at least one of oral, vaginal, duction of at least one enzyme that is required for a biosyn uterine, rectal, nasal, peritoneal, Ventricular, or intestinal. The thetic pathway of a metabolite needed for replication or pro delivery may include inhalation, depot injections, implants, tein synthesis, without sacrificing viability of the 50 or other mode of delivery by way of an apparatus. microorganism (e.g., aro genes, pab genes, purgenes, etc.). In an embodiment, a composition includes a time-release See, for example, U.S. Pat. No. 4,837,151, which is incorpo formulation. In at least one embodiment, the composition rated herein by reference. Standard methods for making non includes at least one of an Suspension, mixture, Solution, Sol, reverting mutants include, but are not limited to, introducing clathrate, colloid, emulsion, microemulsion, aerosol, oint transposable elements, site directed mutagenesis, conjuga 55 ment, capsule, micro-encapsule, powder, tablet, Suppository, tional crossing, or other form of mutagenesis. The modified cream, device, paste, resin, liniment, lotion, ampule, elixir, microorganism is produced by transduction, transformation, spray, syrup, foam, pessary, tincture, detection material, poly or other means. mer, biopolymer, buffer, adjuvant, diluent, lubricant, disinte In an embodiment, the at least one therapeutic agent is able gration agent, Suspending agent, solvent, light-emitting to translocate to at least one other location in the biological 60 agent, colorimetric agent, glidant, anti-adherent, anti-static tissue or subject. For example, in an embodiment, the at least agent, Surfactant, plasticizer, emulsifying agent, flavor, gum, one modified microorganism delivers at least one therapeutic Sweetener, coating, binder, filler, compression aid, encapsu agent to the gastro-intestinal region of a Subject, where it is lation aid, preservative, granulation agent, spheronization absorbed and utilized at a distant location in the same subject agent, stabilizer, adhesive, pigment, Sorbent, nanoparticle, (e.g., lungs, heart, etc.). In an embodiment, the at least one 65 microparticle, or gel. therapeutic agent translocates systemically. In an embodi In an embodiment, the composition includes a lyophilized ment, the at least one therapeutic agent translocates selec formulation. In an embodiment, the composition forms at US 8,852,916 B2 27 28 least part of a food product. In an embodiment, the composi organism expresses a measurable material upon exposure to tion is formulated to be included with at least part of one or at least one byproduct of the modified microorganism. For more of a food product, lip balm, lotion, ointment, Sunscreen example, as set forth in Hansen, et al., a modified microor or Sunblock, perfume, aftershave, shampoo or other hair ganism expresses green fluorescent protein in the presence of products, nail polish, dentures or other oral implants, contact tetracycline, which is produced by a naturally occurring bac lens or other ocular implants, orifice insert, orifice spray or terial strain. Id. As indicated, testing was conducted by inocu inhaler, Sutures, Surgical staples, dental floss, stents, shunts, lating soil with the biosensor microorganisms, and detecting bandages, absorbable mesh, or oral consumable. the biosensor microoganisms by flow cytometry. Id. For The formulation of any of the compositions described example, induced biosensor bacteria were isolated using fluo herein may beformulated neat or may be combined with one 10 rescence-activated cell sorting (FACS) and examined by epi or more acceptable carriers, diluents, excipients, and/or fluorescence microscopy. Id. vehicles such as, for example, buffers, Surfactants, preserva In an embodiment, the at least one detection material tives, solubilizing agents, isotonicity agents, and stablilizing includes at least one of a radioactive, luminescent, colorimet agents as appropriate. A pharmaceutically acceptable carrier, ric or odorous Substance. In an embodiment, the detection for example, may be approved by a regulatory agency of the 15 material includes at least one of a diamagnetic particle, fer state and/or Federal government such as, for example, the romagnetic particle, paramagnetic particle, Super paramag United States Food and Drug Administration (US FDA) or netic particle, particle with altered isotope, or other magnetic listed in the U.S. Pharmacopeia or other generally recognized particle. In an embodiment, the detection material includes at pharmacopeia for use in animals, and more particularly in least one RNA or DNA device. In an embodiment, the detec humans. Conventional formulation techniques generally tion material or a precursor thereof is encoded by the at least known to practitioners are described in Remington: The Sci one heterologous genetic element encoding at least one thera ence and Practice of Pharmacy, 21 Edition, Lippincott Wil peutic agent or environmental medium treatment agent. In an liams & Wilkins, Baltimore, Md. (2000), which is herein embodiment, the detection material includes the at least one incorporated by reference. therapeutic agent or environmental medium treatment agent, Acceptable pharmaceutical carriers include, but are not 25 or a metabolite thereof. limited to, the following: Sugars, such as lactose, glucose and In an embodiment, the detection material includes at least Sucrose; starches, such as corn Starch and potato starch; cel one nucleic acid device, such as a DNA or RNA device. In an lulose, and its derivatives. Such as Sodium carboxymethyl embodiment, the nucleic acid device includes a single input cellulose, ethyl cellulose, bovine serum albumin, keyhole single output RNA device based on the assembly of three limpet hemocyanin, tetanus toxoid, cellulose acetate, and 30 functional components: a sensor made of an RNAaptamer; an hydroxymethylcellulose; polyvinylpyrrolidone; cyclodextrin actuator made of a hammerhead ribozyme; and a transmitter and amylose; powdered tragacanth; malt, gelatin, agar and made of a sequence that couples the sensor and actuator pectin; talc; oils, such as mineral oil, polyhydroxyethoxylated components. See, for example, Win and Smolke, Science, castor oil, peanut oil, cottonseed oil, safflower oil, Sesame oil, vol. 322, pp. 456-460 (2008), which is incorporated herein by olive oil, corn oil and soybean oil; polysaccharides, such as 35 reference. Such devices distributed between two primary alginic acid and acacia; fatty acids and fatty acid derivatives, conformations: one in which the input cannot bind the sensor, Such as Stearic acid, magnesium and sodium Stearate, fatty and the other in which the input can bind the sensor as a result acid amines, pentaerythritol fatty acid esters; and fatty acid of competitive hybridization events within the transmitter monoglycerides and diglycerides; glycols, such as propylene component. Id. Input binding shifts the distribution to favor glycol, polyols. Such as glycerin, Sorbitol, mannitol and poly 40 the input-bound conformation as a function of increasing ethylene glycol; esters, such as ethyl oleate and ethyl laurate; input concentration and is translated to a change in the activ buffering agents, such as magnesium hydroxide, aluminum ity of the actuator, where a “ribozyme-active' state results in hydroxide and sodium benzoate/benzoic acid; water, isotonic self-cleavage of the ribozyme. Id. saline: Ringer's solution; ethyl alcohol; phosphate buffer In an embodiment, the detection material includes the at Solutions; other non-toxic compatible Substances employed 45 least one therapeutic agent, environmental medium treatment in pharmaceutical compositions. The pharmaceutical compo agent, or metabolite thereof. sitions are generally formulated as sterile, Substantially iso In an embodiment, the at least one environmental medium tonic and in full compliance with all Good Manufacturing includes at least one of a solid, liquid, or gas. In an embodi Practice (GMP) regulations of the U.S. Food and Drug ment, the at least one environmental medium includes at least Administration. 50 one of water, soil, food product, or air or other gas. In an In an embodiment, the composition further comprises at embodiment, the at least one environmental medium includes least one detection material associated with the at least one at least one of ground water, Surface water, effluent, or waste modified microorganism. In an embodiment, the at least one water. In an embodiment, the water includes at least one of a detection material includes at least one taggant, contrast lake, river, stream, sludge, slurry, sewage, ocean, fountain, or agent, sensor, or electronic identification device. In an 55 other water. In an embodiment, the at least one environmental embodiment, the at least one electronic identification device medium includes water contained in an at least partially includes at least one radio frequency identification device. enclosed space (e.g., septic tank, lagoon, dam, wastewater In an embodiment, the at least one sensor includes at least treatment vessel, etc.). one biosensor. In an embodiment, the at least one biosensor In an embodiment, the at least one environmental medium includes at least one modified microorganism. In an embodi 60 includes at least one of a structure or device. In an embodi ment, the at least one modified microorganism detects the at ment, the structure includes at least one of metal, concrete, least one agent. For example, in an embodiment, a biosensor cement, textiles, fabric, wood, mineral ore, or rock. In an microorganism expresses green fluorescent protein (or embodiment, the device includes at least one of a patch, another measurable material) upon exposure to the at least bandage, shunt, wound dressing, splint, computer mouse, one agent. See, for example, Hansen et al., App. EnV. Micro 65 telephone, mobile phone, writing instrument, article of cloth biol. Vol. 67, no. 1, pp. 239-244 (2001), which is incorporated ing, blanket, pen-type injection device, other injection device, herein by reference. In an embodiment, the biosensor micro medical instrument, or other article of manufacture. US 8,852,916 B2 29 30 In an embodiment, the at least one modified microorgan Muller and Lingens, Abstract, Ang Chem Int Ed English, Vol. ism degrades or converts at least one hydrocarbon source. For 25, no.9, pp. 779-789 (2003), which is incorporated herein by example, alkanes, alkenes, alkynes, polyalkenes, poly reference. alkynes, chlorinated, Volatile, or aliphatic hydrocarbons are In an embodiment, at least one modified microorganism is common contaminants in soil, or ground water. Such hydro contacted with the at least one substrate. Such as an environ carbons are a common constituent in solvents, degreasers, mental medium (e.g., Soil, water, etc.) to be treated. For and other compounds. Other non-limiting examples of hydro example, the at least one modified microorganism can be carbon compounds include chlorinated aliphatic hydrocar contacted with the at least one substrate in combination with bons, chlorinated aromatic hydrocarbons, or non-chlorinated at least one of pumping and treating, air sparging, drilling, oil aromatic hydrocarbons. Non-limiting examples of hydrocar 10 vapor extraction with activated carbon, etc. In an embodi ment, at least a partial vacuum is maintained within the Sub bon contaminants or other contaminants that can be utilized strate to be treated in order to confine the hydrocarbon source, by at least one modified microorganism include methylene or assistan anaerobic modified microorganism. In an embodi chloride, 1,1-dichloroethane, chloroform, 1,2-dichloropro ment, a venting system provides additional oxygenation for pane, dibromochloromethane, 1,1,2-trichloroethane, 2-chlo 15 degradation by the at least one modified microorganism. roethylvinyl ether, tetrachloroethene (PCE), chlorobenzene, In an embodiment, at least one modified microorganism is 1,2-dichloroethane, 1,1,1-trichloroethane, bromodichlo capable of metabolizing or co-metabolizing iron or manga romethane, trans-1,3-dichloropropene, cis-1,3-dichloropro nese from at least one Substrate. In an embodiment, at least pene, bromoform, benzene, toluene, ethylbenzene, Xylenes, one metal is oxidized and precipitated from the at least one chloromethane, bromomethane, vinyl chloride, chloroet Substrate. In an embodiment, the at least one metal is precipi hane, 1,1-dichloroethene, trans-1,2-dichloroethene, trichlo tated by means including but not limited to pH change, redox roethene (TCE), dichlorobenzenes, cis-1,2-dichloroethene, potential change, metal reduction, or other means provided dibromomethane, 1,4-dichlorobutane, 1,2,3-trichloropro by the at least one modified microorganism. In an embodi pane, bromochloromethane, 2,2-dichloropropane, 1,2-dibro ment, chelated iron is released by the at least one modified moethane, 1,3-dichloropropane, bromobenzene, chlorotolu 25 microorganism. enes, trichlorobenzenes, trimethylbenzenes, trans-1,4- In an embodiment, the at least one substrate includes at dichloro-2-butene, butylbenzenes, methyl tertiary butyl ether, least one of rock, or mineral ore. In an embodiment, the polychlorinated biphenyl, or polycyclic aromatic hydrocar mineral ore includes, but is not limited to, iron ore, copper bon. In another example, a particular contaminant includes, ore, Zinc ore, nickel ore, uranium ore, gold ore, silver ore, or 30 other ores. For example, precious metals can be biooxidized but is not limited to, heavy metals such as arsenic, antimony, from rock or mineral ore directly, or indirectly by the addition beryllium, cadmium, chromium, copper, lead, mercury, iron, of an additive (e.g., hydrocarbon), and allowing microorgan manganese, radium, nickel, selenium, silver, thallium, or isms to oxidize the iron, Sulfur, or other elements Surrounding Zinc. In another example, a particular contaminant includes, the precious metals (e.g., gold, silver, etc.). See, for example, but is not limited to, nitrogen-based aromatic compounds, 35 U.S. Pat. No. 6,875,356, which is incorporated herein by pesticides, esters, ethers, aldehydes, amines, dioxins, herbi reference. In an embodiment, at least one modified microor cides, ketones, phenols, alcohols, Sulfur-containing com ganism capable of bio-oxidizing compounds from rock or pounds, ethylene dibromide, chlorophnolic compounds, mineral ore is utilized to administer at least one agent (e.g., chlorate, cyanide, halogenated compounds, radioactive com carbon, calcium, other minerals, etc.). In an embodiment, the pounds, and other contaminants. 40 at least one ore is leached to cause the dissolution of the metal Bioremediation of an environmental medium, by removing from the ore. In an embodiment, the leached ore is bio contaminant hydrocarbons, heavy metals, or other substances oxidized with a lixiviant, and the precious metals are recov from environmental media can be performed by utilizing ered from the lixiviant. hydrocarbon utilizing bacteria (e.g., methanotropic bacteria) In an embodiment, the precious metals are extracted from or other microorganisms. For example, microbial oxidation 45 the mineral ore by the at least one modified microorganism, of alkanes (C-C) have been shown to include a number of and at least one nutrient that encourage plant growth is deliv chemical processes, including terminal oxidation, and methyl ered by the at least one modified microorganism. As ketone formation. See, for example, Blevins and Perry, J. described herein, in an embodiment the metal is extracted Bacteriol. Vol. 112, no. 1, pp. 513-518 (1972), which is incor from the ore by direct application of the at least one modified porated herein by reference. The terminally oxygenated prod 50 microorganism, or through batch-processing amounts of the uct may be catabolized further by alpha oxidation (i.e. ore in an at least partially enclosed space (e.g., bioreactor). removal of one carbon at each step), or by beta Oxidation. In an embodiment, the at least one modified microorgan Methyl ketones can be metabolized to the alpha-hydroxy ism includes an endophytic microorganism. For example, ketone, as with acetone oracetol, or the methyl ketone can be endophytic microorganisms are commonly associated with subterminally oxidized to an acetate ester, which is cleaved to 55 plant roots, or other plant parts. Endophytes can also be yield acetate and a primary alcohol. Id. identified by amplification (e.g., culturing, or (RT-)PCR In an example, methane-utilizing bacteria can degrade amplification of nucleic acids) or direct sequencing of nucleic TCE metabolically or cometabolically, either by introduction acids. into the environmental medium directly or by introduction to In an embodiment, the at least one environmental medium batches of hydrocarbon-laden environmental medium in an at 60 treatment agent includes at least one plant hormone. In an least partially enclosed space (e.g., a bioreactor). In an embodiment, the at least one agent includes at least one of an embodiment, the at least one modified microorganism utilizes auxin, abscisic acid, cytokinin, ethylene, gibberellin, brassi the at least one hydrocarbon as a food source, and oxygen (or nolide, Salicyclic acid, jasmonate, polyamine, plant peptide another element) as an electron acceptor. In an embodiment, hormone, nitric oxide, Strigolactone, or other compound. microorganisms utilize enzymes that are capable of replacing 65 Plant hormones are capable of regulating various aspects of halogen Substituents in aliphatic and aromatic compounds plant growth, plant differentiation, and plant development. with hydroxyl groups or hydrogen atoms. See, for example, For example, plant hormones regulate formation of flowers, US 8,852,916 B2 31 32 stems, and leaves; shedding of leaves; development of fruit, auxotrophic) microorganism. As described herein, in an and ripening of fruit, cold tolerance, pathogenic tolerance, embodiment the at least one modified microorganism is at overall growth, propagation, reproduction, as well as other least one of responsive or resistant to the at least one antibi processes. In an embodiment, the plant hormone includes a otic. naturally occurring plant hormone. In an embodiment, the 5 In an embodiment, the at least one modified microorgan plant hormone includes a synthetic or artificial hormone. In ism is administered directly to the at least one biological an embodiment, at least one modified microorganism is tissue where the at least one therapeutic agent is desired. In an capable of producing multiple plant hormones. embodiment, the at least one modified microorganism is Methods of Administration of Modified Microorganisms to Biological Tissues 10 administered indirectly at another location, and the at least At least one embodiment disclosed herein includes one or one modified microorganism is translocatable to the at least more methods for administering at least one therapeutic agent one biological tissue where the at least one therapeutic agent to at least one biological tissue, comprising providing a com is desired. The translocation can be conferred in a number of position to at least one biological tissue; wherein the compo ways, including but not limited to homing by the modified sition includes at least one modified (e.g., auxotrophic) 15 microorganism (e.g., adhesion molecules, etc.), or external microorganism including at least one heterologous genetic intervention (e.g., magnetic particles, electrical signaling, element encoding at least one therapeutic agent. Various etc.). In an embodiment, more than one modified microor embodiments of compositions have been described herein. ganisms are administered to at least one biological tissue For example, in an embodiment a method of administering where the at least one therapeutic agent is desired, and at least at least one therapeutic agent to at least one biological tissue 20 one of the more than one modified microorganisms remains in comprises: providing a composition to at least one biological the at least one biological tissue, while others of the group tissue; wherein the composition includes at least one aux may translocate to another location or biological tissue. For otrophic microorganism including at least one pH inducible example, certain recombinant strains of Lactobacillus (e.g., heterologous genetic element encoding at least one therapeu L. reuteri) are capable of consistently and accurately reaching tic agent formulated for at least one biological tissue, and 25 and adhering to target locations on the mucosa of the host, and including at least one genetic element inducible to initiate expressing heterologous proteins. See, for example, U.S. Pat. death of the at least one auxotrophic microorganism; and the No. 6,100.388, which is incorporated herein by reference. composition further including at least one metabolite For example, several Lactobacillus strains have been required by the at least one auxotrophic microorganism. shown to translocate from the stomach into the intestinal tract As described herein, in an embodiment, the at least one 30 without lysis. See, for example, Grangette, et al. Infect. and treatment or therapy can be associated with preventative or Immun. Vol. 72, no. 5, pp. 2731-2737 (2004), and Wado prophylactic treatment, responsive treatment, or both. In an Ikowski, et al. Infect. and Immun. vol. 56, no. 5, pp. 1030 embodiment, the at least one composition provides an effec 1035 (1998), each of which is incorporated herein by refer tive amount of at least one therapeutic agent in relation to at CCC. least one disease, condition, symptom or disorder. In an 35 In another example, a host microorganism cell that embodiment, the effective amount of at least one therapeutic includes a recombinant vaccine virus with a modified thymi agent is provided in relation to at least one disease, condition, dine kinase gene and modified hemagglutinin gene allows for symptom, or disorder. directing the microorganism to a particular biological tissue In an embodiment, the effective amount of at least one location (e.g., immunoprivileged sites, tumors, etc.) for deliv therapeutic agent is provided in relation to at least one infec- 40 ery of particular products. See, for example, U.S. Patent tion. In an embodiment, the effective amount of at least one Application No. 2005003 1643, which is incorporated herein therapeutic agent is provided in relation to at least one of by reference. inflammatory bowel disease, enteritis, colitis, cancer, gastro In another example, a host microorganism cell (e.g., an intestinal disorder, spondyloarthropathy, HIV/AIDS, anaerobic cell) preferably colonizes avascular compartments Crohn's disease, ulcerative colitis, acute colitis, mucosal 45 of tumors for delivery of anti-tumor products. See, for lesions, gastritis, Menetier's syndrome, gastro-esophageal example, U.S. Pat. No. 7,344,710, which is incorporated reflux, peptic or duodenal ulcer, dyspepsia, dental caries, herein by reference. vaginal candidiosis, allergic reaction, lactose intolerance, In an embodiment, the at least one therapeutic agent is lowering of cholesterol, atherosclerosis, prevention of infec provided in an effective amount to shorten or terminate the tion, lowering blood pressure, diarrhea, fever, anemia, anor- 50 life span of a subject (e.g., insects). For example, published exia, abdominal cramps, autoimmune disease, metabolic reports indicate that the duration of an insect’s lifespan can be defects, diabetes, promoting wound healing, decreasing scar cut in half by placing Wolbachia wMelPop in the insect. See, formation, obesity, or malnutrition. for example, Kambris, et al., Science vol. 326, pp. 134-136 In an embodiment, the infection includes at least one of (2009), which is incorporated herein by reference. In an vaginal infection, oral infection, dental infection, urogenital 55 embodiment, Wolbachia or another microorganism is utilized infection, ear infection, eye infection, tonsillitis, ulcer, intes with at least one embodiment described hererin, for delivery tinal blockage or infection, skin infection, nail infection, of at least one therapeutic agent. Thus, in an embodiment, the sinus infection, urinary tract infection, kidney infection, phar composition including the modified microorganism is pro yngitis, laryngitis, or bronchitis. vided to an insect (e.g., fly, mosquito, etc.) in order to control In an embodiment, the at least one metabolite includes an 60 the insect population. antimicrobial agent. In an embodiment, the at least one In an embodiment, the method further comprises adminis metabolite includes an antibiotic. In an embodiment, the at tering at least one antacid, proton pump inhibitor, alkaline least one modified microorganism is resistant to the at least Substance, or other Substance approximately prior to, during, one metabolite. In an embodiment, the method further com or Subsequent to administering the at least one composition to prises administering at least one antibiotic to the at least one 65 the at least one biological tissue, particularly in the case of biological tissue or environmental medium prior to, during, or therapeutic agents that are pH dependent. In an embodiment, Subsequent to administering the at least one modified (e.g., the method further comprises administering at least one pro US 8,852,916 B2 33 34 drug or precursor compound to the at least one biological monkey, horse, cow, pig, chicken, shellfish, fish, turkey, tissue. Various prodrugs or precursor compounds are dis llama, alpaca, bison, buffalo, ape, primate, ferret, wolf, fox, closed herein. coyote, deer, rabbit, guinea pig, yak, elephant, tiger, lion, In an embodiment, the method further comprises adminis cougar, chinchilla, mink, reindeer, elk, camel, fox, elk, deer, tering at least one antibiotic to the at least one biological raccoon, donkey, or mule. In an embodiment, the at least one tissue prior to, during, or Subsequent to administering the at Subject includes at least one anthozoan species. In an embodi least one auxotrophic microorganism. In an embodiment, the ment, the at least one subject includes at least one of a sea at least one auxotrophic microorganism is responsive to the at anemone, coral, mollusk, fish, whale, dolphin, porpoise, Seal, least one antibiotic. In an embodiment, the at least one aux otter, beaver, seabird, gull, pelican, albatross, duck, Swan, or otrophic microorganism is resistant to the at least one antibi 10 goose. In an embodiment, the at least one subject includes at otic. In an embodiment, the at least one antibiotic includes least one insect (e.g., fly, mosquito, beetle, moth, butterfly, one or more ofciprofloxacin, penicillin, amplicillin, amikacin, etc.). In an embodiment, the at least one Subject includes at gentamicin, kanamycin, neomycin, netilmicin, Streptomycin, least one arachnid. In an embodiment, the at least one subject tobramycin, paromomycin, geldanamycin, herbimycin, lora includes at least one crustacean. carbef, ertapenem, doripenem, imipenem, meropenem, 15 In an embodiment, the Subject includes a plant. In an cefadroxil, cefazolin, cefalotin, cefalexin, cometabolite, cefa embodiment, the at least one biological tissue includes one or mandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, more of a stalk, stem, leaf, root, plant, or tendril. In an cefditoren, cefoperaZone, cefotaxime, cefpodoxime, ceftazi embodiment, the at least one biological tissue includes at least dime, cefibuten, ceftizoxime, ceftriaxone, cefepime, ceftobi one food product. In an embodiment, the at least one food prole, teicoplanin, Vancomycin, azithromycin, clarithromy product includes one or more animal, plant, fungal or other cin, dirithromycin, erythromycin, roXithromycin, food product. In an embodiment, the food product includes troleandomycin, tellithromycin, spectinomycin, aztreonam, meat. In an embodiment, the at least one biological tissue azlocillin, carbenicillin, cloxacillin, dicloxacillin, fluclox includes at least one cell mass or wound. acillin, mezlocillin, meticillin, nafcillin, oxacillin, piperacil In an embodiment, the at least one composition is self lin, ticarcillin, bacitracin, colistin, polymyxin B, enoxacin, 25 administered by the at least one Subject. In an embodiment, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nor the at least one composition is ingestable by the at least one floxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxa Subject. In an embodiment, the at least one composition is cin, mafenide, prontosil, Sulfacetamide, Sulfamethizole, Sul ingested by the at least one subject. In an embodiment, the at fanilamide, Sulfasalazine, Sulfisoxazole, trimethoprim, least one biological tissue includes at least one implantable or trimethoprim-sulfamethoxazole, demeclocycline, doxycy 30 transplantable biological tissue. In an embodiment, the at cline, minocycline, Oxytetracycline, tetracycline, arsphe least one biological tissue is transplanted or implanted into at namine, chloramphenicol, clindamycin, lincomycin, etham least one subject. In an embodiment, the at least one biologi butol, fosfomycin, fusidic acid, furazolidone, isoniazid, cal tissue is from at least one donor or recipient. In an embodi lineZolid, metronidazole, mupirocin, nitrofurantoin, platen ment, the at least one biological tissue includes at least one simycin, pyrazinamide, quinupristin, rifampicin, thiam 35 bodily orifice of a subject. phenicol, tridazole, or other antibiotic. In an embodiment, the subject is afflicted with or suspected In at least one embodiment, the at least one biological of being afflicted with at least one symptom, affliction, dis tissue is located at least in one of in vitro, in Vivo, in situ, in order, disease or condition. As described herein, the at least utero, ex vivo, in planta, or in silico. In an embodiment, the at one disease or condition may include one or more of a patho least one biological tissue includes at least one of one mucosal 40 genic infection, parasitic infection, autoimmune disease, sep Surface. In an embodiment, the at least one biological tissue sis, systemic inflammatory response syndrome, septic shock, includes at least one of cartilage, skin, Scalp, hair, nail, nail multiple organ dysfunction syndrome, allergic reaction, or bed, teeth, eye, ear, ovary, Oviduct, tongue, tonsil, adenoid, cancer. In at least one embodiment, the at least one inflam liver, bone, pancreas, Stomach, duct, valve, Smooth muscle, matory disease or condition includes one or more of anaphy appendix, blood vessel, bone marrow, blood, lymph, heart, 45 laxis, viral infection, bacterial infection, plasmodium infec lung, brain, breast, kidney, bladder, urethra, ureter, gallblad tion, protozoan infection, nematode infection, or other worm der, uterus, prostate, testes, vas deferens, fallopian tubes, infection. In at least one embodiment, the at least one inflam large intestine, Small intestine, esophagus, oral cavity, nasal matory disease or condition includes malaria. In at least one cavity, otic cavity, connective tissue, muscle tissue, adipose embodiment, the parasitic infection includes at least one tissue, placental tissue, fetal tissue, or mucosa-associated 50 infection or infestation of one or more of a phytoparasite, lymphoid tissue (MALT). Zooparasite, ectoparasite, endoparasite, or one or more of In an embodiment, the at least one auxotrophic microor parasitic cysts, larvae, or eggs. ganism is in physical or chemical communication in vitro One embodiment relates to one or more methods of modu with one or more cells of the at least one biological tissue lating the activity of intracellular signaling molecules. Any of prior to in vivo administration of the at least one auxotrophic 55 the methods disclosed herein may include detecting in the microorganism to the at least one biological tissue. Subject, or tissues, at least one level of at least one biological In at least one embodiment, the at least one biological signaling molecule that is associated with a disease, disorder, tissue is at least partially located in a Subject. In an embodi or condition described herein. ment, a subject includes, but is not limited to, a vertebrate or Detection of one or more of the biological signaling mol invertebrate, including a fish, reptile, mammal, amphibian, or 60 ecules can be by any method known in the art, including but bird. In at least one embodiment, the subject includes at least not limited to analyzing one or more biological tissues or one human. In an embodiment, the at least one subject fluids from the Subject. Analyzing one or more biological includes at least one of livestock, pet, Zoo animal, undomes fluids can be performed by any of a variety of methods known ticated herd animal, wild animal, aquatic plant or animal, or in the art, including but not limited to utilizing one or more of product animal. 65 thin-layer chromatography, mass spectrometry, nuclear mag In an embodiment, the at least one subject includes at least netic resonance, polymerase chain reaction, reverse tran one of a sheep, goat, frog, dog, cat, rat, mouse, Vermin, scriptase, Northern blot, Western blot, microscopy, flow US 8,852,916 B2 35 36 cytometry, antibody binding, enzyme-linked immunosorbent In an embodiment, the at least one therapeutic agent modu assay, radioactive absorption or release, microfluidic analy lates at least one immune response. In an embodiment, the at sis, nucleic acid chip array analysis, protein chip array analy least one immune response includes at least one allergic or sis, chemical sensor analysis (including arrays), biosensor autoimmune response. analysis, cell counting, or cell sorting. 5 In an embodiment, the at least one therapeutic agent In at least one embodiment, the at least one biological induces apoptosis in one or more cells of the at least one signaling molecule includes but is not limited to, one or more biological tissue (e.g., blood cells, tumor cells, bone cells, nucleic acid, amino acid, peptide, polypeptide, protein, gly etc.). In an embodiment, the at least one therapeutic agent copeptide, glycoprotein, glycolipid, lipopolysaccharide, pep modulates at least one inflammation response. In an embodi tidoglycan, proteoglycan, lipid, metalloprotein, liposome, or 10 ment, the at least one therapeutic agent modulates the viabil ity, proliferation, or metastasis of at least one tumor cell. carbohydrate. Carbohydrates may include, but not be limited As set forth herein, the compositions disclosed are formu to, oligosaccharides, glycans, glycosaminoglycans, or lated by standard practice. In certain instances, in order to derivatives thereof. account for bioavailability, a formulation may be provided in In at least one embodiment, the at least one biological 15 rapid release, extended release or slow-release form. Like signaling molecule includes but is not limited to at least one wise, liposomes, microsomes, or other vehicles or composi cytokine, chemokine, cellular receptor, intracellular second tion modifications allow for regulating the dosage by increas messenger, protease, kinase, enzyme, cellular receptor ing or decreasing the rate of composition delivery, ligand, transcription factor, or hormone. maintenance, decomposition, clearance, or other factors. For A treatment regimen may include a therapeutically effec example, one particular therapeutic agent may have bioavail tive amount of one or more compositions described herein ability properties that require it to be modified by standard that includes modulators or analogs thereof. The treatment techniques so that it can be administered simultaneously with regimen may further include a schedule of changes in the another therapeutic agent. Similarly, in the instance where dosage of the composition to maintain a desired level of one multiple therapeutic agents are included in a single composi or more molecules related to a symptom, affliction, disorder, 25 tion, it may be necessary to modify one or more of the thera disease, or condition in one or more biological tissues or peutic agents by standard techniques. subjects. Such treatment may be individualized for the bio In at least one embodiment the one or more biological logical tissue or Subject. signaling molecules are detected by one or more recognition Treating or treatment that includes administration of at molecules specific to the one or more biological signaling least one of the compositions included herein may prevent or 30 molecules. The recognition molecules may include, but not delay the onset of symptoms, complications, or biochemical be limited to, an antibody, affibody, DNA-recognition mol indicia of a disease, affliction, symptom, condition, or disor ecule, aptamer, or other molecule. der, or alleviate the symptoms, arrest, or inhibit further devel An antibody may include an anti-idiotypic antibody, a het opment of the disease, symptom, affliction, condition, or dis eroantibody, multiple antibodies, one or more antibody frag order. Treatment or administration of at least one composition 35 ments, one or more antibody derivatives, one or more anti described herein may be prophylactic to prevent or delay the bodies linked together, chimeric antibodies, humanized onset of a disease, disorder, symptom, affliction, or condition, antibodies, human antibodies, recombinant antibodies, Syn or prevent the manifestation of clinical or Subclinical Symp thetic antibodies, or others. toms thereof, or therapeutic Suppression or alleviation of Antibodies or fragments thereof may be generated against symptoms after the manifestation of the disease, condition, or 40 an agent, such as a receptor or ligand, using standard meth disorder. ods, for example, such as those described by Harlow & Lane A treatment regimen may be continuous and uninterrupted, (Antibodies: A Laboratory Manual, Cold Spring Harbor which indicates that there is no break in the treatment regimen Laboratory Press; 1 edition 1988), which is herein incorpo during the treatment period. Continuous, uninterrupted rated by reference). Alternatively, an antibody fragment administration of a combinational composition includes that 45 directed against an agent may be generated using phage dis the combination may be administered during the entire treat play technology (See, e.g., Kupper, et al. BMC Biotechnol ment period, e.g., at least once daily or on a continuous and ogy Vol. 5, No. 4, (2005), which is herein incorporated by uninterrupted basis. The treatment regimen may be given to reference). An antibody or fragment thereof could also be maintain an in vivo therapeutic level or a determined cyclic prepared using in silico design (See e.g., Knappik et al., J. level of the one or more agents of the at least one composition. 50 Mol. Biol. Vol. 296, pp. 57-86 (2000), which is herein incor It is expected that the treatment period may vary depend porated by reference). In addition or instead of an antibody, ing, for example, on the symptoms to be treated. Physician the assay may employ another type of recognition element, evaluation along with patient interaction will assist in the Such as a receptor or ligand binding molecule. Such a recog determination of the duration of treatment. Adjustments in the nition element may be a synthetic element like an artificial treatment regimen may depend upon the individuals medical 55 antibody or other mimetic. (See e.g., U.S. Pat. No. 5,804,563 history, or genetic or proteomic information. (Synthetic receptors, libraries and uses thereof), U.S. Pat. No. At least one embodiment relates to one or more methods 6,797.522 (Synthetic receptors), U.S. Pat. No. 6,670,427 based on a genetic or proteomic profile of the Subject. In (Template-textured materials, methods for the production and addition, medical evaluation regarding genetic profiling or use thereof), and U.S. Pat. No. 5,831,012, U.S. Patent Appli genetic testing can be provided as a current determination of 60 cation 20040018508 (Surrogate antibodies and methods of genetic risk factors, or as part of the Subjects medical history. preparation and use thereof); and Ye and Haupt, Anal Bioanal Genetic profiling or genetic testing can be used to design a Chem. Vol. 378, pp. 1887-1897, (2004); Peppas and Huang, treatment regimen and thus determine an optimal level indi Pharm Res. Vol. 19, pp. 578-587 (2002), each of which is vidualized for the Subject. A physician may use the genetic herein incorporated by reference). profile or genetic testing information to determine a genetic 65 In some instances, levels of particular biological signaling basis for needed treatment based on baseline or physiological molecules may be assayed in a bodily fluid or tissue using gas levels of inflammatory agents. or liquid chromatography with or without mass spectrometry. US 8,852,916 B2 37 38 A bodily fluid may include blood, lymph, saliva, urine, Sweat, enzymes and separated on anagarose gel. The separated DNA ascites, serum, urogenital Secretion, bone marrow, a tissue is transferred to a membrane and the fragments are visualized secretion or excretion, or other fluid. using hybridization analysis and gene specific probes. A level of one or more biological signaling molecules may A variety of PCR related methods may be used for genetic also be assayed in a bodily fluid or tissue using a recombinant profiling and may be used to detect both known and unknown cell based assay or sensor. A sensor may include, for example mutations and polymorphisms (See e.g., Tawata, et al., Comb. a chemical sensor, biosensor, protein array, or microfluidic Chem. High Throughput Screen. Vol. 3, pp. 1-9 (2000), which device. is herein incorporated by reference). For known mutations Prior to determining a treatment regimen, additional infor and polymorphisms, specific PCR oligonucleotide probes are mation regarding the physiological status of the Subject or 10 designed to bind directly to the mutation or polymorphism or biological tissue may be gathered and assessed. For example, proximal to the mutation or polymorphism. For example, information may be collected on a Subject's medical history PCR may be used in combination with RFLP. In this instance, or familial history, including genetic or proteomic informa a DNA fragment or fragments generated by PCR with primers tion. The individualized medical evaluation can include a on either side of the mutation or polymorphism site are treated genetic profile of the Subject regarding genes, genetic muta 15 with restriction enzymes and separated by agarose gel elec tions or genetic polymorphisms that indicate risk factors that trophoresis. The fragments themselves may be detected using affect risk of disease, or disease state. The medical evaluation an intercalating dye such as, for example, ethidium bromide. can include information in a population database on disease An aberrant banding pattern may be observed if mutations risks, available drugs and formulations, documented popula exist within the restriction sites. PAGE may be used to detect tion responses to drugs and formulations, or condition in single base differences in the size of a fragment. relation to any possible therapeutic treatment derived from Alternatively, PCR may be used in combination with DNA population databases. sequencing for genetic profiling. For example, PCR primers In an embodiment, one or more polymorphisms are deter may be designed that bind to either side of a potential muta mined prior to administration of at least one composition tion site on the target DNA and generate a PCR fragment that described herein, which could allow for such composition to 25 spans a potential mutation site. The PCR fragment is either be tailored to a particular Subject's genetic makeup. directly sequenced or Subcloned into a cloning vector and A genetic polymorphism or mutation may indicate how a Subsequently sequenced using standard molecular biology biological tissue or subject will respond to a particular treat techniques. ment regimen. Genomic DNA used in genetic profiling may Alternatively, a mutation or polymorphism may be be isolated from any biological sample which contains the 30 screened using comparative genomic hybridization (CGH) DNA of that subject or tissue, including but not limited to (See e.g., Pinkel & Albertson, Nat. Gen. Vol. 37:S11-S17 blood, saliva, cheek Swab, epithelium, or other tissue. For (2005), which is herein incorporated by reference). In this example, genomic DNA may be extracted from whole blood instance, “normal' genomic DNA and test genomic DNA are or from isolated peripheral blood leukocytes isolated by dif differentially labeled and hybridized to metaphase chromo ferential centrifugation from whole blood using a commercial 35 somes or DNA microarrays. The relative hybridization signal kit (See e.g., QIAmp DNA Blood Mini Kit, Qiagen, Valencia, at a given location is proportional to the relative copy number Calif.) according to the manufacturers instructions. of the sequences in the reference and test genomes. Arrays Medical evaluation of the subject or tissue for genetic or may be generated using DNA obtained from, for example, proteomic profiling or genetic or proteomic testing may be bacterial artificial chromosomes (BACs) or PCR. provided as a current determination of genetic risk factors in 40 Analysis of one or more single nucleotide polymorphism the Subject or tissue, or as part of the Subject's medical his (SNP) may be used for genetic profiling. A SNP is a DNA tory. Genetic profiling or genetic testing may be determined sequence variation in which a single nucleotide in the by using a variety of methods including but not limited to genomic sequence differs between members of a species (or restriction landmark genomic scanning (RLGS), Southern between paired chromosomes of an individual). For a varia blot analysis combined with restriction fragment length poly 45 tion to be considered a SNP it must occur in at least 1% of the morphism (RFLP), fluorescence in situ hybridization (FISH), population. Most SNPs do not affect protein function, and/or enzyme mismatch cleavage (EMC) of nucleic acid heterodu are not responsible for a disease state, but they may serve as plexes, ligase chain reaction (LCR) or polymerase chain reac biological markers for pinpointing an altered protein or dis tion (PCR) based methods. Analysis of one or more single ease on the human genome map as they are often located near nucleotide polymorphisms (SNPs) may also be used for 50 a gene found to be associated with a certain disease. Occa genetic profiling. sionally, a SNP may actually affect protein function and/or Restriction fragment landmark genomic scanning (RLGS) cause a disease and, therefore, can be used to search for and may be used to scan an entire mammalian genome. As such, isolate a specific gene, e.g., a T to C mutation in the CYP17 genomic DNA is digested with restriction enzymes to gener gene which affects enzyme function. The pattern of SNPs in ate large DNA fragments. The fragments are separated on an 55 a Subject's genomic DNA may be compared with information agarose gel, digested with one or more restriction enzymes in databases in an association study to determine effect on within the agarose gel, and then separated in a second dimen protein function and/or risk of disease development. SNPs sion by polyacrylamide gel electrophoresis (PAGE) (See e.g., may be identified using PCR and DNA sequencing as Tawata, et al., Comb. Chem. High Throughput Screen. Vol. 3, described above. Alternatively, SNP genotyping may be done pp. 1-9 (2000), which is herein incorporated by reference). 60 using high throughput array analysis (See e.g., Applied Bio The DNA may be labeled prior to digestion, or the fragments Systems, ABI PRISM, 3100 Genetic Analyzer with 22-cm may be stained nonspecifically as with an intercalating dye, Capillary Array; Syvanen, et al., Nat. Genet. Vol. 37, pp. for example. The resulting pattern may be compared with S5-S10 (2005) which is herein incorporated by reference). A pre-established norms to detect genetic mutations. growing number of web-based databases are available for Restriction fragment length polymorphism (RFLP) is 65 finding information regarding SNPs and protein function similar to restriction fragment landmark genomic scanning in and/o disease associations (See e.g., International HapMap that the genomic DNA is digested with specific restriction Project on the worldwide web at //snp.cshl.org: Nature 449: US 8,852,916 B2 39 40 851-861, 2007; National Center Biotechnology Information one offire, flood, contamination, drought, deforestation, tem (NCBI) Single Nucleotide Polymorphisms, on the worldwide perature change, or habitat change. web at ncbi.nlm.nih.gov/projects/SNP/, which is herein In an embodiment, the method further comprises adminis incorporated by reference). tering at least one antibiotic to the at least one environmental Methods of Administration of Modified Microorganisms medium prior to, during, or Subsequent to administering theat to Environmental Media least one composition. Various antibiotics are described In an embodiment, a method of administering at least one herein. environmental medium treatment agent to at least one envi In an embodiment, the at least one modified microorgan ronmental medium comprises providing at least one compo ism is sensitive or responsive to the at least one antibiotic. In sition to at least one environmental medium; wherein the at 10 least one composition includes at least one modified micro an embodiment, the at least one modified microorganism is organism including at least one heterologous genetic element resistant to the at least one antibiotic. encoding at least one environmental medium treatmentagent; In an embodiment, the method further comprises obtaining and at least one genetic element inducible to initiate death of at least one microorganism associated with the at least one the at least one modified microorganism. In an embodiment, 15 environmental medium, and modifying it to produce the at the at least one composition includes an effective amount of least one auxotrophic microorganism. In an embodiment, the the at least one environmental medium treatment agent. method further comprises obtaining genetic sequence infor As described herein, the effective amount of at least one mation from the at least one microorganism. In an embodi environmental medium treatment agent is provided in rela ment, the method further comprises amplifying the at least tion to at least one intended outcome. For example, the effec one microorganism prior to, during, or Subsequent to modi tive amount of at least one environmental medium treatment fying the at least one microorganism. agent is provided in relation to establishment or re-establish In an embodiment, the method further comprises detecting ment of organisms in the at least one environmental medium. at least one of the presence, amount, concentration, or loca In an embodiment, the effective amount of at least one envi tion of at least one of the at least one modified microorganism, ronmental medium treatment agent is provided in relation to 25 the therapeutic agent or environmental medium treatment revitalization of the at least one environmental medium asso agent, or at least one metabolite utilizable by the at least one ciated with at least one of fire, flood, contamination, drought, modified microorganism Subsequent to administration of the deforestation, temperature change, chemical waste, agricul composition. In an embodiment, the method further com tural waste or run-off, municipal waste, landfill gas or run-off prises selecting for administration an amount or type of com nuclear waste or run-off, or habitat change. 30 position. In an embodiment, the method further comprises In an embodiment, the at least one environmental medium treatment agent includes at least one plant growth factor, selecting for administration an amount or type of at least one nutrient, or other compound to the at least one environmental inducer or repressor of the heterologous genetic element medium. For example, in an embodiment, the at least one encoding at least one environmental medium treatment agent. environmental medium treatment agent assists in facilitating 35 In an embodiment, the method further comprises selecting for plant growth, biodegradation of biomass, remediation of con administration an amount or type of at least one of an inducer taminated or polluted environmental media, or other out or repressor of the at least one genetic element inducible to COCS. initiate death of the at least one modified microorganism. In an embodiment, at least one precursor compound is In an embodiment, microorganisms are modified to pro administered to the at least one environmental medium. The 40 duce at least one therapeutic agent (which may include an precursor compound can be in the form of the at least one agent used as a responsive therapy, or prophylactic therapy, environmental medium treatment agent, as described herein, etc.). The modified microorganisms are retained in vivo by or it can be administered prior to, or during administration of administration of at least one required factor as part of the the at least one composition. In an embodiment, the at least modified microorganism composition. one environmental medium treatment agent converts the at 45 In an embodiment, the method further comprises adminis least one precursor compound into an active state. In an tering metabolite Subsequent to administration of the compo embodiment, the at least one environmental medium treat sition. As such, if metabolite is administered as part of the ment agent translocates to another environmental medium. In initial composition, then the Subsequent administration of an embodiment, the at least one environmental medium treat metabolite will be in addition to the metabolite administered ment agent is formulated to translocate to another environ 50 as part of the composition. In embodiments where the mental medium. In an embodiment, the at least one environ metabolite is not administered as part of the initial composi mental medium treatment agent remains in the at least one tion, then the subsequent administration of metabolite will be environmental medium to which it is administered. For instead of the earlier administration. example, an environmental medium treatment agent admin In an embodiment, an oral composition is administered to istered to a water source (such as a lake or river) can translo 55 a subject. In an embodiment, resident microbes are depleted cate to the Surrounding soil by way of osmosis, evaporation, by administering at least one antibiotic prior to, during, or fluid travel, or other mechanism. Subsequent to administration of the modified microorganism In an embodiment, the composition provides an effective composition. For example, ciproflaxocin dosed at moderate amount of at least one environmental medium treatmentagent levels for 5 days causes a reduced abundance of about 30% of in relation to the at least one intended outcome. For example, 60 the bacterial taxa in the human colon. See, for example, the intended outcome includes, but is not limited to, estab Dethlefsen etal, PLoS Biology vol. 6, pp. 2383-2400 (2008), lishment or re-establishment of organisms (e.g., plants, ani which is incorporated herein by reference. In an embodiment, mals, microorganisms) in the at least one environmental the modified microorganisms include live, commensal bacte medium where the composition is administered. In an ria, archea, fungi, or other microbe given orally. As published, embodiment, the environmental medium treatmentagent pro 65 commensal bacteria are able to colonize the intestine follow vides an effective amount for revitalization of the environ ing depletion of microorganisms by antibiotic treatment. See, mental medium following destruction associated with at least for example, Wadolkowski, et al. Inf. Imm., Vol. 56, pp. US 8,852,916 B2 41 42 1030-1035 (1988); and Rao, et al., PNAS, vol. 102, no.34, pp. sequence, or by decreasing the strength of the promoter by 11993-1 1998 (2005), each of which are incorporated herein introducing nucleotide changes in the consensus promoter by reference. sequence (or by variations in the spacer sequence). In an embodiment, at least one antacid or proton pump In an embodiment, gene constructs that direct protein inhibitor (e.g. Pantozol, Altana Pharma BV. Hoofdorp, Neth expression can be integrated into bacterial or other microbial erlands) and a chocolate acid binder (e.g. Questran, Zambon, chromosomal DNA by homologous recombination using Amersfoort, Netherlands) are administered prior to, during, methods described in Steidler et al, Ibid. or Subsequent to administration of the modified microorgan In an embodiment, the modified microorganism is engi ism composition in order to improve viability during passage neered or selected for by utilizing specific nutrient or metabo through the stomach. See, for example, Braat et al. Clin. 10 lite requirements. For example deletion of the thymidine syn Gastroenterol. Hepatol. Vol. 4, pp. 754-759 (2006), which is thase gene in Lactococcus lactis (L. Lactis) by homologous incorporated by reference. For example, a bacterium Such as recombination using recombinant DNA plasmids results in L. Eschericia coli (E. coli), Nissile 1917 can be engineered lactis clones that require thymidine for growth. See, for using rDNA methods to produce proteins or peptides. See, for example, Steidler et al., Ibid. Since thymidine is present only example, Rao etal, Ibid., and U.S. Pat. No. 7.341,860; each of 15 at low levels in human colon, L. lactis thymidine auxotrophs which is incorporated herein by reference. Survive less than two days following oral administration to In an embodiment, the modified microorganism composi human volunteers. See, for example, Braat et al., Ibid. tion requires gene expression for production ordelivery of the However, thymidine auxotrophs can Survive longer than at least one therapeutic agent. Expression of genes encoding 200 hours in vitro when thymidine (10 uM) is provided in the at least one therapeutic agent (e.g., protein, microbicide, anti media. Thus, one can control the growth and Survival of gen, tolerogen, etc.) is directed, for example, by a constitutive thymidine auxotrophs in vivo by dosing and scheduling or inducible bacterial or other microbial promoter. For administration of thymidine prior to, during, or Subsequent to example, in an embodiment, the acid inducible P170, P3, or administration of the modified microorganism composition. P1 promoter (isolated from Lactoccus) can be fused to genes For example, in an embodiment, bacterial (or other microbial) encoding microbicide proteins and gene expression can be 25 auxotrophs are derived from standard bacterial strains (or induced with low pH. See, for example, Madsen, et al., Ibid. other microbial strain) by deleting or mutating genes encod In an embodiment, the arabinose promoter (P) can be ing enzymes or other proteins essential for bacterial (or other fused to heterologous genes encoding therapeutic proteins, or microbial) metabolism and growth. Methods for creating other therapeutic agents. Coexpression of araC, an arabinose mutations, insertions and deletions (such as homologous operon regulatory protein, and provision of L-arabinose regu 30 recombination, recombinant DNA techniques, insertional lates expression of genes fused to the P promoter. See, for mutagenesis, targeted gene deletion, etc.) in essential genes example, U.S. Pat. No. 7,341,860, Ibid. are described at, for example, U.S. Pat. No. 5,643,771, and In an embodiment, gene constructs employ a constitutive Biswas etal, J. Bacteriology, vol. 175, pp. 3628-3635 (1993); promoter including, for example, promoter sequences each of which is incorporated herein by reference. For derived from the bacterial thymidine synthase gene which 35 example, mutation or deletion of the B-aspartate semialde continuously express at least one therapeutic agent, such as a hyde dehydrogenase gene (Asd) in bacteria or other microbe cytokine. See, for example, Steidler et al. Nature Biotechnol precludes synthesis of diaminopimelic acid (DAP), an essen ogy Vol. 21, pp. 785-789 (2003), which is incorporated herein tial cell wall constituent that is not present in animal tissues. by reference. For example, gene constructs directing expres Without exogenous DAP, bacteria or other microbe that have sion of at least one therapeutic agent (e.g. antigenic, micro 40 a mutated or deleted Asd gene will undergo cell death and bicidal or tolerogenic protein, etc.) can be incorporated into lysis (See, for example, U.S. Pat. No. 7,341,860, Ibid.), but expression plasmids with drug-resistance selectable markers providing DAP by oral administration allows Asd mutants to (e.g. amplicillin resistance marker, B-lactamase, or chloram grow, colonize and Survive on mucosal Surfaces in vivo. phenicol resistance marker) and transfected or electroporated Measurement of modified microorganisms present in vivo into bacteria, or other microbes. See, for example, Sambrook 45 can be done using the quantitative polymerase chain reaction et al. Molecular Cloning: A Laboratory Manual, second ed., (PCR) and primers specific for the microbial strain and the Cold Spring Harbor Laboratory Press, N.Y. (1989), which is gene expression construct. For example, in an embodiment, incorporated by reference herein. stool samples from Subjects given L. Lactis, engineered to In an embodiment, a propionate inducible expression sys express human IL-10, is assayed with PCR primers specific tem is utilized, in order to provide a relatively homogenous 50 for the 16S ribosomal RNA of L. lactis and the human IL-10 expression in individual microorganism cells, and allow for expression construct (See, for example, Braat et al., Ibid.). highly regulatable expression. See, for example, Lee and In an embodiment, the number of colony forming units Keasling, App. Env. Microbiol. Vol. 71, no. 11, pp. 6856-6862 (CFU) of L. Lactis is assessed by culturing stool samples on (2005), which is incorporated herein by reference. For microbiological plates containing selective media. For example, expression vectorpPro, described by Lee and Keas 55 example, fecal samples are Suspended in minimal media and ling, is capable of being regulated at the single cell level over then selected on plates coated with antibodies specific for L. a wide range of inducer concentrations in a dose-dependent Lactis. Next, media containing essential nutrients is overlaid manner. Id. Furthermore, since bacterial cells are permeable and the bacterial colonies arising are counted to determine the to the inducer proprionate (which is metabolized by 2-MC by CFU present in the fecal sample (See, for example, Steidler et native chromosomal expression), regulatable and consistent 60 al, Ibid.). In an embodiment, PCR is conducted with colonies induction in all cells of the culture is attainable. and primers specific for the bacteria or other microorganism, In certain instances, repression is equally important as and gene expression construct (e.g. 16S ribosomal RNA and induction (for example, in instances where the gene product is IL-10) to verify the identity of the colonies (See, for example, particularly toxic or difficult to maintain in the host microor Braat etal, Ibid.). ganism). Since protein synthesis depends on translational 65 In an embodiment, bacteria or other microorganisms are efficiency as well as promoter strength, background expres modified to deliver at least one therapeutic agent, while sion may be reduced by using a weaker ribosome binding site requiring an essential metabolite not usually present in bio US 8,852,916 B2 43 44 logical tissues of the Subject (or present at low concentra 6,610,529, which is incorporated herein by reference. The tions). In vivo provision of essential nutrients or metabolites frequency and duration of arabinose pulsing determines the required by the bacteria, or other microorganisms allows for expression of genes (and their corresponding proteins) in the control of the survival and colonization of the bacterial or gene network, while the dose and Schedule of oral arabinose other microbial delivery, and allows for regulation of the 5 administration determines the timing and duration of expres schedule or dose of the at least one therapeutic agent. For sion of the therapeutic agent and Suicide gene, IL-10 and rel example, thymidine synthase mutants of L. lactis (Thy A. L. F, respectively. In an embodiment, synthetic gene networks Lactis) are dependent on exogenous thymidine for growth in with optimal pulse intervals of approximately 10-40 minutes vitro and in vivo. and optimal pulse lengths of approximately 20-30 minutes In an embodiment, in vitro, no viable Thy A. L. Lactis are 10 are utilized. In an embodiment, gene networks with optimal present after culture 72 hours in rich media devoid of thymi pulse intervals and pulse lengths of approximately 2-12 hours dine, but in cultures containing 10 uM thymidine, the are also utilized (see Friedland et al., Ibid.). For example, microbes survive beyond 200 hours. In vivo, only 4% of gene networks encoding IL-10 might require approximately Thy A. L. Lactis auxotrophs survive after 4 hours in the mam two 10-minute pulses of arabinose separated by an interval of malian intestine with only endogenous thymidine present 15 20 minutes to optimally induce IL-10 expression, but rel F (thymidine concentration is less than 0.075uM in humanileal expression and cell death would only ensue following two lavage; See, for example, Steidler et al., Ibid.). 2-hour pulses with arabinose separated by 2 hours. In an In an embodiment, production of at least one therapeutic embodiment, gene networks incorporate multiple inducers agent, for example, IL-10, by bacterial or other microbial (e.g., arabinose, anhydrotetracycline and IPTG), and the auxotrophs are controlled by a constitutive promoter, Such as expression of multiple genes in the network depends, for the thymidine synthase promoter. Production of IL-10 example, on the order, length and interval of pulsing with depends in part on the growth and survival of the bacterial or each of the inducers. See, Friedland et al., Ibid. other microbial auxotroph. In an embodiment, microorganisms are modified to include In an embodiment, an inducible promoter Such as the P/ at least one lethal or Suicide gene to regulate population araC promoter/regulator System (see, for example, U.S. Pat. 25 growth, and to prevent possible dissemination into the envi No. 7,341,860, Ibid.), is used in conjunction with arabinose to ronment (e.g. via feces). For example, in an embodiment, the regulate the production of IL-10. In an embodiment, produc relF gene is controlled by a regulated promoter Such as plac tion and delivery in situ is regulated by dosing and scheduling that is repressed by a regulator protein, LacIq, unless isopro of arabinose administration, and by controlling bacterial or pyl B-D-thiogalactoside (IPTG) is provided. To control other microbe Survival and growth through dosing and Sched 30 growth of a modified microbial strain in vivo one can admin uling of thymidine administration. In an embodiment, the ister IPTG to induce relF expression and cause cell death. amount of protein delivered can be monitored by immunoas Alternatively the relF gene can be controlled by the arabinose say of fecal samples. For example, human IL-10 derived from promoter/repressor. In this example expression of relF is feces samples can be measured by enzyme linked immun regulated by the C2 repressor which, in turn, is regulated by osorbent assay (ELISA; Steidler etal, Ibid.). ELISA reagents 35 the presence of arabinose. Thus, when arabinose levels are and protocols for numerous cytokines including IL-10 are reduced C2 repressor levels decline and relF is expressed available, for example, from Invitrogen Corp., Carlsbad, leading to bacterial cell death (see, for example, U.S. Pat. No. Calif. 6,610,529, which is incorporated herein by reference). In an embodiment the production of a therapeutic agent, In an embodiment, mutually dependent strains of bacteria Such as IL-10 and a Suicide factor, Such as Rel F. are con 40 that signal via Small molecules such as acyl-homoserine lac trolled by a synthetic gene network engineered into a micro tones (acyl-HSL), are modified to express heterologous target organism. For example, oral administration of an inducer genes only when sufficient numbers of both bacterial strains molecule controls expression of IL-10. A synthetic gene net (and acyl-HSL) are present. See, for example, Brenner et al. work responsive to pulses of a metabolite (e.g., arabinose), is PNAS, vol. 104, pp. 17300-17304 (2007), which is incorpo utilized, as described in Friedland et al., Science, vol.324, pp. 45 rated herein by reference. For example, in an embodiment, 1199-1202 (2009), which is incorporated herein by reference. two E. coli strains, modified to produce specific acyl-HSL, In an embodiment, a gene network is constructed by combin control the expression of target genes for each another. ing transcriptional and translational regulatory elements. For In an embodiment, target gene expression is controlled by example in an embodiment, the P. promoter, a transacti provision of exogenous acyl-homoserine lactone to the indi Vating noncoding RNA, a cis repressor sequence RNA, and a 50 vidual microbial Strains. For example, administration of T7 RNA polymerase gene may be combined in a synthetic 3-oxododecanoyl-HSL (3OC12HSL) and butanoyl-HSL gene network to control the expression of multiple proteins as (C4HSL) in vivo can be used to control target protein produc shown by Friedland et al., Ibid. In an embodiment, the regu tion of E. coli strains engineered to be responsive to specific latory elements cause one particular product to be produced acyl-HSL molecules (see, for example, Brenner et al., Ibid.). with a first induction event (e.g., exposure to arabinose); a 55 In addition, Survival of the engineered bacterial strains can be second particular product to be produced with a second controlled by acyl-HSL-regulated expression of toxin (ccdB) induction event; a third particular product to be produced with and antitoxin (ccd A) genes, which cause cell death, or allow a third induction event, etc. In this manner, the synthetic survival, respectively. See, for example, Balagadde etal, Mol. system allows for exhibiting the number of induction events Sys. Biol. Vol. 4, pp. 1-8 (2008), which is incorporated herein that have occurred, or "counting induction events. Id. 60 by reference. In an embodiment, the toxin-antitoxin includes In an embodiment, atherapeutic agent (e.g., IL-10) may be at least one of masEF, chpBIK, relBE, yefM-yoeB, dinJ-yaf, delivered to a patients intestine by ingestion of modified E. or ecna-ecnB. See, Engleberg-Kulka, et al., PLOS Genetics, coli containing a plasmid encoding a synthetic gene network vol. 2, no. 10, 1518-1526 (2006), which is incorporated that responds to multiple pulses of an inducer molecule (e.g., herein by reference. arabinose) by the production of IL-10. The gene network may 65 Coadministration of multiple bacterial strains that express also contain Suicide genes (e.g. relF) that will cause cell death different target genes can be used to co-deliver multiple thera when they are expressed. See, for example, U.S. Pat. No. peutic agents. For example, localized co-expression of IL-10 US 8,852,916 B2 45 46 and transforming growth factor beta by two bacterial strains bility, while the metabolite (e.g., histidine or leucine) regu can provide localized immunoregulatory therapy for inflam lates growth and Survival of the yeast strain. For example, in matory bowel disease. an embodiment, engineered S. boulardii is used for vaccina In an embodiment, a bacterial strain that expresses an anti tion, wherein the strain requires leucine and histidine, and gen derived from a viral pathogen, for example. E7 antigen 5 produces a heterologous protein. In an embodiment, the het from human papilloma virus type 16, is coadministered with erologous protein includes but is not limited to, hepatitis B a second bacterial strain that produces interleukin-12 (IL-12) Surface antigen encoded on a yeast episomal plasmid contain to promote immunization. In an embodiment, the two bacte ing LEU2, and under the control of the ADH1 constitutive rial strains are auxotrophs that are mutually dependent on promoter. In an embodiment, dosing and scheduling of histi each other for essential metabolites, such as amino acids or 10 dine administration regulates the growth and Survival of the DAP engineered S. boulardi and, in turn, the production and deliv In an embodiment, one or both auxotrophic strains are ery of hepatitis B surface antigen. In an embodiment, yeast or Sustained in vivo by administering exogenous metabolites other microbial strains are engineered to express at least one (e.g. histidine, arabinose, DAP, acyl-HSL, thymidine) to the Suicide gene. For example, yeast or other microbe Strain host organism. In an embodiment, expression of the E7 gene 15 transformed with a recombinant plasmid encoding a nuclease and IL-12 is directed by the same or a different promoter. The (e.g. Serratia marcescens nuclease A) under the control of the promoter may be constitutive or inducible. For example, in an S. cerevesiae ADH2 promoter, undergoes cell death when the embodiment, E7 and IL-12 are fused to the lac operon pro nuclease A gene is expressed. Ordinarily, the ADH2 promoter moter (P) which is inducible with IPTG or lactose. is repressed by glucose, and when glucose levels are depleted, In an embodiment, bacterial or other microbial survival and 20 the ADH2 promoter/nuclease A gene is expressed, resulting target protein expression is regulated by administration of the in cell death. See, for example, Balan et al. Yeast, Vol. 22, pp. same metabolite. For example, in an embodiment, the P. 203-212 (2005) which is incorporated herein by reference. promoter is used to regulate expression of a polycistronic Thus, in a glucose poor environment (e.g. feces, intestine, transcript (see, for example, Brenner etal, Ibid.) that encodes soil, etc.) death of the modified yeast or other microorganism a target protein and a Survival factor. Providing lactose (or 25 strain is induced. IPTG) to a subject hosting the bacterial or other microbial In an embodiment, co-administration of at least two strains strain will not only sustain survival of the strain but also of modified auxotrophic microbes is performed, for example, induce expression of the target protein. in order to provide essential nutrients to each other. For Engineered or modified microorganisms, such as fungi, example, in an embodiment, two yeast (Saccharomyces cer derived from generally regarded as safe (GRAS) species can 30 evesiae) auxotrophs, one that requires lysine, and a second be auxotrophic for essential metabolites and express protein that requires adenine, are modified or engineered to over encoding genes under the control of regulated promoters. For produce adenine and lysine, respectively. Conventional meth example, in an embodiment, Saccharomyces cerevesiae (S. ods. Such as mutation, selection and genetic crosses, can be cerevesiae) and Saccharomyces boulardii (S. boulardii) are employed to generate the mutants. See, for example, Shou et GRAS organisms that are mutated and selected (See Abos- 35 al, PNAS, vol. 104, pp. 1877-1882 (2007), which is incorpo ereh et al. Res. J. Agric. Biol. Sci., vol. 2, pp. 478-482 (2006) rated herein by reference. which is incorporated by reference herein) to obtain clones In an embodiment, growth of each of the yeast strains is that require amino acids (e.g., leucine, tryptophan, lysine, dependent on the other strain to supply an essential metabo arginine), purines, or pyrimidines (e.g., adenine, thymidine). lite (i.e., adenine or lysine). Coadministration of live yeast Growth and survival of yeast auxotrophs in vitro and in vivo 40 strains that are mutually dependent on each other for survival is dependent on an essential metabolite (e.g., adenine), and if allows prolonged Survival and colonization of both strains on adenine is not present (or present at low levels) in a subject mucosal Surfaces (e.g. intestinal, vaginal, nasal, oral, bron hosting the strain, then growth of the yeast auxotroph can be chial, etc.). In an embodiment, the yeast strains are modified regulated by administering adenine to the Subject. For to express enzymes essential for overproduction of adenine example, the dose and Schedule of adenine administration can 45 and lysine under the control of regulated promoters such as control the proliferation and survival of the yeast adenine the CUP-1 promoter derived from the metallothionein gene. auxotrophs. (See, for example, Glazer et al., Ibid.) In an embodiment, In an embodiment, yeast or other microbial strains are transcription of genes fused to CUP-1 is induced by providing modified to express at least one therapeutic agent (e.g., pro metalions such as Cu" and Zn". For example, ZnCl2 can be tein). In an embodiment, yeast cloning vectors used for pro- 50 given orally to induce expression of ADE4 and LYS21, tein expression include but are not limited to yeast integrative enzymes that mediate over-production of adenine and lysine, plasmids, yeast episomal plasmids, and yeast centromeric respectively, by modified yeast residing, for example, in the plasmids that contain selectable markers based on metabolite colon (see, for example, Shou et al., Ibid.). Withdrawl of requirements. In an embodiment, genes used as selectable ZnCl2 from the diet lowers Zn" levels, reduces expression of markers include but are not limited to LEU2, URA3, and 55 ADE4 and LYS21, and reduces production of adenine and HIS3, which encode enzymes needed to biosynthesize leu lysine which, in turn, will lead to death of the engineered cine, uracil and histidine (for example, yeast vectors for pro yeast Strains. tein expression are described in Glazer et al. Microbial Bio In an embodiment, a microorganism, Subh as a bacterial technology: Fundamentals of Applied Microbiology, 2" strain, is genetically modified to produce atherapeutic agent, edition, Cambridge University Press (2007), which is incor- 60 Such as an anti-fungal peptide. In an embodiment, the bacte porated herein by reference. In an embodiment, transcription rial strain delivers the anti-fungal peptide to at least one of heterologous genes is directed by a constitutive promoter biological tissue, such as plant leaves, stems, or roots to (e.g., ADH1, TDH3) or a regulated promoter (e.g., GAL1, prevent the growth, inhibit the growth or reduce the viability ADH2, PHO5, CUP-1, etc.). of fungal plant pathogens. In an embodiment, the bacterial In an embodiment, an engineered or modified yeast Strain 65 strain is also modified with an inducible genetic element to that requires two metabolites, for example, leucine and histi cause death of the bacteria when a factor is provided to induce dine, a selectable marker (e.g. LEU2) provides plasmid sta a Suicide gene. The composition including the modified US 8,852,916 B2 47 48 microorganism and induction factor, is applied sequentially al., Appl. Envir. Microbiol. vol. 66, pp. 4673-4678 (2000), to the plants, and optionally their environment (e.g., Soil or which is incorporated herein by reference. For example, water), by spraying, dusting, or sprinkling the composition. modified microorganism compositions may be applied in the For example, bacteria known to inhabit the phylloplane field by spraying, soaking, injection into the soil, seed coat (the surface of plant leaves) or the rhizosphere (the soil sur ing, seedling coating, or the like. For example, bacterial Sus rounding plant roots) may be used as microbial hosts for pensions for application to plants may be approximately 10 plasmid expression vectors directing the expression of an to 10" cells/ml and the volume applied per hectacre may be anti-fungal peptide. For example, Pseudomonas fluorescens approximately 10 ml to 100,000 ml or more. In an embodi strain SBW25 inhabits the leaves and roots of sugar beet ment, administration of bacterial Suspensions to a plant part plants and is competent for transformation with recombinant 10 DNA plasmids. See, for example, Zhang, et al., Microbiol. may be at approximately 10 to 10° cells/cm. vol. 152, pp. 1867-1875 (2006), which is incorporated herein In an embodiment, a method comprises administering at by reference. In an embodiment, Pfluorescens may be trans least one composition to at least one environmental medium. formed with a DNA plasmid that encodes an anti-microbial In an embodiment, the at least one composition includes at peptide or toxin and directs expression of the anti-microbial 15 least one modified microorganism including at least one het peptide or toxin. See, for example, U.S. Pat. No. 5,017,373: erologous genetic element encoding at least one environmen and U.S. Pat. No. 7,510,852, each of which is incorporated tal medium treatment agent for at least one environmental herein by reference. medium, and at least one genetic element inducible to initate In one example, a recombinant DNA plasmid encoding an death in the at least one modified microorganism. In an antifungal peptide, PW2, and methods for its cloning and embodiment, the at least one heterologous genetic element construction are described in U.S. Pat. No. 7,550,558, which encodes at least one plant hormone for the at least one envi is incorporated herein by reference. As indicated in the patent, ronmental medium. peptide PW2 has anti-fungal activity against the following In an embodiment, a modified microorganism includes at plant fungal pathogens: Colletotrichum gossypii, Cepha least one heterologous genetic element encodes at least one losporioides, Alternaria macrospora, Colletotrichum Ora, 25 plant growth factor, for example, acetoin (3-hydroxy-2-bu Bipolaris Sorokiniana, Dreschslera tritici, Phoma Sorghina, tanone) and 2,3-butanediol in the environmental medium Pyricularia grisea, Colletotrichum, Gloeosporioides, (e.g., soil), for example, in order to promote the growth of Rhizoctonia Solani and Fusarium Solani. plants. According to published studies, naturally occurring In an embodiment, genetically modified Pfluorescens are plant-growth promoting rhizobacteria, which are capable of further modified to contain a Suicide gene that may be induced 30 producing acetoin, increase leaf growth in Arabidopsis at any time by providing a factor to the Pfluorescens, thus thaliana by more than 100% relative to control bacteria not killing the bacteria, and stopping the production of the anti capable of producing acetoin. See, for example, Ryu et al. fungal peptide. In an embodiment, Pfluorescens may contain PNAS USA, vol. 100, pp. 4927-4932 (2003), which is incor a Rel F gene that induces death of the bacterium when it is porated herein by reference. As described herein, in an expressed. See, for example, U.S. Pat. No. 6,610,529, which 35 embodiment, the genetic element(s) necessary to produce the is incorporated herein by reference. In an embodiment, the plant growth factors are provided as at least one heterologous Rel F gene is controlled by a regulated promoter Such as plac genetic element to at least one modified microorganism that that is repressed by a regulator protein, LacIq, unless isopro also includes at least one genetic element inducible to initiate pyl B-D-thiogalactoside (IPTG) is provided. For example, to death in the at least one modified microorganism (e.g., a lethal control growth of the modified microbial strain, IPTG (avail 40 or Suicide gene). able from Sigma-Aldrich Corp., St. Louis, Mo.) may be In an embodiment, a bacterium that lives in the soil, e.g. applied to the plant or soil areas where the composition has Streptomyces lividans, is transformed with at least one been applied or translocated, and induce Rel F expression, genetic element (i.e. gene, promoter element, regulatory Subsequently inducing modified microorganism cell death. In sequence, etc.) that encodes at least one enzyme required to an embodiment, IPTG can be applied by spraying, dusting, 45 produce at least one plant growth factor. For example, S. sprinkling or the like. An IPTG Solution containing approxi lividans (ATCC #69441; available from American Type Cul mately 3 mM IPTG is used to induce expression of the ture Collection, Manassas, Va.) is modified by transformation repressed pLac promoter. See, for example, Sambrook, with a plasmid (e.g. pl.J702) containing at least one enzyme Fritsch & Maniatis, Molecular Cloning: A Laboratory gene required to produce at least one plant growth factor (e.g., Manual, Second Edition (1989) Cold Spring Harbor Labora 50 acetoin, gibbrellin, 2,3-butanediol, etc.). Transformation of tory Press, Cold Spring Harbor, N.Y. which is incorporated the microorganism can be conducted utilizing standard pro herein by reference. cecures. See, for example, Wang et al., J. Biotech., vol. 113, Modified Pfluorescens expressing the anti-fungal peptide, pp. 131-144 (1990); and Sambrooketal, Molecular Cloning: PW2, may be applied to protect and treat cotton plants A Laboratory Manual, second ed., Cold Spring Harbor Labo infected with a pathogenic fungus, e.g., Alternaria mac 55 ratory Press, N.Y. (1989), each of which is incorporated rospora. For example, Pfluorescens may be applied to its herein by reference. natural habitat such as the rhizosphere or phyllosphere of the For example, bacteria are genetically engineered to express plant where it can grow and produce PW2. the acetolactate synthase (AlsS) and acetolactate decarboxy In an embodiment, the modified microorganism is capable lase (AlsD) genes which act on pyruvate, a common metabo of colonizing the plant. In an embodiment, the modified 60 lite, to yield acetoin. See, for example, Taghavi et al. Appl. microorganism is not capable of colonizing the plant, and Envir. Microb. Vol. 75, no. 3, pp. 748-757 (2009), which is repeated application of the composition is required. For incorporated herein by reference. Cloning and expression of example genetically engineered Pseudomonas Strains are an Alss gene in bacteria can be conducted utilizing standard stable within the rhizosphere of wheat and barley plants for procecures. See, for example, Smith et al., PNAS USA, Vol. approximately 1 month at a level of approximately 1.0x10 to 65 86, pp. 4179-4183 (1989); and Swindell et al. Appl. Envir. 23x10 CFU/cm of root and may stably express recombinant Microbiol. Vol. 62, no. 7, pp. 2641-2643 (1996), each of genes for approximately 29 days. See, for example, Shim et which is incorporated herein by reference. US 8,852,916 B2 49 50 In an embodiment, expression of gene(s) encoding the mental medium. For example, in an embodiment, the at least AlsS and AlsD enzymes is directed by at least one constitutive one modified microorganism is sprayed onto Soil as a spore or inducible bacterial promoter. For example, in an embodi suspension in wateratarate of approximately 10°-10 colony ment, the inducible lac operon promoter is fused to at least forming units (CFU) per gram of soil. For example, as shown one gene encoding at least one enzyme (e.g. Alss. AlsD), and by published studies, modified S. lividans innoculated at gene expression is induced with lactose or isopropyl B-D- approximately 107 CFU/gm soil and grown at 25° C. survive thiogalactoside. See, for example, Rao, et al., PNAS USA vol. for at least approximately 90 days. See, for example, Craw 102, no. 34, pp. 11993-11998 (2005), which is incorporated ford et al. Appl. Envir. Microbiol., vol. 59, pp. 508-518 herein by reference. (1993), which is incorporated herein by reference. In an In an embodiment, the arabinose promoter (pBAD) is 10 embodiment, the survival of S. lividans is measured by dilut fused to the at least one heterologous genetic element. For ing in water and spreading on plates containing at least one example, coexpression of araC, an arabinose operon regula antibiotic to which the microorganism has been rendered tory protein, and providing L-arabinose regulates expression resistant. For example, S. lividans strain TK23 is modified to of genes fused to the pBAD promoter. See, for example, U.S. be resistant to spectinomycin, while S. lividans transformed Pat. No. 7,341,860, which is incorporated herein by refer 15 with the plasmid, pIJ702 is resistant to thiostrepton. See, for ence. In an embodiment, at least one heterologous genetic example, Crawford et al., Ibid. element employs at least one constitutive promoter, for In an embodiment, a method includes administering at example, at least one promoter sequence derived from the least one composition including at least one modifiec micro bacterial thymidine synthase gene is used to continuously organism encoding at least one environmental medium treat express at least one enzyme or plant peptide hormone. See, ment agent, such as a plant peptide hormone, to at least one Steidler et al, Nat. Biotech. vol. 21, pp. 785-789 (2003), environmental medium (e.g., soil, water, etc.). In an embodi which is incorporated herein by reference. ment, the at least one modified microorganism includes at In an embodiment, the at least one heterologous genetic least one inducible genetic element to initiate death of the at element directing expression of the at least one enzyme or least one modified microorganism. plant peptide hormone is incorporated in at least one expres 25 In an embodiment, the at least one modified microorgan sion plasmid, or other vector. In an embodiment, the at least ism encodes at least one plant peptide hormone (e.g., one expression plasmid, or other vector, includes at least one POLARIS), and at least one genetic element inducible to drug-resistance selectable marker (e.g. amplicillin resistance initiate death in the at least one modified microorganism (e.g., marker, B-lactamase, or chloramphenicol resistance marker). Suicide factor, such as rel F), and each is controlled by a In an embodiment, the at least one expression plasmid or 30 synthetic gene network of the microorganism. As shown in other vector is transfected or electroporated into at least one published studies, application of an inducer molecule regu microorganism. Routine methods for gene transfer and com lates expression of POLARIS, a 36 amino acid peptide that ponents relating to selectable markers are described, for functions in root growth and leaf vascularization. See, for example, in Sambrook et al. Molecular Cloning: A Labora example, Casson et al. Plant Cell, vol. 14, pp. 1705-1721 tory Manual, second ed., Cold Spring Harbor Laboratory 35 (2002), which is incorporated herein by reference. In an Press, N.Y. (1989), which is incorporated herein by refer embodiment, the synthetic gene network is responsive to ence). pulses of a metabolite (e.g., arabinose). See, for example, In an embodiment, the at least one heterologous genetic Friedland et al., Science, vol. 324, pp. 1199-1202 (2009), element that directs protein expression can be integrated into which is incorporated herein by reference. microorganism chromosomal DNA by homologous recom 40 In an embodiment, the gene network is constructed by bination by using standard procedures, for example, as combining transcriptional and translational regulatory ele described in Steidler et al, Ibid. ments. For example, in an embodiment, the pBAD promoter, In an embodiment, the modified microorganism described a transactivating noncoding RNA, a cis repressor sequence herein, e.g. S lividans, is additionally transformed with at RNA, and a T7 RNA polymerase gene are combined in a least one genetic element inducible to initiate death in the at 45 synthetic gene network to control the expression of multiple least one modified microorganism. For example, in an proteins as shown by Friedland et al., Ibid. embodiment, the at least one inducible genetic element In an embodiment, at least one modified microorganism encodes at least one lethal or Suicide gene under the control of (e.g. Pseudomonas putida) including at least one heterolo an inducible promoter. For example, bacteria may be engi gous genetic element encoding at least one environmental neered with the rel F gene to provide additional containment 50 medium treatment agent, for example, a plant peptide hor of the modified bacteria. In an embodiment, expression of the mone (e.g., POLARIS), to at least one environmental medium relF gene is controlled by an inducible promoter. Such as (e.g., soil, water, food products). In an embodiment, the at pLac, that is repressed by an inducer, LacIq, unless isopropyl least one modified microorganism includes at least one plas B-D-thiogalactoside (IPTG) is provided (see Rao etal, Ibid.). mid encoding a synthetic gene network that responds to mul For example, in order to initiate death in the at least one 55 tiple pulses of an inducer molecule (e.g., arabinose, acid, modified microorganism, IPTG is administered to induce relF etc.), by producing the at least one environmental medium expression and cause cell death of the microorganism. In an treatment agent (e.g., POLARIS). The gene network further embodiment, the rel F gene is expressed under the control of includes at least one genetic element inducible to initiate the arabinose promoter/repressor system. For example, in an death in the at least one modified microorganism (e.g., a embodiment, expression of relF is regulated by the C2 repres 60 Suicide gene. Such as rel F), that induces bacterial cell death Sor which, in turn, is regulated by the presence of arabinose. when it is expressed. See, for example, U.S. Pat. No. 6,610, When arabinose levels are reduced, C2 repressor levels 529, which is incorporated herein by reference. decline and relF is expressed leading to microorganism cell In an embodiment, the frequency or duration of arabinose death. See, for example, U.S. Pat. No. 6,610,529, which is application determines the expression of the inducible incorporated herein by reference. 65 genetic element (and the corresponding protein(s)) in the In an embodiment, modified S. lividans bacteria capable of gene network, and the dose and schedule of arabinose appli producing acetoin are administered to at least one environ cation to the Soil, or other environmental medium, determines US 8,852,916 B2 51 52 the timing and duration of expression of POLARIS and rel F. microorganism; the reservoir 520 configured to receive, As published, synthetic gene networks have optimal pulse retain, and dispense at least a portion of the at least one intervals of approximately 10-40 minutes and optimal pulse composition; and at least one component 530 configured to lengths of approximately 20-30 minutes. Id. In an embodi administer the at least one composition to at least one bio ment, gene networks with optimal pulse intervals and pulse logical tissue. lengths of approximately 2-12 hours are also described. Id. As indicated in FIG. 6, in an embodiment 600, the at least Other optimal pulse intervals can be determined for a particu one composition further includes at least one pharmaceuti lar system, according to the published guidelines. Id. cally-acceptable carrier or excipient. In an embodiment 610, In an embodiment, a gene networks incorporates multiple the at least one first constituent of the composition and the at inducers (e.g., arabinose, anhydrotetracycline and IPTG). 10 least one second constituent of the composition are located in and the expression of multiple genes in the network depends different reservoirs of the delivery device. In an embodiment on the order, length, and interval of pulsing with each of the 620, the device is implantable. In an embodiment 630, the inducers. Id. device is implanted into a subject. In an embodiment 640, the In an embodiment, at least one modified microorganism device is external to a subject. In an embodiment 650, the including at least one heterologous genetic element encoding 15 device includes at least one component configured to admin at least one environmental medium treatment agent for the at ister the at least one composition to at least one biological least one environmental medium includes a Substrate-regu tissue includes one or more ports. In an embodiment 660, the lated Suicide gene system that responds to at least one envi at least one of the one or more ports includes at least one outlet ronmental inducer, Such as a Xenobiotic or pollutant. For port or at least one inlet port. In an embodiment 670, each example, in an embodiment, the at least one modified micro reservoir includes at least one separate port. In an embodi organism includes three genetic elements: 1) a regulated pro ment 680, the delivery device includes at least one reservoir moter (e.g., plac) fused to a Suicide gene (e.g., gef), and 2) a including at least one inducer formulated to induce at least TOL plasmid promoter (e.g., Pm) fused to the lad gene (en one promoter operably coupled to the at least one genetic coding the Lac repressor), and 3) XylS2, a gene encoding a element inducible to initiate death of the at least one aux positive regulator of Pm that interacts with 3-methylben 25 otrophic microorganism. Zoate. See, for example, Ramos, et al., Biotech. Vol. 12, pp. As indicated in FIG. 7, the delivery device further com 1349-1356 (1994), which is incorporated herein by reference. prises 700 one or more controllable output mechanisms oper In an embodiment, Soil bacteria, for example, Pputida, modi ably linked to the one or more ports to control dispensing of fied to produce POLARIS under the control of a constitutive at least a portion of the at least one composition from the at promoter (for example, the bacterial thymidine synthase pro 30 least one reservoir. In an embodiment 710, the at least one moter, according to Steidler et al., Ibid.) is transformed with a controllable output mechanism includes at least one of a DNA cassette containing the plac promoter fused to the gef micropump, valve, or actuator. In an embodiment 720, the gene and with a plasmid that contains the Pm promoter fused valve includes at least one of a one-way valve, or pressure to lac I gene and encoding the XylS2 positive regulator of Pm. settable valve. In an embodiment 730, the actuator includes at For example, genetic constructs and construction of recom 35 least one of a piezoelectric actuator, electrostatic actuator, binant bacterial strains can be performed utilizing routine thermal actuator, shape-memory alloy actuator, bioactuator, procedures. Some of which are described in Jensen et al. Appl. or magnetic actuator. In an embodiment 740, the at least one Env. Micro., vol. 59, pp. 3713-3717 (1993), which is incor controllable output mechanism includes at least one thermal porated herein by reference. In an embodiment, modified P or nonthermal gate in communication with the at least one putida encoding POLARIS, and including at least one genetic 40 outlet of the at least one reservoir. In an embodiment 750, the element inducible to initiate death of the at least one micro delivery device further comprises at least one control cir organism, is grown in the presence of 3-methylbenzoate. In cuitry configured to control the at least one controllable out an embodiment, when 3-methylbenzoate is combined with put mechanism. In an embodiment 760, the at least one con the XylS2 gene product, positive regulation of Pm results, and trol circuitry is configured to control the release of the at least leads to production of the Lac repressor which, in turn, 45 two constituents of the composition. In an embodiment 770, represses the expression of the toxic gene, gef. According to the at least one of the rate of release, amount of release, or published studies, P. putida Strains including a Substrate time of release of the at least two constituents of the compo regulated toxic gene system inoculated at approximately 10 sition are different for each constituent. In an embodiment CFU/gm to nonsterile soil containing 0.08% (wt/vol.) 3-me 780, the at least one control circuitry is configured to generate thylbenzoate are present at approximately 10 CFU/gm of 50 and transmit an electromagnetic control signal configured to soil after approximately 14 days. See, for example, Jensen et control the at least one controllable output mechanism. In an al, Ibid. However, in control experiments without 3-methyl embodiment 790, the at least one control circuitry is config benzoate only approximately 10 CFU/gm of soil survived ured to control the at least one controllable output mechanism after 14 days. Id. for time-release of at least a portion of the at least one com Delivery Devices 55 position from the at least one reservoir. In an embodiment, a delivery device comprising a compo As indicated in FIG. 8, in an embodiment 800, the at least sition described herein are disclosed. As indicated in FIG. 5, one control circuitry is configured for variable programming a delivery device 500 comprises a housing 505 including at control of the at least one controllable output mechanism. In least one reservoir containing at least one composition, the at an embodiment 810, the at least one control circuitry is con least one composition including at least one first constituent 60 figured to control release of the composition or a portion including at least one auxotrophic microorganism including thereof in response to a signal from a sensor. In an embodi at least one pH inducible promoter operably coupled to at ment 815, the at least one control circuitry is configured to least one heterologous genetic element encoding at least one control release of at least a portion of the composition in therapeutic agent, and at least one genetic element inducible response to a signal from a sensor. In an embodiment 820, the to initiate death of the at least one auxotrophic microorgan 65 at least one control circuitry is configured to control release of ism; and 515 at least one second constituent including at least at least one inducer formulated to activate at least one pro one metabolite required by the at least one auxotrophic moter operably coupled to the at least one genetic element US 8,852,916 B2 53 54 inducible to initiate death of the at least one auxotrophic nucleic acid, oligosaccharide, polysaccharide, glycopeptide, microorganism. In an embodiment 830, the delivery device glycolipid, lipoprotein, sphingolipid, glycosphingolipid, gly further comprises at least one injector. In an embodiment 840, coprotein, peptidoglycan, lipid, carbohydrate, metallopro the delivery device further comprises at least one transducer. tein, proteoglycan, chromosome, adhesion molecule, cytok In an embodiment 850, the delivery device further comprises ine, chemokine, immunoglobulin, antibody, antigen, platelet, at least one receiver. In an embodiment 860, the at least one extracellular matrix, blood plasma, cell wall, hormone, receiver is configured to receive information from at least one organic compound, inorganic compound, salt, or cell ligand. distal or remote sensor. In an embodiment 870, the receiver is In an embodiment 1030, the at least one detection material is configured to obtain release instructions or authorization to responsive to at least one of glucose, lactate, urea, uric acid, release the at least one composition. In an embodiment 875, 10 glycogen, oxygen, carbon dioxide, carbon monoxide, ketone, the receiver is configured to receive programming instruc nitric oxide, nitrous oxide, alcohol, alkaloid, opioid, cannab tions or data for the controller. In an embodiment 880, the inol, endorphin, epinephrine, dopamine, serotonin, nicotine, delivery device further comprises at least one transmitter. In amphetamine, methamphetamine, anabolic steroid, hydroc an embodiment 885, the at least one transmitter is configured odone, hemoglobin, heparin, clotting metabolite, tumor anti to transmit information regarding one or more of the date, 15 gen, pH, albumin, ATP NADH, FADH2, pyruvate, sulfur, time, presence or approximate quantity of one or more of at mercury, lead, creatinine, cholesterol, alpha-fetoprotein, least a portion of the at least one composition, at least one chorionic gonadotropin, estrogen, progesterone, testoster constituent thereof, or at least one product thereof; at least one one, thyroxine, melatonin, calcitonin, antimullerian hor cell or Substance associated with the at least one biological mone, adiponectin, angiotensin, cholecystokinin, corticotro tissue; at least one metabolite associated with the at least one phin-releasing hormone, erythropoietin, bilirubin, creatine, biological tissue; or at least one microorganism associated follicle-stimulating hormone, gastrin, ghrelin, glucagon, with the at least one biological tissue. In an embodiment 890, gonadotropin-releasing hormone, inhibin, growth hormone, the delivery device further comprises at least one circuit. growth hormone-releasing hormone, insulin, human placen As indicated in FIG.9, in an embodiment 900, the delivery tal lactogen, oxytocin, orexin, luteinizing hormone, leptin, device further comprises at least one power source. In an 25 prolactin, Somatostatin, thrombopoietin, cortisol, aldoster embodiment 910, the delivery device further comprises at one, estradiol, estriol, estrone, leukotriene, brain natriuretic least one detection material. In an embodiment 915, the deliv peptide, neuropeptide Y. histamine, Vitamin, mineral, endot ery device further comprises at least one reservoir for con helin, renin, enkephalin, DHEA, DHT, alloisoleucine, toxic trolled release of the at least one detection material. In an Substance, illegal Substance, therapeutic agent, or any embodiment 920, the at least one detection material includes 30 metabolite thereof. at least one of a radioactive, luminescent, colorimetric or As indicated in FIG. 11, in an embodiment 1100, the deliv odorous substance. In an embodiment 930, the at least one ery device further comprises at least one memory mechanism detection material includes at least one of a taggant, contrast for storing instructions for generating and transmitting an agent, sensor, or electronic identification device. In an electromagnetic control signal. In an embodiment 1110, the embodiment 940, the at least one electronic identification 35 delivery device further comprises at least one imaging appa device includes at least one radio frequency idientification ratus capable of imaging the approximate quantity within a device. In an embodiment 950, theat least one sensor includes treatment region of one or more of the at least one composi at least one biosensor. In an embodiment 960, the at least one tion, at least one constituent thereof, or at least one product biosensor includes at least one modified microorganism. In an thereof; at least one cell or substance associated with the at embodiment 970, the at least one sensor receives information 40 least one biological tissue; or the at least one metabolite, or at associated with at least one oftemperature, pH, inflammation, least one product thereof. In an embodiment 1120, the deliv presence of at least one inducer, amount of at least one ery device further comprises at least one reservoir for con inducer, presence of at least one repressor, amount of at least trolled release of at least one inducer or repressor of the at one repressor, or biological response to the at least one com least one heterologous genetic element encoding at least one position. In an embodiment 980, the at least one detection 45 therapeutic agent. In an embodiment 1130, the delivery material includes at least one of a diamagnetic particle, fer device further comprises at least one reservoir for controlled romagnetic particle, paramagnetic particle, Super paramag release of at least one inducer or repressor of the at least one netic particle, particle with altered isotope, or other magnetic genetic element inducible to initiate death of the at least one particle. In an embodiment 990, the at least one detection modified microorganism. In an embodiment 1140, the deliv material is responsive to the presence of at least one of the at 50 ery device further comprises at least one memory location for least one composition, at least one constituent thereof, or at recording information. In an embodiment 1150, the at least least one product thereof; at least one cell or Substance asso one memory location is configured to record information ciated with the at least one biological tissue; the at least one regarding at least one sensor. In an embodiment 1160, the at metabolite, or at least one product thereof, or the at least one least one memory location is configured to record informa therapeutic agent, or a by-product thereof. 55 tion regarding at least one of a sensed condition, history, or As indicated in FIG. 10, in an embodiment 1000, the at performance of the device. In an embodiment 1170, the at least one detection material is responsive to the approximate least one memory location is configured to record informa quantity of at least one of the at least one composition, at least tion regarding one or more of the date, time, presence or one constituent thereof, or at least one product thereof; at least approximate quantity of at least one of the administered com one cell or Substance associated with the at least one biologi 60 position, constituent thereof, or product thereof the at least cal tissue; or the at least one metabolite, or at least one product one administered metabolite, or product thereof; or at least thereof. In an embodiment 1010, the at least one detection one cell or Substance associated with the at least one biologi material is responsive to the approximate number of micro cal tissue. organisms producing the at least one therapeutic agent. In an As indicated in FIG. 12, in an embodiment 1200, the at embodiment 1020, the at least one detection material is 65 least one cell or Substance associated with the at least one responsive to at least one of enzyme, acid, amino acid, pep biological tissue includes at least one of an organic or inor tide, polypeptide, protein, oligonucleotide, nucleic acid, ribo ganic Small molecule, nucleic acid, amino acid, peptide, US 8,852,916 B2 55 56 polypeptide, protein, glycopeptide, glycoprotein, glycolipid, device further comprises at least one reservoir for controlled lipopolysaccharide, peptidoglycan, proteoglycan, lipid, lipo release of at least one inducer or repressor of the at least one protein, sphingolipid, glycospingolipid, metalloprotein, genetic element inducible to initiate death of the at least one metal, liposome, chromosome, nucleus, acid, base, buffer, modified microorganism. In an embodiment 1350, the deliv protic solvent, aprotic solvent, carbohydrate, energy, arabi ery device further comprises a controller configured to nose, lactose, maltose. Sucrose, glucose, Xylose, Xylan, nisin, respond to the at least one sensor. In an embodiment 1360, the L-arabinose, allolactose, D-glucose, D-xylose, D-galactose, at least one sensor is configured to sense information related ampicillin, tetracycline, penicillin, pristinamycin, retinoic to at least one of the at least one biological tissue, the thera acid, interferon, galactose, rhamnose, fructose, melibiose, peutic agent, or the composition or a consitutuent thereof. In starch, inunlin, lipopolysaccharide, arsenic, cadmium, hydro 10 an embodiment 1370, the information related to the at least carbon, chromium, mercury, antibiotic, oxygen, carbon diox one biological tissue includes at least one oftemperature, pH, ide, carbon monoxide, nitrogen, nitric oxide, Vitamin, min inflammation, or other characteristic. eral, nitrous oxide, nitric oxide synthase, Sulfur, gas, As indicated in FIG. 14, in an embodiment, a delivery cytokine, chemokine, immunoglobulin, antibody, antigen, device 1400 comprises a housing 1410 including at least one extracellular matrix, cell ligand, Zwitterionic material, cat 15 reservoir containing at least one composition, the at least one ionic material, oligonucleotide, nanotube, piloxymer, trans composition including at least one modified microorganism fersome, gas, element, contaminant, radioactive particle, hor including at least one heterologous genetic element encoding mone, virus, enzyme, oligonucleotide, ribonucleic acid, at least one environmental medium treatment agent, and at oligosaccharide, polysaccharide, adhesion molecule, plate least one genetic element inducible to initiate death of the at let, blood plasma, whole blood, cell wall, salt, cell ligand, least one modified microorganism; the reservoir 1420 config lactate, urea, uric acid, glycogen, ketone, alcohol, alkaloid, ured to receive, retain, and dispense at least a portion of the at opioid, cannabinol, endorphin, epinephrine, dopamine, sero least one composition, and at least one component 1430 con tonin, nicotine, amphetamine, methamphetamine, anabolic figured to administer the at least one composition to at least steroid, hydrocodone, hemoglobin, heparin, clotting metabo one environmental medium. lite, tumor antigen, pH, albumin, ATP, NADH, FADH2, pyru 25 As indicated in FIG. 15, in an embodiment 1500, at least Vate, mercury, lead, creatinine, cholesterol, alpha-fetopro one composition further comprises at least one metabolite tein, chorionic gonadotropin, estrogen, progesterone, required by the at least one modified microorganism. In an testosterone, thyroxine, melatonin, calcitonin, antimullerian embodiment 1510, the device including at least one compo hormone, adiponectin, angiotensin, cholecystokinin, corti nent configured to administer the at least one composition to cotrophin-releasing hormone, erythropoietin, bilirubin, cre 30 at least one environmental medium includes one or more atine, follicle-stimulating hormone, gastrin, ghrelin, gluca ports. In an embodiment 1520, the at least one of the one or gon, gonadotropin-releasing hormone, inhibin, growth more ports includes at least one outlet port or at least one inlet hormone, growth hormone-releasing hormone, insulin, port. In an embodiment 1530, each reservoir includes at least human placental lactogen, oxytocin, orexin, luteinizing hor one separate port. In an embodiment 1535, the delivery device mone, leptin, prolactin, Somatostatin, thrombopoietin, corti 35 includes at least one reservoir including at least one inducer Sol, aldosterone, estradiol, estriol, estrone, leukotriene, brain formulated to induce at least one promoter operably coupled natriuretic peptide, neuropeptide Y, histamine, Vitamin, min to the at least one genetic element inducible to initiate death of eral, endothelin, renin, enkephalin, DHEA, DHT, alloisoleu the at least one auxotrophic microorganism. In an embodi cine, toxic Substance, illegal Substance, agent, hydrocarbon, ment 1538, the delivery device further comprises one or more arsenic, gold, silver, cadmium, strontium, mercury, lead, 40 controllable output mechanisms operably linked to the one or other heavy metals, chromium, antibiotic, gas, or any by more ports to control dispensing of at least a portion of the at products thereof, plant cell, animal cell, fungal cell, blood least one composition from the at least one reservoir. In an cell, muscle cell, nerve cell, fibroblast, adipose cell, stem cell, embodiment 1540, the at least one controllable output mecha pluripotent cell, epithelial cell, skin cell, neoplastic cell, nism includes at least one of a micropump, Valve, injector, or tumor cell, cell mass, or other biological tissue or organ cell. 45 actuator. In an embodiment 1550, the valve includes at least As indicated in FIG. 13, in an embodiment 1300, the deliv one of a one-way valve, or pressure settable valve. In an ery device further comprises at least one information trans embodiment 1560, the actuator includes at least one of a mission mechanism configured to transmit information piezoelectric actuator, electrostatic actuator, thermal actua recorded by the at least one electronic memory location. In an tor, shape-memory alloy actuator, bioactuator, or magnetic embodiment 1310, the device is located in or is substantially 50 actuator. In an embodiment 1570, the at least one controllable in the form of one or more of a spray apparatus, pump appa output mechanism includes at least one thermal or nonther ratus, bioreactor, or drilling apparatus. In an embodiment mal gate in communication with the at least one outlet of the 1320, the device is located in or is substantially in the form of at least one reservoir. In an embodiment 1580, the delivery one or more of a patch, oral inhaler, nasal spray or other device further comprises at least one control circuitry config orifice spray, orifice insert, chewing gum, iontophoretic appa 55 ured to control the at least one controllable output mecha ratus, oral consumable, ocular or otic dropper or spray, Stent, nism. shunt, consumer product, food product, food package, lip As indicated in FIG. 16, in an embodiment 1600, the at balm, lotion, ointment, Sunscreen or Sunblock, perfume, least one control circuitry is configured to control the at least aftershave, shampoo or other hair products, nail polish, den one controllable output mechanism for time-release of at least tures or other oral implants, contact lens or other ocular 60 a portion of the at least one composition from the at least one implants, orifice insert, orifice spray or inhaler, Sutures, Sur reservoir. In an embodiment 1610, the at least one control gical staples, dental floss, stents, shunts, bandages, absorb circuitry is configured for variable programming control of able mesh, or oral consumable. In an embodiment 1330, the the at least one controllable output mechanism. In an embodi delivery device further comprises at least one reservoir for ment 1620, the at least one control circuitry is configured to controlled release of at least one inducer or repressor of the at 65 control the release of at least a portion of the composition. In least one heterologous genetic element encoding at least one an embodiment 1625, the at least one control circuitry is therapeutic agent. In an embodiment 1340, the delivery configured to control the release of at least a portion of the US 8,852,916 B2 57 58 composition in response to a signal from a sensor. In an embodiment 1840, the at least one detection material includes embodiment 1628, the at least one control circuitry is config at least one of a diamagnetic particle, ferromagnetic particle, ured to control release of at least one inducer formulated to paramagnetic particle, Super paramagnetic particle, particle activate attleast one promoter operably coupled to the at least with altered isotope, or other magnetic particle. In an embodi one genetic element inducible to initiate death of the at least ment 1850, the at least one detection material is responsive to one modified microorganism. In an embodinient 1630, the the presence of at least one of the at least one composition, at delivery device further comprises at least one reservoir for least one constituent thereof, or at least one product thereof; at controlled release of at least one inducer or repressor of the at least one cell or Substance associated with the at least one least one heterologous genetic element encoding at least one environmental medium; at least one metabolite associated environmental medium treatment agent. In an embodiment 10 1650, the delivery device further comprises at least one trans with the environmental medium; or at least one organism ducer. In an embodiment 1660, the delivery device further associated with the at least one environmental medium. In an comprises at least one receiver. In an embodiment 1670, the at embodiment 1860, the at least one organism associated with least one receiver is configured to obtain release instructions the at least one environmental medium includes at least one of or authorization to release the at least one composition. In an 15 a plant, animal, fungus, or microorganism. In an embodiment embodiment 1680, the receiver is configured to receive pro 1870, the at least one detection material is responsive to the gramming instructions or data for the controller. In an approximate quantity of one or more of the at least one com embodiment 1685, the receiver is configured to receive infor position, at least one constituent thereof, or at least one prod mation from at least one distal or remote sensor. In an embodi uct thereof; at least one cell or substance associated with the ment 1690, the delivery device further comprises at least one at least one environmental medium; at least one metabolite transmitter. associated with the at least one environmental medium; or at As indicated in FIG. 17, in an embodiment 1700, the at least one organism associated with the at least one environ least one transmitter is configured to transmit information mental medium. regarding one or more of the date, time, presence or approxi As indicated in FIG. 19, in an embodiment 1900, the at mate quantity of one or more of the at least one composition, 25 least one organism associated with the at least one environ at least one constituent thereof, or at least one product thereof; mental medium includes at least one of a plant, animal, fun at least one cell or Substance associated with at least one gus, or microorganism. In an embodiment 1910, the at least environmental medium; at least one metabolite associated one detection material is responsive to the approximate num with the at least one environmental medium; or at least one ber of microorganisms producing the at least one treatment organism associated with the at least one environmental 30 agent, or an environmental medium Substance. In an embodi medium. In an embodiment 1710, the delivery device ment 1920, the environmental medium substance includes includes at least one reservoir including at least one inducer one or more of an enzyme, acid, amino acid, peptide, formulated to induce at least one promoter operably coupled polypeptide, protein, oligonucleotide, nucleic acid, ribo to the at least one genetic element inducible to initiate death of nucleic acid, oligosaccharide, polysaccharide, glycopeptide, the at least one modified microorganism. In an embodiment 35 1720, the delivery device further comprises at least one power glycolipid, lipoprotein, sphingolipid, glycosphingolipid, gly source. In an embodiment 1730, the delivery device further coprotein, peptidoglycan, lipid, carbohydrate, metallopro comprises at least one detection material. In an embodiment tein, proteoglycan, chromosome, adhesion molecule, cytok 1740, the delivery device further comprises at least one res ine, chemokine, immunoglobulin, antibody, antigen, ervoir for controlled release of the at least one detection 40 extracellular matrix, cell wall, hormone, organic compound, material. In an embodiment 1750, the at least one detection inorganic compound, salt, cell ligand, glucose, lactate, urea, material includes at least one of a taggant, contrast agent, uric acid, glycogen, oxygen, carbon dioxide, carbon monox sensor, or electronic idenfication device. In an embodiment ide, ketone, nitric oxide, nitrous oxide, alcohol, alkaloid, 1760, the at least one electronic identification device includes opioid, cannabinol, endorphin, epinephrine, dopamine, sero at least one radio frequency identification device. In an 45 tonin, nicotine, amphetamine, methamphetamine, pH, albu embodiment 1770, wherein the at least one sensor includes at min, ATP, NADH, FADH2, pyruvate, sulfur, mercury, lead, least one biosensor. In an embodiment 1780, the at least one creatinine, cholesterol, estrogen, progesterone, testosterone, biosensor includes at least one modified microorganism. In an calcitonin, ghrelin, glucagon, inhibin, growth hormone, embodiment 1790, the at least one sensor receives informa growth hormone-releasing hormone, insulin, Vitamin, min tion associated with at least one oftemperature, pH, presence 50 eral, DHEA, DHT, alloisoleucine, toxic substance, illegal of at least one inducer, amount of at least one inducer, pres Substance, hydrocarbon, arsenic, gold, silver, cadmium, ence of at least one repressor, amount of at least one repressor, strontium, mercury, lead, other heavy metals, chromium, or environmental response to administration of the at least antibiotic, gas, or any by-products thereof, a microorganism, one composition. plant cell, animal cell, fungal cell, plant, animal, fungus, or As indicated in FIG. 18, in an embodiment 1800, wherein 55 other organism. In an embodiment 1925, the at least one the at least one sensor is configured to sense information detecton material is responsive to the approximate number of related to at least one of the environmental medium, the microorganisms producing the at least one environmental environmental treatment medium agent, or the composition medium treatment agent. In an embodiment 1930, the deliv or a constituent thereof. In an embodiment 1810, the infor ery device further comprises at least one memory mechanism mation related to the at least one environmental medium 60 for storing instructions for generating and transmitting an includes at least one of temperature, pH, soil content, water electromagnetic control signal. In an embodiment 1940, the content, mineral content, organic or inorganic matter content, delivery device further comprises at least one imaging appa or other characteristic. In an embodiment 1820, the delivery ratus capable of imaging the approximate quantity within a device further comprises a controller configured to respond to treatment region of one or more of the at least one composi the at least one sensor. In an embodiment 1830, the at least 65 tion, at least one constituent thereof, or at least one product one detection material includes at least one of a radioactive, thereof; at least one organism associated with the at least one luminescent, colorimetric or odorous Substance. In an environmental medium; at least one metabolite associated US 8,852,916 B2 59 60 with the at least one environmental medium; or at least one or be part of a system, as described herein. Furthermore, in an cellor Substance associated with the at least one environmen embodiment, the bioreactor 140 includes gas injectors (e.g., tal medium. oxygen, hydrocarbon, etc.) to facilitate remediation of the at As indicated in FIG. 20, in an embodiment 2000, the deliv least one environmental medium, or to increase production of ery device further comprises at least one memory location for the at least one environmental medium treatment agent. For recording information. In an embodiment 2010, the at least example, flow through or batch type reactors may be used. one memory location is configured to record information Flow rates into the system can be adjusted depending on the regarding at least one sensor. In an embodiment 2020, the at levels of contaminants or other compounds in the flow stream. least one memory location is configured to record informa In an embodiment, cycles are alternated between microbial tion regarding at least one of a sensed condition, history, or 10 growth with contaminant degradation and production of theat performance of the device. In an embodiment 2030, the at least one environmental medium treatment agent. For least one memory location is configured to record informa example, in an embodiment, the modified microorganisms tion regarding one or more of the date, time, presence or are provided with at least one metabolite (e.g., air, oxygen, approximate quantity of dispensing of one or more of the at hydrocarbon, carbohydrate, etc.), which encourages produc least one composition, at least one constituent thereof, or at 15 tion of the at least one environmental medium treatmentagent least one product thereof; at least one cell or Substance asso and, in Some instances, growth of the microorganisms. Sub ciated with the at least one environmental medium; at least sequently, the environmental medium 110 to be treated is one organism associated with the at least one environmental passed through the reactor for administration of the at least medium; or at least one metabolite associated with the at least one environmental medium treatment agent and, in some one environmental medium. In an embodiment 2040, the instances, bioremediation of the at least one environmental delivery device further comprises at least one information medium. transmission mechanism configured to transmit information As indicated in FIG. 2, in an embodiment, the environmen recorded by the at least one electronic memory location. In an tal medium 110 is brought to the bioreactor 240 for treatment, embodiment 2050, the device is located in or is substantially or treated environmental medium 110 is removed from the in the form of one or more of a patch, tarp, insert, mesh, 25 bioreactor, for example by a vehicle 250. In an embodiment, netting, or at least one disposable or biodegradable product. the environmental medium 110 is treated in a system that In an embodiment 2060, the device is located in or is substan provides the environmental medium to be treated, or collects tially in the form of one or more of a food package, disposable the already treated environmental medium 260. package, bioreactor, spray apparatus, pump apparatus, or As indicated in FIG. 44, and as disclosed in other figures drilling apparatus. In an embodiment 2070, the delivery 30 and text herein, in an embodiment, a delivery device 4400, device further comprises at least one reservoir for controlled includes a housing 4402 including at least one reservoir 4405 release of at least one inducer or repressor of the at least one containing at least one composition disclosed herein, and at heterologous genetic element encoding at least one environ least one component 4415 configured to administer the at mental medium treatment agent. In an embodiment 2080, the least one composition to at least one Substrate (e.g., biological delivery device further comprises at least one reservoir for 35 tissue, environmental medium, etc.). In an embodiment, the controlled release of at least one inducer or repressor of the at delivery device includes one or more ports. For example, the least one genetic element inducible to initiate death of the at one or more ports includebutare not limited to at least an inlet least one modified microorganism. port 4.425, an outlet port 4420, or both. In an embodiment, at With regard to the figures, FIG. 1 illustrates at least one least one controllable output mechanism 4435 is operably treatment area 100 that includes at least one environmental 40 linked to the one or more ports to control dispensing of at least medium 110, for administration of at least one environmental a portion of the at least one composition. In an embodiment, medium treatment agent. In an embodiment, the treatment the delivery device further comprises at least one control area corresponds to remediation of at least one compound, circuitry 4440 configured to generate and transmit an electro Such as an element or contaminant, from the environmental magnetic control signal configured to control the at least one medium. In an embodiment, the at least one composition 45 controllable output mechanism 4435. In an embodiment, the including at least one modified microorganism is capable of delivery device further comprises at least one reservoir 4475 converting at least one contaminant or other compound in the for controlled release of at least one inducer or repressor of environmental medium into elements, or other extractable the at least one heterologous genetic element encoding at components (e.g., converting hydrocarbon or converting least one environmental medium treatment agent. In an nitrogen-containing compounds). In an embodiment, the at 50 embodiment, the delivery device further comprises at least least one environmental medium treatment agent binds to at one reservoir 4450 for controlled release of at least one least one compound in the environmental medium (e.g., bind inducer or repressor of the at least one genetic element induc ing elements, polymers, etc.). In an embodiment, the treat ible to initiate death of the at least one modified microorgan ment area corresponds to an area deficient in nutrients (e.g., ism. In an embodiment, the delivery device further comprises lawn, garden, golf course, other agricultural area). For 55 a transducer 4470. In an embodiment, the delivery device example, as shown, a treatment area 100 may include at least further comprises a transmitter 4460. In an embodiment, the one environmental medium 110 in proximity to an industrial delivery device further comprises a receiver 4480. In an area 120 or other area of high concentration of pollutants embodiment, the delivery device further comprises a power (e.g., gas station 130). In an embodiment, the environmental source 4410. medium treatment agent provides at least one plant growth 60 In an embodiment, the delivery device further comprises at factor or other agent to encourage revitalization of the area least one detection material 4490. In an embodiment, the 170. In at least one embodiment, the at least one environmen detection material includes at least one of a taggant, contrast tal medium 110, is treated directly (e.g., drilling, spraying, agent, sensor 4470, or electronic identification device. In an sparging, patching or tarping) with the at least one environ embodiment, the delivery device optionally includes separate mental medium treatment agent. 65 reservoirs 4430 for separate constituents of the composition In an embodiment, the at least one delivery device is in the (e.g., metabolite, or other constituent). In an embodiment, the form of a bioreactor 140. The bioreactor 140 can stand alone delivery device further comprises a controller 4485 config US 8,852,916 B2 61 62 ured to respond to the at least one sensor 4470. In an embodi erologuous genetic elements is located on a separate vector ment, the delivery device further comprises an imaging appa from another element. In an embodiment, at least one of the ratus 4465. In an embodiment, the delivery device further multiple htereologous genetic elements is under the control of comprises a memory mechanism 4445 for storing instruc a promoter that is different from another element. tions for generating and transmitting an electromagnetic con In an embodiment, the at least one modified microorgan trol signal. In an embodiment, the delivery device further ism includes at least one genetic element for one or more comprises at least one memory location 4455 for recording monooxygenase enzymes. For example, monooxygenase information. In an embodiment, the delivery device further enzymes are utilized in biological cells for degradation of comprises an information transmission mechanism 4.478 food or pharmaceutical drugs. In soil microbes, monooxyge configured to transmit information recorded by the at least 10 nase enzymes are utilized for degradation of contaminants. At one electronic memory location. least one gene has been isolated that encodes a methane Computer-Implemented Methods and Systems Thereof monooxygenase enzyme (MMO) associated with the first In an embodiment, the method includes isolating at least step in methane oxidation by at least some methanotropic one microorganism from the at least one Substrate, modifying bacteria. See, for example, U.S. Pat. No. 5,316,940, which is the at least one microorganism, and placing the modified 15 incorporated herein by reference. For example, soluble meth microorganism back into the at least one original Substrate, or ane monooxygenase (SMMO) constitutive mutant strains of into at least one other Substrate. In an embodiment, modifying M trichosporium (ATCC 55314) are capable of degrading the at least one microorganism includes obtaining at least chlorinated hydrocarbons in the presence of copper, which is Some genetic sequence from the at least one microorganism, normally suppressed in the presence of copper. Id. In an and altering at least a portion of the genetic sequence of the at embodiment described herein, Such mutants, or other modi least one microorganism. In an embodiment, modifying theat fied microorganisms, are further modified to produce at least least one microorganism includes chemically altering the at one agent (e.g., carbon, calcium, plant growth factors, etc.). In least one microorganism. In an embodiment, the at least one another embodiment, the cytochromes OmcA or Omc3 are modified microorganism is amplified prior to placing into at included in the at least one modified microorganism. See, for least one Substrate. In an embodiment, the amplification of 25 example, Myers and Myers, App. Env. Microbiol. Vol. 68, no. the at least one modified microorganism includes amplifying 11, pp. 5585-5594 (2002), which is incorporated by refer the modified microorganism in vitro. ence. In an embodiment, the monooxygenase enzymes are Isolating and sequencing microorganisms from Substrates involved in the biotransformation of at least one agent, (e.g., biological tissues, environmental media, etc.) can be including a therapeutic agent or environmental contaminant. done according to standard techniques. For example, Samples 30 See, for example, Hoensch, et al., Gut, vol. 20, pp. 666-672 of the Substrate are isolated and used to inoculate appropriate (1979), which is incorporated herein by reference. cultures, or microorganisms can be sequenced directly from Furthermore, strains of Candida tropicalis are capable of the substrate sample. See, for example, U.S. Pat. No. 5,888, growing on fatty acids or alkanes as the sole source of carbon 396, which is incorporated herein by reference. For example, and energy due to their cytochrome P450 monooxygenase rapid detection and quantitative assessment of specific micro 35 enzymes. See, for example, Eschenfeldt, et al., App Env bial species in environmental samples can be conducted uti Microbiol. vol. 69, no. 10, pp. 5992-5999 (2003), which is lizing species-specific PCR primer sets. See, for example, incorporated herein by reference. Thus, either prokaryotic Wilson, et al., Abstract, J. Micro. Meth., vol.39, no. 1, pp. and eukaryotic microorganisms can be modified to utilize 59-78 (1999). For example, PCR primers can be developed hydrocarbons as an energy source. for the 16S RNA gene, which is a highly variable region. 40 In an embodiment, at least one computer-implemented These primers can be paired with a universal set of PCR algorithm is utilized to predictat least one modified microor primers chosen from highly conserved neighboring ganism population Suited for the particular Substrate, or sequences in the same gene. Id. Amplification products can be agent. In an embodiment, at least one computer-implemented verified and validated by utilizing, for example, hemi-nested algorithm is utilized to predetermine multiple populations of PCR and with ligase chain reaction (LCR) techniques. Id. 45 microorganisms for modification for a particular Substrate or As indicated by various testing measures, microorganism agent. production of various proteins or other agents is measurable. The disclosure further provides kits including at least one For example, incorporation of radiolabeled leucine or thymi composition or method disclosed herein. Any particular kit dine into protein indicates that the mean turnover time for may also contain instructional material teaching the method bacteria at approximately 22°C. is approximately 4.3 days. 50 ologies and uses of the composition or method, as described See, for example, Baath, Abstract, Biol. Fertil. Soils, Vol. 17. herein. pp. 147-153 (1994); Buesing and Gessner, Abstract, Microb. The methods and therapeutic compositions are further Ecol. Vol. 45, pp. 29.1-301 (2003), each of which is incorpo described with reference to the following examples; however rated herein by reference. In an embodiment, microbial it is to be understood that the methods and compositions are growth rates can be estimated by measuring protein synthesis. 55 not limited to such examples. Id. In an embodiment, production of the at least one agent by As indicated in FIG. 21, in an embodiment, a system 2100 the at least one modified microorganism can be measured by comprises at least one computer device 2105, at least one radiolabeling a component (e.g., amino acid), as described. delivery device 2110 configured to retain and dispense at least Id. a portion of at least one composition to at least one biological In an embodiment, the modified microorganism includes at 60 tissue; 2115 and a recordable medium including one or more least one heterologous genetic element, which may includean instructions that when executed on the computing device element isolated from another species or another organism cause the computing device to regulate dispensing of at least (e.g., plant oranimal). In an embodiment, the modified micro a portion of the at least one composition, 2120 wherein the at organism includes multiple heterologous genetic elements. In least one composition includes at least one first constituent an embodiment, at least one of the multiple heterologous 65 including at least one auxotrophic microorganism including genetic elements is located on a separate locus from another at least one pH inducible promoter operably coupled to at element. In an embodiment, at least one of the multiple het least one heterologous genetic element encoding at least one US 8,852,916 B2 63 64 therapeutic agent, and including at least one genetic element tissue indicators prior to, during, or Subsequent to adminis inducible to initiate death of the at least one auxotrophic tering the at least one composition to the at least one biologi microorganism; and at least one second constituent including cal tissue. In an embodiment 2440, the information related to at least one metabolite required by the at least one aux one or more biological tissue indicators includes information otrophic microorganism. from at least one of an assay, image, or gross assessment of As indicated in FIG. 22, in an embodiment 2200, the at the at least one biological tissue prior to, during, or Subse least one computing device includes at least one computing quent to administering the at least one composition. In an device located on or in the at least one delivery device. In an embodiment 2450, the assay includes at least one technique embodiment 2210, the at least one computing device includes including spectroscopy, microscopy, electrochemical detec at least one computing device located remotely from the at 10 tion, polynucleotide detection, histological examination, least one delivery device. In an embodiment 2220, the at least biopsy analysis, fluorescence resonance energy transfer, elec one computing device includes one or more of a desktop tron transfer, enzyme assay, electrical conductivity, isoelec computer, workstation computer, or computing system. In an tric focusing, chromatography, immunoprecipitation, immu embodiment 2230, the at least one computing system noseparation, aptamer binding, filtration, electrophoresis, includes one or more of a cluster of processors, a networked 15 computer, a tablet personal computer, a laptop computer, a immunoassay, or radioactive assay. In an embodiment 2460, mobile device, a mobile telephone, or a personal digital assis the at least one image includes one or more images acquired tant computer. In an embodiment 2240, the system further by at least one of laser, holography, X-ray crystallography, comprises one or more instructions that when executed on the optical coherence tomography, computer-assisted tomogra at least one computing device cause the at least one comput phy scan, computed tomography, magnetic resonance imag ing device to generate at least one output to a user. In an ing, positron-emission tomography scan, ultrasound, X-ray, embodiment 2250, wherein the at least one output includes at electrical-impedance monitoring, microscopy, spectrometry, least one graphical illustration of one or more of the at least flow cytommetry, radioisotope imaging, thermal imaging, one composition, at least one constituent thereof, or at least infrared visualization, multiphoton calcium-imaging, pho one product thereof; at least one cell or substance associated 25 tography, or in silico generation. with the at least one biological tissue; the at least one metabo As indicated in FIG. 25, in an embodiment 2500, one or lite, or at least one product thereof; at least one property of the more instructions for receiving information related to one or delivery device; or at least one property of dispensing the at more biological tissue indicators relate to one or more of least one delivery device. In an embodiment 2260, wherein administering the at least one composition, at least one con the at least one output includes at least one protocol for 30 generating the at least one auxotrophic microorganism. In an stituent thereof, or at least one product thereof administering embodiment 2270, wherein the at least one output includes at the at least one metabolite, cell or tissue formation, cell or least one protocol for making the at least one composition. In tissue growth, cell or tissue apoptosis, cell or tissue necrosis, an embodiment 2280, wherein the at least one output includes cell division, cytoskeletal rearrangement, cell or tissue secre at least one protocol for administering the at least one com 35 tion, cell or tissue differentiation, status of the at least one position to the at least one biological tissue. microorganism of the at least one composition, status of the at As indicated in FIG. 23, in an embodiment 2300, the user least one composition, status of the at least one therapeutic includes at least one entity. In an embodiment 2310, the entity agent, status of the at least one metabolite, or depletion of the includes at least one person, or computer. In an embodiment at least one metabolite. In an embodiment 2510, the at least 2320, the output includes output to a user readable display. In 40 one biological tissue is located in at least one of in situ, in an embodiment 2340, the user readable display includes a vitro, in Vivo, in utero, in planta, in silico, or ex vivo. In an human readable display. In an embodiment 2350, the user embodiment 2520, the at least one biological tissue is at least readable display includes one or more active displays. In an partially located in at least one subject. In an embodiment embodiment 2360, the user readable display includes one or 2530, the at least one subject includes at least one of an more passive displays. In an embodiment 2370, the user read 45 invertebrate or vertebrate animal. In an embodiment 2540, the able display includes one or more of a numeric format, at least one subject includes at least one of a reptile, mammal, graphical format, or audio format. In an embodiment 2380, amphibian, bird, or fish. In an embodiment 2550, the at least the system further comprises one or more instructions for one subject includes at least one human. In an embodiment making the at least one composition. In an embodiment 2390. 2560, the at least one subject includes at least one plant. In an the system further comprises one or more instructions for 50 embodiment 2570, one or more instructions for obtaining inducing the at least one pH inducible promoter operably genetic sequence information from at least one microorgan coupled to at least one heterologous genetic element encod ism isolated from the at least one biological tissue. ing at least one therapeutic agent. In an embodiment 2395, the As indicated in FIG. 26, in an embodiment 2600, the sys system further comprises one or more instructions for induc tem further comprises comprising one or more instructions ing the at least one genetic element inducible to initiate death 55 for modifying the at least one microorganism isolated from of the at least one auxotrophic microorganism. In an embodi the at least one biological tissue. In an embodiment 2610, the ment 2398, the composition further comprises at least one system further comprises one or more instructions for ampli repressor operably coupled to the at least one heterologous fying the at least one microorganism isolated from the at least genetic element encoding at least one therapeutic agent. one biological tissue. In an embodiment 2620, the system As indicated in FIG. 24, in an embodiment 2400, the sys 60 further comprises one or more instructions for reinstating the tem further comprises one or more instructions for selecting at least one microorganism isolated from the at least one the composition, or a constituent thereof. In an embodiment biological tissue Subsequent to modification. In an embodi 2420, the system further comprises one or more instructions ment 2630, the system further comprises one or more instruc for administering the at least one composition or a constituent tions for predeterming at least one microorganism type for thereof to at least one biological tissue. In an embodiment 65 modifying to produce at least one therapeutic agent based on 2430, the system further comprises one or more instructions at least one feature of the at least one biological tissue. In an for receiving information related to one or more biological embodiment 2640, the at least one feature of the at least one US 8,852,916 B2 65 66 biological tissue includes at least one property of one or more tissue in response to the one or more biological tissue indica microorganism populations associated with the at least one tors. In an embodiment 2940, the receiving information biological tissue. related to one or more biological tissue indicators includes As indicated in FIG. 27, a computer-implemented method information from at least one of an assay, image, or gross 2700, comprises 2710 one or more instructions for regulating 5 assessment of the at least one biological tissue prior to, dur dispensing at least one composition from at least one delivery ing, or Subsequent to administering the at least one composi device to at least one biological tissue, the at least one com tion. In an embodiment 2950, the assay includes at least one position including at least one auxotrophic microorganism technique including spectroscopy, microscopy, electrochemi including at least one pH inducible heterologous genetic ele cal detection, polynucleotide detection, histological exami ment encoding at least one therapeutic agent formulated for at 10 nation, biopsy analysis, fluorescence resonance energy trans least one biological tissue, and including at least one genetic fer, electron transfer, enzyme assay, electrical conductivity, element inducible to initiate death of the at least one aux isoelectric focusing, chromatography, immunoprecipitation, otrophic microorganism; and at least one metabolite required immunoseparation, aptamer binding, filtration, electrophore by the at least one auxotrophic microorganism. In an embodi sis, immunoassay, or radioactive assay. In an embodiment ment 2720, the computer-implemented method further com- 15 2960, the at least one image includes one or more images prises generating at least one output to a user. In an embodi acquired by at least one of laser, holography, X-ray crystal ment 2730, the at least one output includes at least one lography, optical coherence tomography, computer-assisted graphical illustration of one or more of the at least one com tomography Scan, computed tomography, magnetic reso position, at least one constituent thereof, or at least one prod nance imaging, positron-emission tomography Scan, ultra uct thereof; the at least one metabolite, or at least one product 20 Sound, X-ray, electrical-impedance monitoring, microscopy, thereof, at least one cell or substance associated with the at spectrometry, flow cytommetry, radioisotope imaging, ther least one biological tissue; at least one property of the at least mal imaging, infrared visualization, multiphoton calcium one delivery device; or at least one property of dispensing the imaging, photography, or in silico generation. at least one delivery device. In an embodiment 2740, the at As indicated in FIG.30, in an embodiment 3000, the one or least one output includes at least one protocol for generating 25 more biological tissue indicators relate to one or more of the at least one auxotrophic microorganism. In an embodi administration of the at least one therapeutic agent, or a ment 2750, the at least one output includes at least one pro constituent thereof, or product thereof administration of the tocol for making the at least one composition. In an embodi at least one composition, or constituent thereof, or product ment 2760, the at least one output includes at least one thereof, administration of the at least one metabolite, admin protocol for administering the at least one composition to the 30 istration of the at least one auxotrophic microorganism, cell at least one biological tissue. or tissue formation, cell or tissue growth, cell or tissue apo As indicated in FIG. 28, in an embodiment 2800, the user ptosis, cell or tissue necrosis, cell division, cytoskeletal rear includes at least one entity. In an embodiment 2810, the entity rangement, cell or tissue secretion, cell or tissue differentia includes at least one person, or computer. In an embodiment tion, status of the at least one microorganism of the at least 2820, the at least one output includes at least one output to a 35 one composition, status of the at least one composition, status user readable display. In an embodiment 2830, the user read of the at least one therapeutic agent, status of the at least one able display includes a human readable display. In an embodi metabolite, or depletion of the at least one metabolite. In an ment 2840, the user readable display includes one or more embodiment 3010, the at least one biological tissue is located active displays. In an embodiment 2850, the user readable in at least one of in situ, in vitro, in vivo, in utero, in planta, in display includes one or more passive displays. In an embodi- 40 silico, or ex vivo. In an embodiment 3020, the at least one ment 2860, the user readable display includes one or more of biological tissue is at least partially located in at least one a numeric format, graphical format, or audio format. In an subject. In an embodiment 3030, the at least one subject embodiment 2870, the computer-implemented method fur includes at least one of an invertebrate or vertebrate animal. In ther comprises one or more instructions for making the at an embodiment 3040, the at least one subject includes at least least one composition. In an embodiment 2880, the com- 45 one of a reptile, mammal, amphibian, bird, or fish. In an puter-implemented method further comprises one or more embodiment 3050, the at least one subject includes at least instructions to dispense the at least one composition or a one human. In an embodiment 3060, the at least one subject constituent thereof to at least one biological tissue. includes at least one plant. In an embodiment 3070, the com As indicated in FIG. 29, in an embodiment 2900, the com puter-implemented method further comprises obtaining puter-implemented method further comprises one or more 50 genetic sequence information from at least one microorgan instructions for dispensing at least one inducer formulated to ism isolated from the at least one biological tissue. induce the at least one genetic element inducible to initiate As indicated in FIG. 31, in an embodiment 3100, the com death of the at least one auxotrophic microorganism. In an puter-implemented method further comprises one or more embodiment 2910, the computer-implemented method fur instructions for modifying the at least one microorganism ther comprises receiving information related to one or more 55 isolated from the at least one biological tissue. In an embodi biological tissue indicators prior to, during, or Subsequent to ment 3110, the computer-implemented method further com administering the at least one composition or a constituent prises one or more instructions for amplifying the at least one thereof, to the at least one biological tissue. In an embodiment microorganism isolated from the at least one biological tis 2920, the computer-implemented method further comprises sue. In an embodiment 3120, the computer-implemented one or more instructions for dispensing the at least one com- 60 method further comprises one or more instructions for rein position or a constituent thereof, to the at least one biological stating the at least one microorganism isolated from the at tissue in response to the one or more biological tissue indica least one biological tissue Subsequent to modification. In an tors. In an embodiment 2930, the computer-implemented embodiment 3130, the computer-implemented method fur method further comprises one or more instructions for dis ther comprises one or more instructions for predeterming at pensing at least one inducer formulated to induce the at least 65 least one microorganism type for modifying to produce at one genetic element inducible to initiate death of the at least least one therapeutic agent based on at least one feature of the one auxotrophic microorganism, to the at least one biological at least one biological tissue. In an embodiment 3140, the at US 8,852,916 B2 67 68 least one feature of the at least one biological tissue includes one composition, at least one constituent thereof, or at least at least one property of one or more microorganism popula one product thereof; the at least one metabolite, or at least one tions associated with the at least one biological tissue. product thereof; at least one cell or Substance associated with As indicated in FIG. 32, in an embodiment, a computer the at least one biological tissue; at least one property of the at program product 3200, comprises 3210 a recordable medium 5 least one delivery device; or at least one property of dispens bearing one or more instructions for regulating dispensing of ing the at least one delivery device. In an embodiment 3390, at least one delivery device, wherein the delivery device the computer program product further comprises one or more includes at least one composition, wherein the at least one instructions for displaying results of the processing. composition includes at least one auxotrophic microorganism As indicated in FIG.34, in an embodiment, a system 3400, including at least one pH inducible heterologous genetic ele 10 comprises 3405 at least one computing device: 3410 at least ment encoding at least one therapeutic agent formulated for at one delivery device configured to retain and dispense at least least one biological tissue, and including at least one genetic one composition to at least one environmental medium; and element inducible to initiate death of the at least one aux 3415 a recordable medium including one or more instructions otrophic microorganism; at least one metabolite required by that when executed on the computing device cause the com the at least one auxotrophic microorganism; and generating at 15 puting device to regulate dispensing of at least a portion of the least one output. In an embodiment 3220, the recordable at least one composition; 3420 wherein the at least one com medium includes a computer-readable medium. In an position includes at least one modified microorganism embodiment 3230, the recordable medium includes a com including at least one heterologous genetic element encoding munications medium. In an embodiment 3240, the computer at least one environmental medium treatment agent; and at program product further comprises one or more instructions least one genetic element inducible to initiate death of the at for receiving information related to one or more biological least one modified microorganism. tissue indicators prior to, during, or Subsequent to adminis As indicated in FIG. 35, in an embodiment 3500, the at tering the at least one composition. In an embodiment 3250, least one computing device includes at least one computing the one or more biological tissue indicators relate to one or device located on or in the at least one delivery device. In an more of administration of the at least one therapeutic agent, 25 embodiment 3510, the at least one computing device includes or a consitutent thereof administration of the at least one at least one computing device located remotely from the at composition, or constituent thereof, or product thereof. least one delivery device. In an embodiment 3520, the at least administration of the at least one metabolite, administration one computing device includes one or more of a desktop of the at least one auxotrophic microorganism, cell or tissue computer, workstation computer, or computing system. In an formation, cell or tissue growth, cell or tissue apoptosis, cell 30 embodiment 3530, the at least one computing system or tissue necrosis, cell division, cytoskeletal rearrangement, includes one or more of a cluster of processors, a networked cell or tissue secretion, cell or tissue differentiation, status of computer, a tablet personal computer, a laptop computer, a the at least one microorganism of the at least one composition, mobile device, a mobile telephone, or a personal digital assis status of the at least one composition, status of the at least one tant computer. In an embodiment 3540, the system further therapeutic agent, status of the at least one metabolite, or 35 comprises one or more instructions that when executed on the depletion of the at least one metabolite. In an embodiment at least one computing device cause the at least one comput 3260, the computer program product further comprises one or ing device to generate at least one output to a user. In an more instructions for obtaining genetic sequence information embodiment 3550, the at least one output includes at least one from at least one microorganism isolated from the at least one graphical illustration of one or more of the at least one com biological tissue. 40 position, at least one constituent thereof, or at least one prod As indicated in FIG.33, in an embodiment 3300, the com uct thereof; at least one cell or substance associated with the puter program product further comprises one or more instruc at least one environmental medium; or the at least one prop tions for modifying the at least one microorganism isolated erty of dispensing the at least one delivery device. In an from the at least one biological tissue. In an embodiment embodiment 3560, the at least one output includes at least one 3310, the computer program product further comprises one or 45 protocol for generating the at least one modified microorgan more instructions for amplifying the at least one microorgan ism. In an embodiment 3570, the at least one output includes ism isolated from the at least one biological tissue. In an at least one protocol for making the at least one composition. embodiment 3320, the computer program product further In an embodiment 3580, the at least one output includes at comprises one or more instructions for reinstating the at least least one protocol for administering the at least one compo one microorganism isolated from the at least one biological 50 sition to the at least one environmental medium. tissue subsequent to modification. In an embodiment 3330, As indicated in FIG. 36, in an embodiment 3600, the user the computer program product further comprises one or more includes at least one entity. In an embodiment 3610, the entity instructions for predeterming at least one microorganism type includes at least one person, or computer. In an embodiment for modifying to produce at least one therapeutic agent based 3620, the output includes an output to a user readable display. on at least one feature of the at least one biological tissue. In 55 In an embodiment 3640, the user readable display includes a an embodiment 3340, the at least one feature of the at least human readable display. In an embodiment 3650, the user one biological tissue includes at least one property of one or readable display includes one or more active displays. In am more microorganism populations associated with the at least embodiment 3660, the user readable display includes one or one biological tissue. more passive displays. In an embodiment 3670, the user read In an embodiment 3350, the output includes at least one 60 able display includes one or more of a numeric format, protocol for making the at least one composition. In an graphical format, or audio format. In an embodiment 3680, embodiment 3360, the output includes at least one protocol the at least one environmental medium includes at least one of for generating the at least one auxotrophic microorganism. In water, soil, food product, or air or other gas. In an embodi an embodiment 3370, the output includes at least one protocol ment 3690, the at least one environmental medium includes at for administering the at least one composition to at least one 65 least one of ground water, Surface water, effluent, or waste biological tissue. In an embodiment 3380, the output includes water. In an embodiment 3695, the water includes at least one at least one graphical illustration of one or more of the at least of a lake, river, stream, sewage, sludge, slurry, sediment, US 8,852,916 B2 69 70 ocean, fountain, or other water. In an embodiment 3696, theat ism. In an embodiment 3850, the at least one output includes least one environmental medium includes at least one of at least one protocol for making the at least one composition. metal, concrete, cement, textiles, fabric, wood, mineral ore, In an embodiment 3860, the at least one output includes at or rock. In an embodiment 3697, the at least one environmen least one protocol for administering the at least one compo tal medium treatment agent includes at least one plant hor sition to the at least one environmental medium. mone. In an embodiment 3698, the at least one planthormone As indicated in FIG. 39, in an embodiment 3900, the user includes at least one of an auxin, abscisic acid, cytokinin, includes at least one entity. In an embodiment3910, the entity ethylene, gibberellin, brassinolide, salicyclic acid, jas includes at least one person, or computer. In an embodiment monate, polyamine, plant peptide hormone, nitric oxide, 3920, the output includes an output to a user readable display. Strigolactone, or other compound. 10 In an embodiment 3930, the user readable display includes a As indicated in FIG. 37, in an embodiment 3700, the sys human readable display. In an embodiment 3940, the user tem further comprises one or more instructions for obtaining readable display includes one or more active displays. In an genetic sequence information from at least one microorgan embodiment 3950, the user readable display includes one or ism isolated from the at least one environmental medium. In more passive displays. In an embodiment 3960, the user read an embodiment 3710, the system further comprises one or 15 able display includes one or more of a numeric format, more instructions for modifying the at least one microorgan graphical format, or audio format. In an embodiment 3970, ism isolated from the at least one environmental medium. In the computer-implemented method further comprises one or an embodiment 3720, the system further comprises one or more instructions for making the at least one composition. In more instructions for amplifying the at least one microorgan an emboeiment 3980, the computer-implemented method ism isolated from the at least one environmental medium. In further comprises one or more instructions to dispense at least an embodiment 3730, the system further comprises one or a portion of the at least one composition or a constituent more instructions for reinstating the at least one microorgan thereof, to at least one environmental medium. ism isolated from the at least one environmental medium As indicated in FIG. 40, in an embodiment 4000, the com subsequent to modification. In an embodiment 3740, the sys puter-implemented method further comprises one or more tem further comprises one or more instructions for prede 25 instructions for dispensing at least a portion of at least one terming at least one microorganism type for modifying to inducer formulated to induce the at least one genetic element produce at least one environmental medium treatment agent inducible to initiate death of the at least one modified micro based on at least one feature of the at least one environmental organism. In an embodiment 4010, the computer-imple medium. In an embodiment 3750, the at least one feature of mented method further comprises receiving information the at least one environmental medium includes at least one 30 related to one or more environmental medium indicators prior property of one or more microorganism populations associ to, during, or Subsequent to administering the at least one ated with the at least one environmental medium. In an composition or a constituent thereof, to the at least one envi embodiment 3760, the system further comprises one or more ronmental medium. In an embodiment 4020, the computer instructions for making the at least one composition. In an implemented method further comprises one or more instruc embodiment 3770, the system further comprises one or more 35 tions for dispensing at least a portion of the at least one instructions for inducing at least one inducible promoter composition or constituent thereof, to the at least one envi operably coupled to at least one heterologous genetic element ronmental medium in response to the one or more environ encoding at least one environmental medium treatmentagent. mental medium indicators. In an embodiment 4030, the com In an embodiment 3780, the system further comprises one or puter-implemented method further comprises one or more more instructions for inducing the at least one genetic ele 40 instructions for dispensing at least a portion of at least one ment inducible to initiate death of the at least one modified inducer formulated to induce the at least one genetic element microorganism. In an embodiment 3790, the system further inducible to initiate death of the at least one modified micro comprises one or more instructions for selecting the compo organism, to the at least one environmental medium in sition, or a constituent thereof. In an embodiment 3795, the response to the one or more biological tissue indicators. system further comprises one or more instructions for admin 45 In an embodiment 4040, the computer-implemented istering the at least one composition or a constituent thereof to method the receiving information related to one or more at least one environmental medium. environmental medium indicators includes information from As indicated in FIG. 38, in an embodiment 3800, a com at least one of an assay, image, or gross assessment of the at puter-implemented method comprises 3810 one or more least one environmental medium prior to, during, or Subse instructions for regulating dispensing of at least one compo 50 quent to administering the at least one composition. sition from at least one delivery device to at least one envi In an embodiment 4050, the assay includes at least one ronmental medium, the at least one composition including at technique including spectroscopy, microscopy, electrochemi least one modified microorganism including at least one het cal detection, polynucleotide detection, histological exami erologous genetic element encoding at least one environmen nation, biopsy analysis, fluorescence resonance energy trans tal medium treatment agent; and at least one genetic element 55 fer, electron transfer, enzyme assay, electrical conductivity, inducible to initiate death of the at least one modified micro isoelectric focusing, chromatography, immunoprecipitation, organism. In an embodiment 3820, the computer-imple immunoseparation, aptamer binding, filtration, electrophore mented method further comprises generating at least one sis, immunoassay, or radioactive assay. output to a user. In an embodiment 3830, the at least one In an embodiment 4060, the at least one image includes one output includes at least one graphical illustration of one or 60 or more images acquired by at least one of laser, holography, more of the at least one composition, at least one constituent X-ray crystallography, optical coherence tomography, com thereof, or at least one product thereof; at least one metabolite puter-assisted tomography scan, computed tomography, utilizable by the at least one modified microorganism; at least magnetic resonance imaging, positron-emission tomography one property of the at least one delivery device; or at least one Scan, ultrasound, X-ray, electrical-impedance monitoring, property of dispensing the at least one delivery device. In an 65 microscopy, spectrometry, flow cytommetry, radioisotope embodiment 3840, the at least one output includes at least one imaging, thermal imaging, infrared visualization, multipho protocol for generating the at least one modified microorgan ton calcium-imaging, photography, or in silico generation. US 8,852,916 B2 71 72 As indicated in FIG. 41, in an embodiment 4100, the one or microorganism of the at least one composition; status of the at more environmental medium indicators relate to one or more least one composition; status of the at least one environmental of administration of the at least one composition, at least one medium treatment agent; or status of at least one metabolite constituent thereof, or at least one product thereof adminis utilizable by the modified microorganism; or depletion of at tration of at least one metabolite utilizable by the at least one least one metabolite utilizable by the modified microorgan modified microorganism; administration of the at least one ism. modified microorganism; status of the at least one modified As indicated in FIG. 43, in an embodiment 4300, the output microorganism of the at least one composition; status of theat includes at least one protocol for making the at least one least one composition; status of the at least one environmental composition. In an embodiment 4310, the output includes at medium treatment agent; or status of at least one metabolite 10 least one protocol for generating the at least one modified utilizable by the at least one modified microorganism; or microorganism. In an embodiment 4320, the output includes depletion of at least one metabolite utilizable by the at least at least one protocol for administering the at least one com one modified microorganism. In an embodiment 4110, the position to at least one environmental medium. In an embodi computer-implemented method further comprises obtaining ment 4330, the output includes at least one graphical descrip genetic sequence information from at least one microorgan 15 tion of one or more of the at least one composition, ism isolated from the at least one environmental medium. In constituent thereof, or product thereof information related to an embodiment 4120, the computer-implemented method at least one organism associated with the at least one envi further comprises one or more instructions for modifying the ronmental medium; or information related to at least one cell at least one microorganism isolated from the at least one or Substance associated with the at least one environmental environmental medium. In an embodiment 4130, the com medium. In an embodiment 4340, the computer program puter-implemented method further comprises one or more product further comprises one or more instructions for instructions for amplifying the at least one microorganism obtaining genetic sequence information from at least one isolated from the at least one environmental medium. In an microorganism isolated from the at least one environmental embodiment 4140, the computer-implemented method fur medium. In an embodiment 4350, the computer program ther comprises one or more instructions for reinstating the at 25 product further comprises one or more instructions for modi least one microorganism isolated from the at least one envi fying the at least one microorganism isolated from the at least ronmental medium Subsequent to modification. In an one environmental medium. embodiment 4150, the computer-implemented method fur In an embodiment 4360, the computer program product ther comprises one or more instructions for predeterming at further comprises one or more instructions to dispense the at least one microorganism type for modifying to produce at 30 least one composition or a constituent thereof to at least one least one environmental medium treatment agent based on at environmental medium. In an embodiment 4370, the com least one feature of the at least one environmental medium. In puter program product further comprises one or more instruc an embodiment 4160, the at least one feature of the at least tions for amplifying the at least one microorganism isolated one environmental medium includes at least one property of from the at least on environmental medium. In an embodi one or more microorganism populations associated with the 35 ment 4380, the computer program product further comprises at least one environmental medium. one or more instructions for reinstating the at least one micro As indicated in FIG. 42, in an embodiment 4200, a com organism isolated from the at least one environmental puter program product comprises 4210 a recordable medium medium subsequent to modification. In an embodiment 4390, bearing one or more instructions for regulating dispensing of the computer program product further comprises one or more at least one delivery device, wherein the delivery device 40 instructions for predetermining at least one microorganism includes at least one composition, wherein the at least one type for modifying to produce at least one environmental composition includes at least one modified microorganism medium treatmentagent based on at least one feature of the at including at least one heterologous genetic element encoding least one environmental medium. In an embodiment 4395, the at least one environmental medium treatment agent; at least at least one feature of the at least one environmental medium one genetic element inducible to initiate death of the at least 45 includes at least one property of one or more microorganism one modified microorganism; and generating at least one populations associated with the at least one environmental output. In an embodiment 4220, the recordable medium medium. includes a computer-readable medium. In an embodiment 4230, the recordable medium includes a communications PROPHETIC EXAMPLES medium. In an embodiment 4233, the computer program 50 product further comprises one or more instructions for Example 1 obtaining genetic sequence information from at least one microorganism isolated from the at least one environmental Compositions Including Modified Microorganisms medium. for the Delivery of a Therapeutic Agent In an embodiment 4235, the computer program product 55 further comprises one or more instructions for displaying Microorganisms are modified to produce at least one thera results of the processing. In an embodiment 4240, the com peutic agent (which may include an agent used as a respon puter program product further comprises one or more instruc sive therapy, or prophylactic therapy, etc.). The modified tions for receiving information related to one or more envi microorganisms are retained in vivo by administration of at ronmental medium indicators prior to, during, or Subsequent 60 least one required factor as part of the modified microorgan to administering the at least one composition. In an embodi ism composition. ment 4250, the one or more environmental medium indicators The composition is given orally, following depleting resi include one or more of administration of the at least one dent microbes by administering ciproflaxocin dosed at mod composition, or constituent thereof, or product thereof. erate levels for 5 days. See, for example, Dethlefsen et al. administration of at least one metabolite utilizable by the 65 PLoS Biology vol. 6, pp. 2383-2400 (2008), which is incor modified microorganism; administration of the at least one porated herein by reference. The modified microorganisms modified microorganism; status of the at least one modified include live, commensal bacteria given orally, and are US 8,852,916 B2 73 74 allowed to colonize the intestine following depletion of resi and transfected or electroporated into bacteria. See, for dent microorganisms by antibiotic treatment. See, for example, Sambrook et al. Molecular Cloning: A Laboratory example, Wadolkowski, et al. Inf. Imm., vol. 56, pp. 1030 Manual, second ed., Cold Spring Harbor Laboratory Press, 1035 (1988); and Rao, et al., PNAS, vol. 102, no. 34, pp. N.Y. (1989), which is incorporated by reference herein. 11993-1 1998 (2005), each of which are incorporated herein 5 by reference. Example 5 At least one antacid or proton pump inhibitor (e.g. Panto Zol. Altana Pharma BV. Hoofdorp, Netherlands) and a cholate Compositions Including Modified Microorganisms acid binder (e.g. Questran, Zambon, Amersfoort, Nether for the Delivery of a Therapeutic Agent lands) are administered inconjunction with administration of the modified microorganism composition in order to improve A propionate inducible expression system is utilized with viability during passage through the stomach. See, for transformed bacteria in order to provide a relatively homog example, Braat et al. Clin. Gastroenterol. Hepatol. Vol. 4, pp. enous expression in individual microorganism cells, and 754-759 (2006), which is incorporated herein by reference. allow for highly regulatable expression. See, for example, 15 Lee and Keasling, App. Env. Microbiol. Vol. 71, no. 11, pp. Example 2 6856-6862 (2005), which is incorporated herein by reference. Expression vector pPro, as described by Lee and Keasling, is Compositions Including Modified Microorganisms capable of being regulated at the single cell level over a wide for the Delivery of a Therapeutic Agent range of inducer concentrations in a dose-dependent manner. Id. Furthermore, since bacterial cells are permeable to the Eschericia coli (E. coli), Nissile 1917 is modified using inducer proprionate (which is metabolized by 2-MC by native rDNA methods to allow the bacteria to produce heterologous chromosomal expression), regulatable and consistent induc proteins or peptides. See, for example, Rao et al., Ibid., and tion in all cells of the culture is attainable. U.S. Pat. No. 7,341,860; each of which is incorporated herein by reference. Expression of genes encoding the therapeutic 25 Example 6 agent (e.g., protein, microbicide, antigen, tolerogen, etc.) is directed by an inducible bacterial promoter (e.g., the acid Compositions Including Modified Microorganisms inducible P170, P3, or P1 promoter (isolated from Lactoccus) for the Delivery of a Therapeutic Agent and inducible by low pH). See, for example, Madsen, et al., Ibid. 30 The bacteria are modified from standard bacterial strains by deleting or mutating genes encoding enzymes or other Example 3 proteins essential for bacterial metabolism and growth by conventional standards (such as homologous recombination, Compositions Including Modified Microorganisms recombinant DNA techniques, insertional mutagenesis, tar for the Delivery of a Therapeutic Agent 35 geted gene deletion, etc.). See, for example, U.S. Pat. No. 5,643,771, and Biswas et al., J. Bacteriology, Vol. 175, pp. Eschericia coli (E. coli), Nissile 1917 is modified using 3628-3635 (1993); each of which is incorporated herein by rDNA methods to allow the bacteria to produce heterologous reference. proteins or peptides. See, for example, Rao et al., Ibid., and Deletion of the thymidine synthase gene in Lactococcus U.S. Pat. No. 7,341,860; each of which is incorporated herein 40 lactis (L. Lactis) by homologous recombination using recom by reference. The arabinose promoter (P) is fused to het binant DNA plasmids results in L. lactis clones that require erologous genes encoding therapeutic proteins or other thera thymidine for growth. See, for example, Steidler et al., Ibid. peutic agents. Coexpression of araC, an arabinose operon Subsequently, the L. lactis thymidine negative mutants are regulatory protein, and provision of L-arabinose regulates selected for by identifying which clones require thymidine expression of genes fused to the P. promoter. See, for 45 for growth (e.g., through media Supplementation). See, for example, U.S. Pat. No. 7,341,860, Ibid., which is incorpo example, Steidler etal, Ibid. Thymidine is present only at low rated herein by reference. levels in human colon, such that L. lactis thymidine auxotro phs survive less than two days following oral administration Example 4 to human volunteers. See, for example, Braat et al., Ibid. 50 However, thymidine auxotrophs survive longer than 200 Compositions Including Modified Microorganisms hours in vitro when thymidine (10 uM) is provided, as pub for the Delivery of a Therapeutic Agent lished. Thus, the growth and survival of thymidine auxotro phs in vivo is regulated by dosing and scheduling administra Eschericia coli (E. coli), Nissile 1917 is modified using tion of thymidine prior to, during, or Subsequent to rDNA methods to allow the bacteria to produce heterologous 55 administration of the L. lactis and thymidine composition. proteins or peptides. See, for example, Rao et al., Ibid., and U.S. Pat. No. 7,341,860; each of which is incorporated herein Example 7 by reference. The heterologous gene constructs employ a constitutive promoter derived from the bacterial thymidine Compositions Including Modified Microorganisms synthase gene which continuously expresses at least one 60 for the Delivery of a Therapeutic Agent therapeutic agent, such as a cytokine. See, for example, Steidler et al. Nature Biotechnology vol. 21, pp. 785-789 Mutation or deletion of the f-aspartate semialdehyde (2003), which is incorporated herein by reference. dehydrogenase gene (Asd) in bacteria precludes synthesis of That is, the heterologous gene construct encoding IL-2 is diaminopimelic acid (DAP), an essential cell wall constituent incorporated into an expression plasmid that also contains 65 that is not present in animal tissues. Without exogenous DAP. drug-resistance selectable markers (e.g. amplicillin resistance bacteria that have a mutated or deleted Asd gene will undergo marker, B-lactamase, or chloramphenicol resistance marker) cell death and lysis (See, for example, U.S. Pat. No. 7,341, US 8,852,916 B2 75 76 860, Ibid.), but providing DAP by oral administration allows Example 11 Asd mutants to grow, colonize and Survive on mucosal Sur faces in vivo. Id. Compositions Including Modified Microorganisms for the Delivery of a Therapeutic Agent Example 8 Production of a suicide factor, Rel F, is regulated by a Compositions Including Modified Microorganisms synthetic gene network engineered into a microorganism. Briefly, oral administration of an inducer molecule (arabi for the Delivery of a Therapeutic Agent nose) controls expression of IL-10. A synthetic gene network 10 responsive to pulses of a metabolite (e.g., arabinose), is uti Measurement and detection of the mutant bacteria that are lized, as described in Friedland et al., Science, Vol. 324, pp. present in vivo is conducted using quantitative polymerase 1199-1202 (2009), which is incorporated herein by reference. chain reaction (PCR) and primers specific for the microbial The gene network is constructed by combining transcrip strain and the gene expression construct. Stool samples from tional and translational regulatory elements. The P. pro Subjects given L. Lactis, modified to express human IL-10, is 15 moter, a transactivating noncoding RNA, a cis repressor detectable with PCR primers specific for the 16S ribosomal sequence RNA, and a T7 RNA polymerase gene are com RNA of L. lactis and the human IL-10 expression construct bined in a synthetic gene network to control the expression of (See, for example, Braat et al, Ibid.). multiple proteins as shown by Friedland et al., Ibid. The regulatory elements cause one particular product to be pro Example 9 duced with a first induction event (e.g., exposure to arabi nose); a second particular product to be produced with a Compositions Including Modified Microorganisms second induction event; a third particular product to be pro for the Delivery of a Therapeutic Agent duced with a third induction event, etc. In this manner, the synthetic system allows for exhibiting the number of induc 25 tion events that have occurred, or “counting induction The number of colony forming units (CFU) of L. Lactis is events. Id. assessed by culturing Stool samples on microbiological plates Thus, the therapeutic agent (e.g., IL-10) is delivered to a containing selective media. Briefly, fecal samples are sus patients intestine by ingestion of modified E. coli containing pended in minimal media and then selected on plates coated a plasmid encoding a synthetic gene network that responds to with antibodies specific for L. Lactis. Next, media containing 30 multiple pulses of an inducer molecule (e.g., arabinose) by essential nutrients (including thymidine) is overlaid and the the production of IL-10. The gene network also contains bacterial colonies arising are counted to determine the CFU suicide genes (e.g. Rel F) that will cause cell death when they present in the fecal sample (See, for example, Steidler et al. are expressed. See, for example, U.S. Pat. No. 6,610,529, Ibid.). which is incorporated herein by reference. The frequency and Since thymidine synthase mutants of L. lactis (Thy A. L. 35 duration of arabinose pulsing determines the expression of Lactis) are dependent on exogenous thymidine for growth in genes (and their corresponding proteins) in the gene network, vitro and in vivo, no viable Thy A. L. Lactis are present after while the dose and schedule of oral arabinose administration culture 72 hours in rich media devoid of thymidine, but in determines the timing and duration of expression of the thera cultures containing 10 uM thymidine, the microbes survive peutic agent and Suicide gene, IL-10 and Rel F. respectively. beyond 200 hours. In vivo, only 4% of Thy A. L. Lactis 40 The synthetic gene networks for therapeutic agent produc auxotrophs Survive after 4 hours in the mammalian intestine tion utilize optimal pulse intervals of approximately 10-40 with only endogenous thymidine present (thymidine concen minutes and optimal pulse lengths of approximately 20-30 tration is less than 0.075 uM in human ileal lavage; See, for minutes, while synthetic gene networks for Suicide gene example, Steidler et al., Ibid.). expression utilize optimal pulse intervals and pulse lengths of 45 approximately 2-12 hours (see Friedland et al., Ibid.). For Example 10 example, gene networks encoding IL-10 utilize approxi mately two 10-minute pulses of arabinose separated by an Compositions Including Modified Microorganisms interval of 20 minutes to optimally induce IL-10 expression, for the Delivery of a Therapeutic Agent while Rel F expression and cell death ensue following two 50 2-hour pulses with arabinose separated by 2 hours. Synthetic gene networks incorporating multiple inducers Production of at least one therapeutic agent, IL-10, by L. (e.g., arabinose, anhydrotetracycline and IPTG), and the lactis (Thy A. L. Lactis) is controlled by an inducible pro expression of multiple genes in the network can be employed moter, PyaraC promoter/regulator system (see, for that utilize variations of the order, length and interval of example, U.S. Pat. No. 7,341,860, Ibid.). The inducible pro 55 pulsing with each of the inducers. See, Friedland et al., Ibid. moter is used in conjunction with arabinose to regulate the production of IL-10 in the microorganism. In an embodiment, Example 12 production and delivery in situ is regulated by dosing and scheduling of arabinose administration, while bacterial Sur Compositions Including Modified Microorganisms vival and growth is regulated through dosing and scheduling 60 for the Delivery of a Therapeutic Agent of thymidine administration. The amount of IL-10 delivered is monitored by immunoas Modified E. coli including a plasmid encoding the Rel F say of fecal samples. For example, human IL-10 derived from gene regulated by a promoter, Such as plac that is repressed feces samples is measured by enzyme linked immunosorbent by a regulator protein, LacIq, unless isopropyl B-D-thioga assay (ELISA; Steidler et al., Ibid.). ELISA reagents and 65 lactoside (IPTG) is provided. Thus, in order to control the protocols for numerous cytokines including IL-10 are avail growth of the modified E. coli strain in vivo, IPTG is admin able, for example, from Invitrogen Corp., Carlsbad, Calif. istered to induce Rel F expression and cause cell death. Alter US 8,852,916 B2 77 78 natively the Rel F gene can be controlled by the arabinose IL-12 are fused to the lac operon promoter (P), which is promoter/repressor. Thus, expression of Rel F is regulated by inducible with IPTG or lactose. the C2 repressor which, in turn, is regulated by the presence of arabinose. When arabinose levels are reduced, C2 repressor Example 15 levels decline, resulting in Rel F expression and leading to 5 bacterial cell death (see, for example, U.S. Pat. No. 6,610, Compositions Including Modified Microorganisms 529, which is incorporated herein by reference). for the Delivery of a Therapeutic Agent Modified fungi, derived from generally regarded as safe Example 13 10 (GRAS) species can be auxotrophic for essential metabolites and express protein-encoding genes under the control of regu Compositions Including Modified Microorganisms lated promoters. For example, in an embodiment, Saccharo for the Delivery of a Therapeutic Agent myces cerevesiae (S. cerevesiae) and Saccharomyces boular dii (S. boulardii) are GRAS organisms that are mutated and 15 selected (see, for example, Abosereh et al. Res. J. Agric. Biol. In an embodiment, mutually dependent strains of bacteria Sci., vol. 2, pp. 478-482 (2006), which is incorporated by that signal via Small molecules, such as acyl-homoserine reference herein) to obtain clones that require amino acids lactones (acyl-HSL), are modified to express heterologous (e.g., leucine, tryptophan, lysine, arginine), purines, or pyri target genes only when sufficient numbers of both bacterial midines (e.g., adenine, thymidine). Growth and Survival of strains (and acyl-HSL) are present. See, for example, Brenner yeast auxotrophs in vitro and in vivo is dependent on an et al., PNAS, vol. 104, pp. 17300-17304 (2007), which is essential metabolite (e.g., adenine), and if adenine is not incorporated herein by reference. Thus, two E. coli strains, present (or present at low levels) in a subject hosting the modified to produce specific acyl-HSL, control the expres strain, then growth of the yeast auxotroph is regulated by sion of target genes for each other, while target gene expres administering adenine to the Subject. For example, the dose sion is controlled by providing exogenous acyl-homoserine 25 and schedule of adenine administration controls the prolif lactone to the individual microbial strains. eration and Survival of the yeast adenine auxotrophs. Yeast cloning vectors used for protein expression include Administration of 3-oxododecanoyl-HSL (3OC12HSL) but are not limited to yeast integrative plasmids, yeast episo and butanoyl-HSL (C4HSL) in vivo is used to regulate target mal plasmids, and yeast centromeric plasmids that contain protein production of the two E. coli Strains (see, for example, 30 selectable markers based on metabolite requirements. Genes Brenner et al., Ibid.). In addition, survival of the two strains is used as selectable markers include but are not limited to regulated by acyl-HSL-regulated expression of a toxin (ccd LEU2, URA3, and HIS3, which encode enzymes needed to B) gene (causes cell death), and an antitoxin (ccd A) gene biosynthesize leucine, uracil and histidine (for example, yeast (allows survival). See, for example, Balagadde et al. Mol. vectors for protein expression are described in Glazer et al. Sys. Biol., vol. 4, pp. 1-8 (2008), which is incorporated herein 35 Microbial Biotechnology: Fundamentals of Applied Micro by reference. The toxin-antitoxin includes at least one of biology, 2" edition, Cambridge University Press (2007), masEF, chpBIK, relBE, yefM-yoeB, dinJ-yafl, or ecna which is incorporated herein by reference. Transcription of ecnB. See, Engleberg-Kulka, et al., PLOS Genetics, Vol. 2, heterologous genes is directed by a constitutive promoter no. 10, 1518-1526 (2006), which is incorporated herein by (e.g., ADH1, TDH3) or a regulated promoter (e.g., GAL1, reference. 40 ADH2, PHO5, CUP-1, etc.). Coadministration of multiple bacterial strains that express Example 16 different target genes are used to co-deliver multiple thera peutic agents. For example, localized co-expression of IL-10 Compositions Including Modified Microorganisms and transforming growth factor beta (TGF-B) by separate 45 for the Delivery of a Therapeutic Agent bacterial strains, provide localized immunoregulatory therapy for conditions such as inflammatory bowel disease. Modified yeast strain that requires two metabolites, for example, leucine and histidine, a selectable marker (e.g. Example 14 LEU2) provides plasmid stability, while the metabolite (e.g., 50 histidine or leucine) regulates growth and Survival of the yeast strain. For example, in an embodiment, modified S. boulardii Compositions Including Modified Microorganisms is used for vaccination, wherein the Strain requires leucine for the Delivery of a Therapeutic Agent and histidine, and produces a heterologous protein, hepatitis B Surface antigen encoded on a yeast episomal plasmid con 55 taining LEU2, and under the control of the ADH1 constitutive A bacterial strain that expresses an antigen derived from a promoter. Dosing and scheduling of histidine administration viral pathogen, for example. E7 antigen from human papil regulates the growth and survival of the modified S. boulardi loma virus type 16, is coadministered with a second bacterial and, in turn, the production and delivery of hepatitis B surface strain that produces interleukin-12 (IL-12) to promote immu antigen. nization. The two bacterial strains are auxotrophs that are 60 mutually dependent on each other for essential metabolites, Example 17 such as amino acids or DAP. One or both auxotrophic strains are sustained in vivo by Compositions Including Modified Microorganisms administering exogenous metabolites (e.g. histidine, arabi for the Delivery of a Therapeutic Agent nose, DAP, acyl-HSL, thymidine) to the host organism. In an 65 embodiment, expression of the E7 gene and IL-12 is directed A modified yeast strain transformed with a recombinant by the same or a different promoter. For example. E7 and plasmid encoding a nuclease (e.g. Serratia marcescens US 8,852,916 B2 79 80 nuclease A) under the control of the S. cerevesiae ADH2 The Pfluorescens strain is also modified with an inducible promoter, undergoes cell death when the nuclease A gene is genetic element (e.g., Rel F) to initiate death of the bacteria expressed. Ordinarily, the ADH2 promoter is repressed by when a factor is provided to induce the suicide gene. The glucose, and when glucose levels are depleted the ADH2 composition including the modified microorganism and promoter/nuclease A gene is expressed, resulting in cell induction factor is applied sequentially to the plants, and death. See, for example, Balan et al. Yeast, Vol. 22, pp. 203 optionally to their environment (e.g., Soil or water), by spray 212 (2005) which is incorporated herein by reference. Thus, ing, dusting, sprinkling, or otherwise applying the composi death of the modified yeast strain is induced in a glucose poor tion to the selected area. Expression of the Rel F gene is environment (e.g. feces, intestine, soil, etc.). controlled by a regulated promoter Such as plac that is 10 repressed by a regulator protein, LacIq, unless isopropyl B-D- Example 18 thiogalactoside (IPTG) is provided. Compositions Including Modified Microorganisms Thus, in order to control growth of the modified microbial for the Delivery of a Therapeutic Agent strain, IPTG (available from Sigma-Aldrich Corp., St. Louis, 15 Mo.) is applied to the plant or soil areas where the composi Co-administration of two yeast (Saccharomyces cereve tion has also been applied or translocated, in Sufficient siae) auxotrophs, one that requires lysine, and a second that amount to induce Rel F expression and leading to death of the requires adenine, are modified to over-produce adenine and microorganism(s). IPTG is applied by spraying, dusting, lysine, respectively. The mutants are generated using conven sprinkling or other application. An IPTG Solution containing tional methods, such as mutation, selection and genetic approximately 3 mMIPTG is used to induce expression of the crosses. See, for example, Shou et al., PNAS, vol. 104, pp. repressed pLac promoter. See, for example, Sambrook, 1877-1882 (2007), which is incorporated herein by reference. Fritsch & Maniatis, Molecular Cloning: A Laboratory Co-administration of live yeast strains that are mutually Manual, Second Edition (1989) Cold Spring Harbor Labora dependent on each other for Survival allows prolonged Sur tory Press, Cold Spring Harbor, N.Y. which is incorporated vival and colonization of both strains on mucosal Surfaces 25 herein by reference. (e.g. intestinal, vaginal, nasal, oral, bronchial, etc.). The yeast strains are modified to express enzymes essential for overpro Example 20 duction of adenine and lysine under the control of regulated promoters, CUP-1 promoter derived from the metallothion Compositions and Methods of Administering ein gene. (See, for example, Glazer et al., Ibid.) 30 Therapeutic Agents to Plants Transcription of genes fused to CUP-1 is induced by pro viding metalions such as Cu" and Zn". For example, ZnCl Pseudomonas known to inhabit the phylloplane (the sur can be given orally to induce expression of ADE4 and face of plant leaves) or the rhizosphere (the soil Surrounding LYS21, enzymes that mediate over-production of adenine plant roots) are used as microbial hosts for plasmid expres and lysine, respectively, by modified yeast residing, for 35 sion vectors directing the expression of an anti-fungal pep example, in the colon (see, for example, Shou et al., Ibid.). tide, PW2. Withdrawal of ZnCl2 from the diet lowers Zn" levels, Modified Pseudomonas strains are stable within the rhizo reduces expression of ADE4 and LYS21, and reduces sphere of wheat and barley plants for approximately 1 month production of adenine and lysine which, in turn, leads to death at a level of approximately 1.0x10 to 23x10 CFU/cm of root of the modified yeast strains. 40 and is capable of stably expressing recombinant genes for approximately 29 days, according to published reports. See, Example 19 for example, Shim et al., Appl. Envir. Microbiol. Vol. 66, pp. 4673-4678 (2000), which is incorporated herein by reference. Compositions and Methods of Administering For example, modified microorganism compositions are Therapeutic Agents to Plants 45 applied in the field by spraying, dusting, soaking, soil injec tion, seed coating, seedling coating, or the like. Bacterial Pseudomonas fluorescens strain SBW25 inhabits the Suspensions for application to plants are approximately 10° to leaves and roots of Sugar beet plants and is competent for 10" cells/ml and the volume applied per hectacre is approxi transformation with recombinant DNA plasmids. See, for mately 10 ml to 100,000 ml or more. Administration of bac example, Zhang, et al., Microbiol. Vol. 152, pp. 1867-1875 50 terial suspensions to a plant part is at approximately 10 to 10° (2006), which is incorporated herein by reference. Pfluore cells/cm. scens is transformed with a DNA plasmid that encodes an anti-microbial peptide, PW2. See, for example, U.S. Pat. No. Example 21 5,017,373; and U.S. Pat. No. 7,510,852, each of which is incorporated herein by reference. PW2, and methods for its 55 Compositions and Methods of Administering cloning and construction are described in U.S. Pat. No. 7,550, Environmental Medium Treatment Agents to 558, which is incorporated herein by reference. As published, Environmental Media PW2 has anti-fungal activity against the following plant fun gal pathogens: Colletotrichum gossypii, Cephalosporioides, A modified microorganism includes at least one heterolo Alternaria macrospora, Colletotrichum Ora, Bipolaris soro 60 gous genetic element encodes at least one plant growth factor, Kiniana, Dreschslera tritici, Phoma sorghina, Pyricularia for example, acetoin (3-hydroxy-2-butanone) and 2,3-bu grisea, Colletotrichum, Gloeosporioides, Rhizoctonia Solani tanediol in the environmental medium (e.g., soil), for and Fusarium Solani. example, in order to promote the growth of plants. According Thus, the modified P. fluorescens strain delivers the anti to published studies, naturally occurring plant-growth pro fungal peptide PW2 to at least one biological tissue, such as 65 moting rhizobacteria, which are capable of producing plant leaves, stems, or roots to prevent the growth, inhibit the acetoin, increase leaf growth in Arabidopsis thaliana by more growth or reduce the viability of fungal plant pathogens. than 100% relative to control bacteria not capable of produc US 8,852,916 B2 81 82 ing acetoin. See, for example, Ryu et al., PNAS, USA, Vol. example, S. lividans strain TK23 is modified to be resistant to 100, pp. 4927-4932 (2003), which is incorporated herein by spectinomycin, while S. lividans transformed with the plas reference. mid, pIJ702 is resistant to thiostrepton. See, for example, Thus, a bacterium that lives in the soil, Streptomyces liv Crawford etal, Ibid. idans, is transformed with at least one genetic element (i.e. 5 gene, promoter element, regulatory sequence, etc.) that Example 22 encodes at least one enzyme required to produce at least one plant growth factor. For example, S. lividans (ATCC #69441: Compositions and Methods of Administering available from AmericanType Culture Collection, Manassas, Environmental Medium Treatment Agents to Va.) is modified by transformation with a plasmid (e.g. 10 Environmental Media pIJ702) containing at least one enzyme gene required to pro duce at least one plant growth factor (e.g., acetoin, gibbrellin, A method includes administering at least one composition 2,3-butanediol, etc.). Transformation of the microorganism including at least one modified microorganism encoding at can be conducted utilizing standard procedures. See, for least one environmental medium treatment agent, such as a example, Wang etal, J. Biotech., vol. 13, pp. 131-144 (1990); 15 plant peptide hormone, to at least one environmental medium and Sambrook et al. Molecular Cloning: A Laboratory (e.g., soil, water, etc.). In an embodiment, the at least one Manual, second ed., Cold Spring Harbor Laboratory Press, modified microorganism includes at least one inducible N.Y. (1989), each of which is incorporated herein by refer genetic element to initiate death of the at least one modified CCC. microorganism. For example, S. lividans bacteria are modified to express Pseudomonas putida is modified to include at least one the acetolactate synthase (AlsS) and acetolactate decarboxy genetic element encoding at least one plant peptide hormone lase (AlsD) genes which act on pyruvate, a common metabo (e.g., POLARIS), and at least one genetic element inducible lite, to yield acetoin. See, for example, Taghavi et al. Appl. to initiate death in the at least one modified microorganism Envir. Microb. Vol. 75, no. 3, pp. 748-757 (2009), which is (e.g., Rel F), and each is controlled by a synthetic gene net incorporated herein by reference. Cloning and expression of 25 work of the microorganism. As shown in published studies, an Alss gene in S. lividans is conducted utilizing standard application of an inducer molecule regulates expression of procedures. See, for example, Smith et al., PNAS, USA, Vol. POLARIS, a 36 amino acid peptide that functions in root 86, pp. 4179-4183 (1989); and Swindell et al. Appl. Envir. growth and leaf vascularization. See, for example, Casson et Microbiol. Vol. 62, no. 7, pp. 2641-2643 (1996), each of al, Plant Cell, vol. 14, pp. 1705-1721 (2002), which is incor which is incorporated herein by reference. 30 porated herein by reference. The synthetic gene network is Expression of genes encoding the Alss and AlsD enzymes responsive to pulses of a metabolite (e.g., arabinose). See, for is directed by the inducible lac operon promoter fused to at example, Friedland et al., Science, Vol. 324, pp. 1199-1202 least one gene encoding at least one enzyme (e.g. AlsS. AlsD), (2009), which is incorporated herein by reference. and gene expression is induced with lactose or isopropyl Thus, the synthetic gene network is constructed by com B-D-thiogalactoside. See, for example, Rao, et al., PNAS, 35 bining transcriptional and translational regulatory elements USA vol. 102, no. 34, pp. 11993-1 1998 (2005), which is including the pBAD promoter, a transactivating noncoding incorporated herein by reference. RNA, a cis repressor sequence RNA, and a T7 RNA poly The expression plasmid includes at least one drug-resis merase gene. See, for example, Friedland et al., Ibid. tance selectable marker (e.g. amplicillin resistance marker, The frequency or duration of arabinose application deter B-lactamase, or chloramphenicol resistance marker). Routine 40 mines the expression of the inducible genetic element (and methods for gene transfer and components relating to select the corresponding protein(s)) in the synthetic gene network, able markers are described, for example, in Sambrook et al. and the dose and schedule of arabinose application to the soil Molecular Cloning: A Laboratory Manual, second ed., Cold determines the timing and duration of expression of Spring Harbor Laboratory Press, N.Y. (1989), which is incor POLARIS and Rel F. As published, synthetic gene networks porated herein by reference. 45 have optimal pulse intervals of approximately 10-40 minutes S. lividans, is additionally transformed with at least one and optimal pulse lengths of approximately 20-30 minutes. genetic element inducible to initiate death in the at least one Id. Additionally, synthetic gene networks with optimal pulse modified microorganism. For example, the at least one induc intervals and pulse lengths of approximately 2-12 hours are ible genetic element encodes at least one lethal or Suicide also described. Id. Other optimal pulse intervals can be deter gene (Rel F) under the control of an inducible promoter, such 50 mined for a particular system, according to the published as plac. The inducible promoter plac is repressed by an guidelines. Id. inducer, LacIq, unless isopropyl B-D-thiogalactoside (IPTG) Thus, synthetic gene networks incorporating multiple is provided (see Rao et al., Ibid.). Thus, in order to initiate inducers (e.g., arabinose, anhydrotetracycline and IPTG). death in the modified S. lividans, IPTG is administered to and the expression of multiple genes in the network depends induce Rel F expression and initiate cell death. 55 on the order, length, and interval of pulsing with each of the The composition including modified S. lividans bacteria inducers. Id. transformed with the genetic element encoding acetoin is sprayed onto Soil as a spore Suspension in water at a rate of Example 22 approximately 10°-10 colony forming units (CFU) per gram of soil. As shown by published studies, modified S. lividans 60 Compositions and Methods of Administering innoculated at approximately 107 CFU/gm soil and grown at Environmental Medium Treatment Agents to 25° C. survive for at least approximately 90 days. See, for Environmental Media example, Crawford et al. Appl. Envir. Microbiol. Vol. 59, pp. 508-518 (1993), which is incorporated herein by reference. Pputida, a bacteria commonly found in Soil, is modified to The survival of S. lividans is measured by diluting in water 65 include three genetic elements: 1) a regulated promoter (e.g., and spreading on plates containing at least one antibiotic to pLac) fused to a Suicide gene (e.g., gef), and 2) a TOL plasmid which the microorganism has been rendered resistant. For promoter (e.g., Pm) fused to the lad gene (encoding the Lac US 8,852,916 B2 83 84 repressor), and 3) XylS2, a gene encoding a positive regulator instances where a convention analogous to “at least one of A, of Pm that interacts with 3-methyl benzoate. See, for B, and C, etc. is used, in general Such a construction is example, Ramos, et al., Biotech: Vol. 12, pp. 1349-1356 intended in the sense one having skill in the art would under (1994), which is incorporated herein by reference. Pputida is stand the convention (e.g., “a system having at least one of A, modified to produce POLARIS under the control of a consti 5 B, and C would include but not be limited to systems that tutive promoter (for example, the bacterial thymidine syn have A alone, B alone, C alone, A and B together, A and C thase promoter, according to Steidler et al., Ibid.) is trans together, B and C together, and/or A, B, and C together, etc.). formed with a DNA cassette containing the plac promoter In those instances where a convention analogous to “at least fused to the gef gene. The strain is also transformed with a one of A, B, or C, etc. is used, in general Such a construction plasmid that contains the Pm promoter fused to lad gene and 10 is intended in the sense one having skill in the art would encoding the XylS2 positive regulator of Pm. Genetic con understand the convention (e.g., “a system having at least one structs and construction of recombinant bacterial strains is of A, B, or C would include but not be limited to systems that conducted by routine procedures, some of which are have A alone, B alone, C alone, A and B together, A and C described in Jensen et al. Appl. Env. Micro. Vol. 59, pp. together, B and C together, and/or A, B, and C together, etc.). 3713-3717 (1993), which is incorporated herein by reference. 15 It will be further understood by those within the art that Modified P. putida encoding POLARIS, and including at typically a disjunctive word and/or phrase presenting two or least one genetic element inducible to initiate death of the P. more alternative terms, whether in the description, claims, or putida, is grown in the presence of 3-methylbenzoate. When drawings, should be understood to contemplate the possibili 3-methylbenzoate is combined with the XylS2 gene product, ties of including one of the terms, either of the terms, or both positive regulation of Pm results, and leads to production of terms unless context dictates otherwise. For example, the the Lac repressor. The Lac repressor represses the expression phrase A or B will be typically understood to include the of the toxic gene, gef. According to published studies, P possibilities of “A” or “B” or “A and B.” putida Strains including a substrate-regulated toxic gene sys With respect to the appended claims, those skilled in the art tem inoculated at approximately 10 CFU/gm to nonsterile will appreciate that recited operations therein may generally soil containing 0.08% (wt/vol.) 3-methyl benzoate are 25 be performed in any order. Also, although various operational present at approximately 10 CFU/gm of soil after approxi flows are presented in a sequence(s), it should be understood mately 14 days. See, for example, Jensen etal, Ibid. However, that the various operations may be performed in other orders in control experiments without 3-methyl benzoate only than those which are illustrated, or may be performed con approximately 10 CFU/gm of soil survived after 14 days. Id. currently. Examples of such alternate orderings may include While particular aspects of the present subject matter 30 overlapping, interleaved, interrupted, reordered, incremental, described herein have been shown and described, it will be preparatory, Supplemental, simultaneous, reverse, or other apparent that, based upon the teachings herein, changes and variant orderings, unless context dictates otherwise. Further modifications may be made without departing from the Sub more, terms like “responsive to.” “related to,” or other past ject matter described herein and its broader aspects and, tense adjectives are generally not intended to exclude Such therefore, the appended claims are to encompass within their 35 variants, unless context dictates otherwise. Scope all such changes and modifications as are within the All publications and patent applications cited in this speci true spirit and scope of the subject matter described herein. It fication are herein incorporated by reference to the extent not will be understood by those within the art that, in general, inconsistent with the description herein and for all purposes terms used herein, and especially in the appended claims as if each individual publication or patent application were (e.g., bodies of the appended claims) are generally intended 40 specifically and individually indicated to be incorporated by as “open’ terms (e.g., the term “including should be inter reference for all purposes. preted as “including but not limited to the term “having What is claimed is: should be interpreted as “having at least, the term “includes’ 1. A composition, comprising: should be interpreted as “includes but is not limited to, etc.). at least two modified microorganisms that are mutually It will be further understood by those within the art that if a 45 dependent and express at least one environmental specific number of an introduced claim recitation is intended, medium treatment agent only after satisfying a popula such an intent will be explicitly recited in the claim, and in the tion threshold, at least one modified microorganism absence of Such recitation no such intent is present. For including at least one heterologous genetic element example, as an aid to understanding, the following appended encoding at least one-environmental medium treatment claims may contain usage of the introductory phrases “at least 50 agent including at least one of nitroreductase; and at one' and “one or more' to introduce claim recitations. How least one modified microorganism including at least one ever, the use of such phrases should not be construed to imply heterologous genetic element encoding at least one envi that the introduction of a claim recitation by the indefinite ronmental medium treatment agent including an articles 'a' or “an limits any particular claim containing enzyme required to produce at least one plant growth Such introduced claim recitation to claims containing only 55 factor; one such recitation, even when the same claim includes the and wherein each of the at least two modified microorgan introductory phrases “one or more' or “at least one' and isms include indefinite articles such as “a” or “an” (e.g., “a” and/or “an at least one genetic element operably coupled to at least one should typically be interpreted to mean “at least one' or “one promoter or enhancer and inducible to initiate death of or more'); the same holds true for the use of definite articles 60 the at least one modified microorganism; and used to introduce claim recitations. In addition, even if a at least one sensor including an RNA aptamer, a hammer specific number of an introduced claim recitation is explicitly head ribozyme actuator, and a transmitter including a recited, those skilled in the art will recognize that such reci sequence that couples the sensor and actuator. tation should typically be interpreted to mean at least the 2. The composition of claim 1, wherein the at least one recited number (e.g., the bare recitation of “two recitations.” 65 genetic element inducible to initiate death of the at least two without other modifiers, typically means at least two recita modified microorganisms includes at least one heterologous tions, or two or more recitations). Furthermore, in those genetic element. US 8,852,916 B2 85 86 3. The composition of claim 1, wherein the at least one genic strain, transgenic microorganism, magnetotactic plant growth factor includes at least one plant hormone. microorganism, anaerobic microorganism or aerobic micro 4. The composition of claim 3, wherein the at least one organism, food grade strain, obligate microorganism, attenu plant hormone includes at least one of an auxin, abscisic acid, ated microorganism Strain, facultative anaerobe, non-inva cytokinin, ethylene, gibberellin, brassinolide, salicyclic acid, sive strain, probiotic, colonizing microorganism, element jasmonate, polyamine, plant peptide hormone, nitric oxide, or modifying microorganism, photosynthetic microorganism. Strigolactone. 21. The composition of claim 20, wherein the at least one 5. The composition of claim 1, wherein the at least one element-modifying microorganism includes at least one of a genetic element inducible to initiate death of the at least two nitrogen-fixing microorganism, nitrifying microorganism, modified microorganisms includes at least one of an induc 10 denitrifying microorganism, hydrocarbon-utilizing microor ible promoter, inducible enhancer, or inducible repressor. ganism, dechlorinating microorganism, or a sulfate-reducing 6. The composition of claim 1, further including at least microorganism. one metabolite utilizable by at least one of the at least two modified microorganisms. 22. The composition of claim 1, wherein the at least one 7. The composition of claim 6, wherein the at least one 15 environmental medium treatment agent is pH dependent. metabolite includes at least one inducer of the heterologous 23. The composition of claim 1, wherein the composition genetic element encoding at least one environmental medium includes at least one time-release formulation. treatment agent. 24. The composition of claim 23, wherein the time-release 8. The composition of claim 6, wherein the at least one formulation includes at least one of a suspension, mixture, metabolite includes at least one repressor of at least one of the Solution, Sol, clathrate, colloid, emulsion, microemulsion, heterologous genetic elements encoding at least one environ aerosol, ointment, capsule, micro-encapsule, cream, paste, mental medium treatment agent. resin, liniment, lotion, foam, polymer, buffer, lubricant, Sus 9. The composition of claim 6, wherein the at least one pending agent, Solvent, stabilizer, or gel. metabolite includes at least one inducer of at least one genetic 25. The composition of claim 1, wherein the at least one element inducible to initiate death of at least one of the two 25 environmental medium treatment agent is encoded by at least modified microorganisms. One VectOr. 10. The composition of claim 6, wherein the at least one 26. The composition of claim 1, further including at least metabolite includes at least one repressor of the at least one one detection material associated with each of the at least two genetic element inducible to initiate death of at least one of modified microorganisms. the two modified microorganisms. 30 27. The composition of claim 26, wherein the at least one 11. The composition of claim 6, wherein the at least one detection material includes at least one, sensor, contrast metabolite includes a time-release formulation. agent, or electronic identification device. 12. The composition of claim 6, wherein the at least one 28. The composition of claim 27, wherein the at least one metabolite is pH-dependent. sensor includes at least one biosensor. 13. The composition of claim 6, wherein the at least 35 metabolite is temperature dependent. 29. The composition of claim 27, wherein the at least one 14. The composition of claim 6, wherein the at least one electronic identification device includes at least one radio metabolite includes the at least one environmental medium frequency identification device. treatment agent. 30. The composition of claim 26, wherein the at least one 15. The composition of claim 6, wherein the at least one 40 detection material includes at least one of a radioactive, lumi metabolite is provided or produced by the at least one envi nescent, colorimetric or odorous Substance. ronmental medium. 31. The composition of claim 26, wherein the detection 16. The composition of claim 6, wherein the at least one material includes at least one of a diamagnetic particle, fer metabolite is provided by at least one biological cell. romagnetic particle, paramagnetic particle, Super paramag 17. The composition of claim 1, wherein the at least one 45 netic particle, particle with altered isotope, or other magnetic genetic element inducible to initiate death of either of the particle. modified microorganisms includes at least one genetic ele 32. The composition of claim 26, wherein the detection ment inducible to initiate programmed cell death. material includes at least one of a DNA or RNA device. 18. The composition of claim 1, wherein the at least one 33. The composition of claim 26, wherein the detection modified microorganism includes at least one of a prokaryote 50 material or a precursor thereof is encoded by the at least one or a eukaryote. heterologous genetic element encoding at least one environ 19. The composition of claim 1, wherein the at least one mental medium treatment agent. modified microorganism includes at least one of bacteria, 34. The composition of claim 26, wherein the detection protozoa, rotifers, algae, archaeon, or fungi. material includes the at least one environmental medium 20. The composition of claim 1, wherein the at least one 55 treatment agent or a metabolite thereof. modified microorganism includes at least one of a non-patho k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,852,916 B2 Page 1 of 1 APPLICATIONNO. : 12/637607 DATED : October 7, 2014 INVENTOR(S) : Roderick A. Hyde et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
In the Claims Column 85, Lines 35-36, Claim 13: “... wherein the at least metabolite is temperature dependent should read -- wherein the at least one metabolite is temperature dependent --
Column 86, Line 31, Claim 27: "... includes at least one, Sensor, contrast agent, ... should read -- includes at least one Sensor, contrast agent, --
Signed and Sealed this Twenty-seventh Day of January, 2015 74-4-04- 2% 4 Michelle K. Lee Deputy Director of the United States Patent and Trademark Office