USOO9689046B2

(12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall et al. (45) Date of Patent: Jun. 27, 2017

(54) SYSTEM AND METHODS FOR THE FOREIGN PATENT DOCUMENTS DETECTION OF MULTIPLE CHEMICAL WO O125472 A1 4/2001 COMPOUNDS WO O169245 A2 9, 2001 (71) Applicants: Robert Matthew Mayall, Calgary (CA); Emily Candice Hicks, Calgary OTHER PUBLICATIONS (CA); Margaret Mary-Flora Bebeselea, A. et al., “Electrochemical Degradation and Determina Renaud-Young, Calgary (CA); David tion of 4-Nitrophenol Using Multiple Pulsed Amperometry at Christopher Lloyd, Calgary (CA); Lisa Graphite Based Electrodes', Chem. Bull. “Politehnica” Univ. Kara Oberding, Calgary (CA); Iain (Timisoara), vol. 53(67), 1-2, 2008. Fraser Scotney George, Calgary (CA) Ben-Yoav. H. et al., “A whole cell electrochemical biosensor for water genotoxicity bio-detection”. Electrochimica Acta, 2009, 54(25), 6113-6118. (72) Inventors: Robert Matthew Mayall, Calgary Biran, I. et al., “On-line monitoring of gene expression'. Microbi (CA); Emily Candice Hicks, Calgary ology (Reading, England), 1999, 145 (Pt 8), 2129-2133. (CA); Margaret Mary-Flora Da Silva, P.S. et al., “Electrochemical Behavior of Hydroquinone Renaud-Young, Calgary (CA); David and Catechol at a Silsesquioxane-Modified Carbon Paste Elec trode'. J. Braz. Chem. Soc., vol. 24, No. 4, 695-699, 2013. Christopher Lloyd, Calgary (CA); Lisa Enache, T. A. & Oliveira-Brett, A. M., "Phenol and Para-Substituted Kara Oberding, Calgary (CA); Iain Phenols Electrochemical Oxidation Pathways”, Journal of Fraser Scotney George, Calgary (CA) Electroanalytical Chemistry, 2011, 1-35. Etesami, M. et al., “Electrooxidation of hydroquinone on simply prepared Au-Pt bimetallic nanoparticles'. Science China, Chem (73) Assignee: FREDSENSE TECHNOLOGIES istry, vol. 56, No. 6, p. 746-754, Jun. 2013. CORP., Calgary, Alberta (CA) Funabashi, H. et al., “Electrochemical evaluation of cellular physi ological status under stress in Escherichia coli with therpoS-lacz, (*) Notice: Subject to any disclaimer, the term of this reporter gene'. Biotechnology and Bioengineering, vol. 90, No. 4. patent is extended or adjusted under 35 May 20, 2005. U.S.C. 154(b) by 210 days. Hou, P. et al., “Electrochemistry of Hydroquinone Derivatives at Metal and Iodine-modified Metal Electrodes'. Chemical Research in Chinese Universities, 2006, 22(4), 493-499. (21) Appl. No.: 14/491,367 Huang, W. et al., “Simultaneous determination of 2-nitrophenol and 4-nitrophenol based on the multi-wall carbon nanotubes Nafion (22) Filed: Sep. 19, 2014 modified electrode'. Analytical and Bioanalytical Chemistry, (2003)375(5), 703-707. (65) Prior Publication Data Ko, F. H., & Monbouquette, H. G., Photometric and Electrochemi cal Enzyme-Multiplied Assay Techniques Using f-Galactosidase as US 2015/OO83612 A1 Mar. 26, 2015 Reporter Enzyme. Biotechnology Progress, 2006, 22(3), 860-865. Luo, L.-Q. et al., “Derivative voltammetric direct simultaneous determination of nitrophenol isomers at a carbon nanotube modified Related U.S. Application Data electrode'. Sensors and Actuators B: Chemical, 2008, 135(1), 61-65. (60) Provisional application No. 61/880,277, filed on Sep. Mathiyarasu, J. et al., “Electrochemical detection of phenol in 20, 2013. aqueous solutions'. Indian Journal of Chemical Technology, vol. 11, Nov. 2004, p. 797-803. (51) Int. Cl. Nematollahi. D. et al., “Cyclic Voltammetric Study of the Oxidation CI2O I/34 (2006.01) of Catechols in the Presence of Cyanide Ion'. Electroanalysis 2004, CI2O I/68 (2006.01) 16(16), p. 1359–1365. (52) U.S. Cl. Ni, Y. et al., “Simultaneous determination of nitrobenzene and CPC ...... CI2O 1/6897 (2013.01): CI2O I/34 nitro-substituted phenols by differential pulse voltammetry and (2013.01); G0IN 2333/938 (2013.01); G0IN chemometrics”. Analytica Chimica Acta 431 (2001), 101-113. 2333/942 (2013.01); G0IN 2458/30 (2013.01) (Continued) (58) Field of Classification Search None Primary Examiner — Suzanne M Noakes See application file for complete search history. Assistant Examiner — Jae W Lee (74) Attorney, Agent, or Firm — Bereskin & Parr (56) References Cited LLP/S.E.N.C.R.L., S.r.l.; Micheline Gravelle U.S. PATENT DOCUMENTS (57) ABSTRACT 5,196,306 A 3, 1993 Bobrow et al. Methods that may be used for the electrochemical detection 5,409,583 A 4/1995 Yoshioka et al. of multiple parameters, including chemical compounds. 6,391,549 B1 5, 2002 Ron et al. Further provided are cells that may be used in the electro 6,750,033 B2 6/2004 LeJeune et al. chemical detection of multiple parameters, including chemi 7,041,209 B1 5, 2006 Pizzariello et al. 7,422,892 B2 9, 2008 LeJeune et al. cal compounds, as well as a kit for the electrochemical 7,704,745 B2 4/2010 Baudenbacher et al. detection of multiple parameters, including chemical com 8,702.959 B2 * 4/2014 Shacham pounds. Diamand ...... GON 27.403 204,403.01 4 Claims, 13 Drawing Sheets US 9,689,046 B2 Page 2

(56) References Cited

OTHER PUBLICATIONS Sripriya, R. et al., “Voltammetric analysis of hydroquinone, ascorbic acid, nitrobenzene and benzyl chloride in aqueous, non-aqueous, micellar and microemulsion media'. Colloid and Polymer Science (2006) 285(1), 39-48. Togo, C. A. et al., “Novel detection of Escherichia coli B-d- glucuronidase activity using a microbially-modified glassy carbon electrode and its potential for faecal pollution monitoring'. Bio technology Letters, (2007) 29(4), 531-537. Tomasic, J. and D. Keglevic, “The Kinetics of Hydrolysis of Synthetic Glucuronic Esters and Glucuronic Ethers by Bovine Liver and Escherichia coli B-Glucuronidase'. Biochem. J. (1973) 133, 789-795 Vijayan, M. & Krishnan, V. (1995). “Electrocatalytic oxidation of hydroquinone on a polypyrrole-coated glassy carbon electrode'. Electroanalysis, 1995, 7(2), 197-198. Yang, Y-L. et al., “Amperometric Determination of 4-Nitrophenol at Multi-Walled Carbon Nanotube-Poly (Diphenylamine) Composite Modified Glassy Carbon Electrode'. Int. J. Electrochem. Sci. 6 (2011) 3902-3912. Yoshimura, F. and H. Nikaido, “Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic Solutes'. Journal of Bacteriology, J. Bacteriology, vol. 152, 1982, p. 636-642. * cited by examiner U.S. Patent Jun. 27, 2017 Sheet 1 of 13 US 9,689,046 B2

HO , -(-)-oh PNPG PDPG

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HO-(S)- NO Ho-KY-OH PNP PDP

FIGURE 1. U.S. Patent Jun. 27, 2017 Sheet 2 of 13 US 9,689,046 B2

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FIGURE 2 U.S. Patent Jun. 27, 2017 Sheet 3 of 13 US 9,689,046 B2

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FIGURE 3 U.S. Patent Jun. 27, 2017 Sheet 4 of 13 US 9,689,046 B2

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FIGURES U.S. Patent Jun. 27, 2017 Sheet 6 of 13 US 9,689,046 B2

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FIGURE 6 U.S. Patent Jun. 27, 2017 Sheet 7 of 13 US 9,689,046 B2

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U.S. Patent Jun. 27, 2017 Sheet 9 of 13 US 9,689,046 B2

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(Wrd) US 9,689,046 B2 1. 2 SYSTEMAND METHODS FOR THE The reporter gene based methodologies for the detection DETECTION OF MULTIPLE CHEMICAL of chemical compounds known to the prior art, including COMPOUNDS those provided by U.S. Pat. No. 6,391,549, are unsuitable for use in instances where it is desirable that multiple chemical RELATED APPLICATION compounds are detected in an assay sample. There exists therefore in the art a need for improved methods for the This application claims benefit of 35. U.S.C. 119 based on detection of chemical compounds. priority of U.S. Provisional Patent Application No. 61/880, 277 filed on Sep. 20, 2013, which is incorporated herein in SUMMARY OF THE DISCLOSURE its entirety. 10 The following paragraphs are intended to introduce the INCORPORATION OF SEQUENCE LISTING reader to the more detailed description that follows and not to define or limited the claimed subject matter of the present A computer readable form of the Sequence Listing disclosure. “21806-P44849US01 SequenceListing..txt” (36,864 bytes), 15 The present disclosure relates to methods for the detection submitted via EFS-WEB and amended on Oct. 23, 2014, is of multiple parameters in a liquid medium. Accordingly, the herein incorporated by reference. present disclosure provides, in at least one aspect, at least one embodiment of a method for simultaneously detecting FIELD OF THE DISCLOSURE two parameters in a liquid medium comprising: (i) providing a liquid medium comprising a first and The present disclosure relates to methods for the detection second parameter; of chemical compounds. More particularly, the present dis (ii) contacting in a vessel the liquid medium with cells and closure relates to methods for detecting chemical com a first and second Substrate to form an assay mixture, pounds using electrochemical means. 25 wherein the first substrate comprises a first electroac tive analyte chemically linked to a first carrier and the BACKGROUND OF THE DISCLOSURE second Substrate comprises a second electroactive ana lyte chemically linked to a second carrier; and wherein The following paragraphs are provided by way of back the cells comprise a chimeric nucleic acid construct ground to the present disclosure. They are not however an 30 comprising in the 5' to 3’ direction of translation as admission that anything discussed therein is prior art or part operably linked components a first nucleic acid con of the knowledge of persons skilled in the art. struct comprising: The reliable and rapid detection of chemical compounds (a) a first promoter inducible by the first parameter; and in a liquid medium is desirable in many industrial processes, (b) a nucleic acid sequence encoding a first reporter and detection methodologies uniquely relating a chemical 35 property of a compound requiring detection to a detectable polypeptide comprising a first hydrolase capable of output signal are manifold. Recently, methods involving the hydrolyzing the first substrate and releasing the first use of reporter genes have been developed to detect the electroactive analyte from the carrier; and presence of chemical compounds. For example, the method a second nucleic acid construct comprising: disclosed in U.S. Pat. No. 6,391,549 permits the detection of 40 (c) a second promoter inducible by the second param a chemical compound using an electrochemical cell and a eter, and reporter polypeptide mediating the production of an electri (d) a nucleic acid sequence encoding a second poly cally active compound. The method may be used to assay a peptide comprising a second hydrolase capable of single sample, or alternatively multiple samples may be hydrolyzing the second Substrate and releasing the assayed simultaneously. However the method is impractical 45 second electroactive analyte from the carrier; where multiple chemical compounds require detection. In (iii) detecting an electrical signal facilitated by the first particular, in order to detect multiple chemical compounds, and second electroactive analyte in the assay mixture, in accordance with the specification of U.S. Pat. No. 6,391, wherein detecting the electrical signal facilitated by the 549, an electrical cell would be inserted in a first sample to first and second electroactive analyte detects the first detect a chemical compound, an assay result would be 50 and second parameters in the liquid medium. obtained, and then the electrical cell would be removed from In another aspect, the present disclosure provides a cell the sample, cleaned and recalibrated for assaying for a comprising a chimeric nucleic acid sequence comprising in second and any Subsequent chemical compounds. Alterna the 5' to 3' direction of translation as operably linked tively, two or more electrical cells are used in order to detect components a first nucleic acid construct comprising: two or more chemical compounds. Thus, one of the draw 55 (a) a first promoter operable in the cell; and backs of the methods disclosed by U.S. Pat. No. 6,391,549, (b) a nucleic acid sequence encoding a first reporter is that in order to detect the presence of multiple chemical polypeptide comprising a first hydrolase capable of compounds in an assay sample, it is required that either hydrolyzing a first Substrate comprising a first electro multiple assay samples are available, and, furthermore, that active analyte and a first carrier to release the first multiple electrochemical cells are employed, or that multiple 60 electroactive analyte from the carrier; and non-simultaneous measurements are conducted. The former a second nucleic acid construct comprising: requires the presence of Sufficient assay sample, and, more (c) a second promoter operable in the cell; and over, requires a complex and impractical electrical compo (d) a nucleic acid sequence encoding a second reporter nent, in particular where a substantial number of different polypeptide comprising a second hydrolase capable of chemical compounds require detection. The latter is a time 65 hydrolyzing a second Substrate comprising a second consuming methodology, and potentially more prone to electroactive analyte and a second carrier to release the operator error. second electroactive analyte from the carrier. US 9,689,046 B2 3 4 In a preferred embodiment, the first promoter is an Other features and advantages of the present disclosure inducible promoter. In a further preferred embodiment, the will become apparent from the following detailed descrip first and the second promoter are inducible promoters. tion. It should be understood, however, that the detailed In yet a further aspect, the present disclosure provides a description, while indicating preferred implementations of mixture comprising two cells, the first cell comprising a first 5 the disclosure, are given by way of illustration only, since chimeric nucleic acid sequence comprising in the 5' to 3' various changes and modifications within the spirit and direction of translation as operably linked components a first scope of the disclosure will become apparent to those of skill nucleic acid construct comprising: in the art from the detailed description (a) a first promoter operable in the first cell; and (b) a nucleic acid sequence encoding a first reporter 10 BRIEF DESCRIPTION OF THE DRAWINGS polypeptide comprising a first hydrolase capable of hydrolyzing a first Substrate comprising a first electro The disclosure is in the hereinafter provided paragraphs active analyte and a first carrier to release the first described in relation to its Figures. The Figures provided electroactive analyte from the carrier; and herein are provided for illustration purposes and are not the second cell comprising a second chimeric nucleic acid 15 intended to limit the present disclosure. sequence comprising in the 5' to 3' direction of translation as FIG. 1 depicts the chemical formulas of certain substrates operably linked components a first nucleic acid construct and conversion thereof into electroactive analytes, notably comprising: the conversion of chlorophenol red-f-D galactopyranoside (c) a second promoter operable in the second cell; and (CPRG) to chlorophenol red (CPR), para-nitrophenol-f-D- (d) a nucleic acid sequence encoding a second reporter 20 glucuronide (PNPG) to para-nitrophenol (PNP), para-diphe polypeptide comprising a second hydrolase capable of nol-3-D-glucopyranoside (PDPG) to para-dihydroxyphenol hydrolyzing a second Substrate comprising a second (PDP). electroactive analyte and a second carrier to release the FIG. 2 depicts a graph of a chronoamperometric mea second electroactive analyte from the carrier. surement of the enzymatic conversion of the CPRG to CPR. In yet a further aspect, the present disclosure relates to a 25 FIG. 3 depicts a graph of a chronoamperometric mea method of detecting a first and second electroactive analyte surement of the enzymatic conversion of PDPG to PDP. in a liquid medium, the method comprising: FIG. 4 depicts a graph of a chronoamperometric mea (i) contacting in a vessel the liquid medium and (a) a first surement of the enzymatic conversion of PNPG to PNP. Substrate comprising a first electroactive analyte FIG. 5 depicts a Voltammogram simultaneously measur chemically linked to a first carrier; (b) a second sub- 30 ing the in vivo enzymatic conversion of CPRG to CPR and strate comprising a second electroactive analyte chemi PDPG to PDP. cally linked to a second carrier; (c) a first reporter FIG. 6 depicts a voltammogram simultaneously measur polypeptide comprising a first hydrolase; and (d) a ing the in vivo enzymatic conversion of PDPG to PDP, and second reporter polypeptide comprising a second PNPG to PNP. hydrolase, to form an assay mixture, wherein the first 35 FIG. 7 depicts a Voltammogram, showing simultaneous hydrolase is capable of hydrolyzing the first substrate detection of PDP PNP and CPR. Arrows indicate the pres and the second hydrolase is capable of hydrolyzing the ence at unique Voltages of each of the electroactive analytes. second substrate to release the first and second sub FIG. 8 depicts a voltammogram measuring the in vivo strate from the first and second carrier; and enzymatic conversion of PDPG to PDP following induction (ii) detecting an electrical signal facilitated by the first and 40 off-glucuronidase expression in E. coli. Various Sweeps are second electroactive analyte in the assay mixture, shown. The ten minute (black), twenty minute (blue), and wherein detecting the electrical signal facilitated by the thirty minute (red) sweeps show the growth of a PDP peak first and second electroactive analyte detects the first in both maximum current and charge over time. and second parameters in the liquid medium. FIG. 9 depicts a voltammogram measuring the in vivo In yet another aspect, the present disclosure also relates to 45 enzymatic conversion of PAPG to PAP following induction a diagnostic kit and provides at least one embodiment of a of B-galactosidase expression in E. coli. Various Sweeps are diagnostic kit for simultaneously detecting two parameters shown. The baseline (black), ten minute (red), twenty min comprising: ute (blue), and thirty minute (green) Sweeps show the growth (i) a cell comprising a chimeric nucleic acid sequence of a PAP peak in both maximum current and charge over comprising in the 5' to 3' direction of translation as 50 time. operably linked components a first nucleic acid con FIG. 10 depicts a Voltammogram simultaneously measur struct comprising: ing the in vivo enzymatic conversion of PAPG to PAP (a) a first promoter operable in the cell; and following induction of B-galactosidase expression and (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of PNPG to PNP following induction of B-glucuronidase hydrolyzing a first Substrate comprising a first elec 55 expression in E. coli. The baseline (black), ten minutes troactive analyte and a first carrier and releasing the (blue), twenty minutes (green), and thirty minutes (red) first electroactive analyte from the carrier; and sweeps show the growth of both PDP and PAP peaks in both a second nucleic acid construct comprising: maximum current and charge over time. (c) a second promoter operable in the cell; and FIG. 11 depicts a differential pulse voltammogram simul (d) a nucleic acid sequence encoding a second reporter 60 taneously measuring the in vivo enzymatic conversion of polypeptide comprising a second hydrolase capable PAPG to PAP following induction of B-galactosidase expres of hydrolyzing a second Substrate comprising a sec sion and PNPG to PNP following induction of B-glucuroni ond electroactive analyte and a second carrier and dase expression in E. coli. The ten minute (black), twenty releasing the second electroactive analyte from the minute (blue), and thirty minute (red) minute Sweeps are carrier, 65 displayed to demonstrate the growth of both PDP and PAP (ii) a first and second Substrate; and reduction peaks in both maximum current and charge over (iii) instructions for use or storage of the kit. time. US 9,689,046 B2 5 6 FIG. 12 depicts a graph showing the maximum current in use of multiple reporter genes capable of facilitating the the oxidation peak of PAP calculated and plotted as a generation of separate electrical signals in response to the function of time. Compared are E. coli cells induced to presence of two or more parameters in a single vessel using produce B-galactosidase and convert PAPG to PAP versus a single electrical cell. Thus a limited amount of assay non-induced E. coli cells. Induced cells are represented by sample is required in order to conduct the assay, and the the blue diamonds and non-induced cells are represented by electrical signal detection component of the assay is straight the red squares. forward and inexpensive to manufacture and operate. Fur FIG. 13 depicts a Voltammogram showing baseline pro thermore the Source assay materials required for the assays duction of PAP in E. coli comprising an inducible promoter are inexpensive and readily available, rendering the assays under non-induced conditions. The ten (black), twenty 10 useful for implementation in a wide range of commercial (blue), and thirty (red) minute Sweeps are displayed to applications. demonstrate the minimal growth of a PAP peak in both Accordingly, the present disclosure provides, in at least maximum current and charge over time. one aspect, at least one embodiment of a method for simultaneously detecting two parameters in a liquid medium DETAILED DESCRIPTION OF THE 15 comprising: DISCLOSURE (i) providing a liquid medium comprising a first and second parameter; Various compositions or processes will be described (ii) contacting in a vessel the liquid medium with cells and below to provide an example of an embodiment of each a first and second Substrate to form an assay mixture, claimed subject matter. No embodiment described below wherein the first substrate comprises a first electroac limits any claimed Subject matter and any claimed subject tive analyte chemically linked to a first carrier and the matter may cover processes, compositions or systems that second Substrate comprises a second electroactive ana differ from those described below. The claimed subject lyte chemically linked to a second carrier; and wherein matter is not limited to compositions or processes having all the cells comprise a chimeric nucleic acid construct of the features of any one composition, system or process 25 comprising in the 5' to 3’ direction of translation as described below or to features common to multiple or all of operably linked components a first nucleic acid con the compositions, systems or processes described below. It struct comprising: is possible that a composition, system or process described (a) a first promoter inducible by the first parameter; and below is not an embodiment of any claimed Subject matter. (b) a nucleic acid sequence encoding a first reporter Any subject matter disclosed in a composition, system or 30 polypeptide comprising a first hydrolase capable of process described below that is not claimed in this document hydrolyzing the first substrate and releasing the first may be the subject matter of another protective instrument, electroactive analyte from the carrier; and for example, a continuing patent application, and the appli a second nucleic acid construct comprising: cants, inventors or owners do not intend to abandon, dis (c) a second promoter inducible by the second param claim or dedicate to the public any such subject matter by its 35 eter, and disclosure in this document. (d) a nucleic acid sequence encoding a second reporter It should be noted that terms of degree such as “substan polypeptide comprising a second hydrolase capable tially”, “about and “approximately' as used herein mean a of hydrolyzing the second Substrate and releasing reasonable amount of deviation of the modified term such second electroactive analyte from the carrier; that the end result is not significantly changed. These terms 40 (iii) detecting an electrical signal facilitated by the first of degree should be construed as including a deviation of the and second electroactive analyte in the assay mixture, modified term if this deviation would not negate the mean wherein detecting the electrical signal facilitated by the ing of the term it modifies. first and second electroactive analyte detects the first As used herein, the wording “and/or” is intended to and second parameters in the liquid medium. represent an inclusive-or. That is, “X and/or Y” is intended 45 to mean X or Y or both, for example. As a further example, DEFINITIONS “X, Y, and/or Z is intended to mean X or Y or Z or any combination thereof. Unless defined otherwise, all technical and scientific All patents and patent applications, and other publica terms used herein have the same meaning as commonly tions, cited herein, whether Supra or infra, including nucleic 50 understood by one of ordinary skill in the art to which the acid and polypeptide sequences from GenBank, SwissPro disclosure pertains. and other databases, are hereby incorporated by reference in The term “carrier as used herein refers to a compound their entirety, where permitted. which can be chemically linked to an electroactive analyte It is further noted that, as used in this specification and the and when linked thereto can be hydrolyzed, e.g. by a appended claims, the singular forms “a”, “an', and “the 55 hydrolase, to form upon hydrolysis an electroactive analyte include plural referents unless the context clearly dictates and the carrier. otherwise. The term “electroactive analyte' as used herein refers to As hereinbefore mentioned, the present disclosure relates a compound capable of being reduced and/or oxidized when to methods for detecting multiple parameters in a liquid a Voltage is applied to an electrical cell and facilitate an sample. The herein provided methods represent a novel and 60 electrical current in such electrochemical cell. efficient means for detecting multiple parameters, including The term “hydrolase' as used herein refers to any and all the presence of multiple chemical compounds. The methods enzymes comprising a sequence of amino acid residues of the present disclosure are particularly advantageous in which is (i) substantially identical to the amino acid that they permit the simultaneous detection of two or more sequences constituting any hydrolase polypeptide set fort compounds in a single vessel using a single electrical cell. 65 herein, including, for example, SEQ.ID NO:2, SEQ. ID To the best of the inventors knowledge, the present disclo NO:4, SEQ.ID NO:6, or (ii) encoded by a nucleic acid Sure provides, for the first time, a methodology involving the sequence capable of hybridizing under at least moderately US 9,689,046 B2 7 8 stringent conditions to any nucleic acid sequence encoding selective hybridization between two complementary nucleic any hydrolase polypeptide set forth herein, but for the use of acid molecules in Solution. Hybridization may occur to all or synonymous codons. a portion of a nucleic acid sequence molecule. The hybrid The term “nucleic acid sequence' as used herein refers to izing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) a sequence of nucleoside or nucleotide monomers consisting nucleotides in length. Those skilled in the art will recognize of naturally occurring bases, Sugars and interSugar (back that the stability of a nucleic acid duplex, or hybrids, is bone) linkages. The term also includes modified or substi determined by the Tm, which in sodium containing buffers tuted sequences comprising non-naturally occurring mono is a function of the Sodium ion concentration and tempera mers or portions thereof. The nucleic acid sequences of the ture (Tm=81.5° C.-16.6 (Log 10 Na+I)+0.41 (% (G+C)- present disclosure may be deoxyribonucleic acid sequences 10 (DNA) or ribonucleic acid sequences (RNA) and may 600/1), or similar equation). Accordingly, the parameters in include naturally occurring bases including adenine, gua the wash conditions that determine hybrid stability are nine, cytosine, thymidine and uracil. The sequences may Sodium ion concentration and temperature. In order to also contain modified bases. Examples of Such modified identify molecules that are similar, but not identical, to a bases include aza and deaza adenine, guanine, cytosine, 15 known nucleic acid molecule a 1% mismatch may be thymidine and uracil, and Xanthine and hypoxanthine. assumed to result in about a 1° C. decrease in Tm, for The herein interchangeably used terms “nucleic acid example if nucleic acid molecules are sought that have a sequence encoding a hydrolase' and “nucleic acid sequence >95% identity, the final wash temperature will be reduced by encoding a hydrolase polypeptide', refer to any and all about 5° C. Based on these considerations those skilled in nucleic acid sequences encoding a hydrolase polypeptide, the art will be able to readily select appropriate hybridization including, for example, SEQ.ID NO:1, SEQ.ID NO:3 and conditions. In preferred embodiments, stringent hybridiza SEQ.ID NO:5. Nucleic acid sequences encoding a hydrolase tion conditions are selected. By way of example the follow polypeptide further include any and all nucleic acid ing conditions may be employed to achieve stringent hybrid sequences which (i) encode polypeptides that are Substan ization: hybridization at 5x sodium chloride/sodium citrate tially identical to the hydrolase polypeptide sequences set 25 (SSC)/5xDenhardt’s solution/1.0% SDS at Tm (based on the forth herein; or (ii) hybridize to any hydrolase nucleic acid above equation) -5°C., followed by a wash of 0.2xSSC/ sequences set forth herein under at least moderately strin 0.1% SDS at 60° C. Moderately stringent hybridization gent hybridization conditions or which would hybridize conditions include a washing step in 3xSSC at 42°C. It is thereto under at least moderately stringent conditions but for understood however that equivalent stringencies may be the use of synonymous codons. 30 achieved using alternative buffers, salts and temperatures. By the term “substantially identical it is meant that two Additional guidance regarding hybridization conditions may polypeptide sequences preferably are at least 70% identical, be found in: Current Protocols in Molecular Biology, John and more preferably are at least 85% identical and most Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et preferably at least 95% identical, for example 96%, 97%, al., Molecular Cloning, a Laboratory Manual, Cold Spring 98% or 99% identical. In order to determine the percentage 35 Harbor Laboratory Press, 1989, Vol. 3. of identity between two polypeptide sequences the amino The term "chimeric' as used herein in the context of acid sequences of Such two sequences are aligned, using for nucleic acid sequences refers to at least two linked nucleic example the alignment method of Needleman and Wunsch acid sequences which are not naturally linked. Chimeric (J. Mol. Biol., 1970, 48: 443), as revised by Smith and nucleic acid sequences include linked nucleic acid Waterman (Adv. Appl. Math., 1981, 2: 482) so that the 40 sequences of different natural origins. For example a nucleic highest order match is obtained between the two sequences acid sequence constituting a bacterial promoter linked to a and the number of identical amino acids is determined nucleic acid sequence encoding a non-bacterial hydrolase between the two sequences. Methods to calculate the per protein is considered chimeric. Chimeric nucleic acid centage identity between two amino acid sequences are sequences also may comprise nucleic acid sequences of the generally art recognized and include, for example, those 45 same natural origin, provided they are not naturally linked. described by Carillo and Lipton (SIAM J. Applied Math. For example a nucleic acid sequence constituting a promoter 1988, 48:1073) and those described in Computational obtained from a particular cell-type may be linked to a Molecular Biology, Lesk, e.d. Oxford University Press, New nucleic acid sequence encoding a polypeptide obtained from York, 1988, Biocomputing: Informatics and Genomics Pro that same cell-type, but not normally linked to the nucleic ects. Generally, computer programs will be employed for 50 acid sequence constituting the promoter, Chimeric nucleic Such calculations. Computer programs that may be used in acid sequences also include nucleic acid sequences com this regard include, but are not limited to, GCG (Devereux prising any naturally occurring nucleic acid sequence linked et al., Nucleic Acids Res., 1984, 12: 387) BLASTP to any non-naturally occurring nucleic acid sequence. BLASTN and FASTA (Altschulet al., J. Molec. Biol., 1990: The term “promoter as used herein means any nucleic 215:403). A particularly preferred method for determining 55 acid sequence element capable of conferring, effecting, the percentage identity between two polypeptides involves initiating, directing, or enhancing transcription of a nucleic the Clustal Walgorithm (Thompson, J. D. Higgines, D G and acid sequence element operably linked to the promoter, Gibson TJ, 1994, Nucleic Acid Res 22(22): 4673-4680 including, without limitation, SEQID NO:7, SEQ.ID NO:8, together with the BLOSUM 62 scoring matrix (Henikoff S SEQ.ID NO:9, SEQ.ID NO:10, SEQ.ID NO:11, SEQ.ID & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 60 NO:12, SEQ.ID NO:13, SEQID NO:14, SEQ.ID NO:15, 10915-10919 using a gap opening penalty of 10 and a gap SEQ.ID NO:16, SEQ.ID NO:17, SEQ.ID NO:18, and extension penalty of 0.1, so that the highest order match SEQ.ID NO:19. A promoter is generally located 5' (i.e. obtained between two sequences wherein at least 50% of the upstream) of the transcribed nucleic acid sequence element. total length of one of the two sequences is involved in the The transcribed nucleic acid sequence, in accordance here alignment. 65 with, corresponds with a coding region, and transcription By “at least moderately stringent hybridization condi thereof, in accordance herewith, effects the production of a tions' it is meant that conditions are selected which promote polypeptide. US 9,689,046 B2 10 The terms “inducible promoter” or “promoter inducible nitrophenol (also known as 4-nitrophenol) (PNP). Further by a parameter', as may be used interchangeably herein, specific suitable electroactive analytes include, without limi refer to a promoter which is activated by the presence of an tation, para-diphenol (also known as 1,4-diphenol and as exogenous stimulus, e.g. the presence of an exogenous hydroquinone) (PDP), ortho-nitrophenol (ONP), 5-bromo parameter. 4-chloro-3-indolyl-?3-D-galactopyranoside (X-GAL), chlo The term “constitutive promoter as used herein refers to rophenol-red (also known as 2-chloro-4-3-(-chloro-4-hy a promoter which provides transcription at a relatively droxyphenyl)-1,1-dioxobenzocoxathiol-3-ylphenol constant rate independent from exogenous stimuli. (CPR) bicarbazolyl), para-aminophenol (PAP), diazapam, The term “reporter polypeptide' as used herein is a ferrocyanide, nitrazepam, and papaverine. In preferred polypeptide that is used in order to mediate detection of a 10 embodiments of the present disclosure, the electroactive parameter. analytes PNP PDP, PAP and CPR are used. The term “parameter refers to the detectable existence of The first and second carrier as used herein are chemical a condition in a liquid medium, including, without limita compounds which when chemically linked to an electroac tion, temperature, light, or the presence of a chemical tive analyte are capable of being enzymatically hydrolyzed compound. 15 by a hydrolase. The first and second carrier selected may be General Implementation chemically identical or may be chemically distinct. Suitable In accordance with at least one embodiment of the present carriers that may be used in accordance herewith are any disclosure, one aspect of the methods herein provided independently selected compounds that can function as a involves contacting, preferably by mixing, in a liquid carrier, including any Sugars and derivatives thereof. In medium comprising two parameters, a first and second preferred embodiments, the Sugars used are glucose or a Substrate, the first Substrate comprising a first carrier and a derivative thereof, including any oligosaccharide glucose first electroactive analyte, and the second Substrate compris linked to another molecule, phospho-glucose, any glucose ing a second carrier and a second electroactive analyte. The 6-phosphate, glucose molecule modified with phosphate, electroactive analyte in accordance herewith is a chemical nitrate, or any other chemical group, or galactose or a compound which when free in a solution to which a voltage 25 derivative thereof, including polygalactose, Xylose, ribose, is applied, is capable of generating an electrical signal. fructose, maltose, Sucrose, chitin, any modified version of Moreover when an electroactive analyte is chemically linked these chemicals, any disaccharide, any oligosaccharide, any to the carrier and in Solution, the application of a Voltage to orientation of these Sugars D or L, any ketoses, aldose, any the solution does not generate a Substantive electrical signal. furanose or pyranose Sugar type of any of the aforemen In accordance herewith, the first and second electroactive 30 tioned Sugars. Further carriers that may be used are glycer analytes are non-identical chemical compounds. In further aldehyde, erythrose, threose, arabinose, lyxose, allose, particularly preferred embodiments, the first and second altrose, mannose, gulose, idose, and talose. In particularly electroactive analytes are selected such that they have dis preferred embodiments, the carriers used in accordance tinct redox properties. By the term “distinct redox proper herewith are B-glucopyranoside, B-galactopyranoside and ties it is meant that when a Voltage is applied to the assay 35 B-D-glucoronide. Further carriers that may be used are mixture in order to detect the electroactive analytes, as mannoses and derivatives thereof fructoses and derivatives hereinafter described. Such Voltage application results in the thereof; fucoses and derivatives thereof; xyloses and deriva generation of an electrical current through reduction and/or tives thereof rhamnoses and derivatives thereof, and chitin oxidation of the first and second electroactive analyte at and derivatives thereof. Sugar linkages may be any beta- or Voltages sufficiently separate from each other so that the 40 alpha-linkage, including beta-1-4, beta-1-6, alpha-1-4 and amperage generated by the first electroactive analyte can be alpha-1-6. detected without substantive interference by the amperage As hereinbefore mentioned, in accordance herewith the generated by the second electroactive analyte. Thus the first and second carrier and first and second electroactive difference in voltage at which the first and second electro analyte are chemically linked to form a first and second active analyte provide an amperage is at least from about 0.1 45 substrate. Preferred substrates used in accordance herewith Volt to about 0.5 Volt, more preferably at about 0.25 Volt to are chlorophenol red-f-D-galactopyranoside (CPRG), para about 0.5 Volt, and in certain preferred embodiments, the nitrophenol-f-D-glucuronide (PNPG), para-di-phenol-f-D- difference in voltage at which the first and second electro glucopyranoside (also known as arbutin) (PDPG) and para active analyte provide an amperage may be as low as 10 mV. aminophenol-f-galactopyranoside (PAPG). The substrate e.g. between 0.01 V and 0.02 V. Electroactive analytes are 50 may be added in any form to the liquid medium, for example further selected in Such a manner that they do not damage or in the form of a pure or Substantially pure compound. In react chemically with the electrode that is used. Suitable another embodiment, the substrate is produced by the cells electroactive analytes that may be used in accordance with of the present disclosure, for example by including in the the present disclosure are any electroactive analytes having chimeric nucleic acid sequence, one or more sequences a voltage range of approximately within +/-2 Volt when in 55 encoding polypeptides capable of mediating the production an aqueous solution. Suitable electroactive analytes include of the substrate in the cells. In a further embodiment in independently selected phenolic compounds; single, double accordance herewith, a first cell produces the Substrate, and or triple ring aromatic compounds; phosphate conjugated a second cell produces the hydrolase. compounds; chlorine or bromine Substituted compounds; In accordance herewith, the liquid medium is further ring compounds comprising a double bond; indole-based 60 mixed with a first reporter polypeptide comprising a first compounds, cyanide based compounds; metal compounds; hydrolase and second reporter polypeptide comprising a metal-conjugated organics (e.g. ferrocene); compounds with second hydrolase, wherein the first hydrolase is capable of oxidizable alcohol groups; Substituted naphthalene com hydrolyzing the first substrate and the second hydrolase is pounds; hydrazide based compounds, including 2-4-(meth capable of hydrolyzing the second substrate. It is preferable ylthio)phenyl)acetohydrazide; and triazole compounds and 65 that the hydrolases are independently selected in Such a their derivatives, including -(4-methyltiobenzyl)-1,2,4-triaz manner that the first hydrolase and the second hydrolase are ole-5-thione; and aromatic nitro compounds, Such as para specific for hydrolysis of the first and second substrate, US 9,689,046 B2 11 12 respectively. With term “specific’ it is meant that hydrolyis glucosidase, oligo-1,6-glucosidase, C-glucosidase, by the first hydrolase of the second substrate is less than 5% B-glucosidase, amylo-C-1,6-glucosidase, glucan-endo-1,3- (w/w), more preferably less than 1% (w/w) and most pref B-glucosidase, GDP-glucosidase, Sucrose-O-glucosidase, erably 0.1% or less, and that the hydrolysis by the second glucan 1.3-3-glucosidase, glucan endo-1,3-O-glucosidase, hydrolase of the first substrate is less than 5% (w/w), more glucan 1,6-O-glucosidase, glucan endo-1,2-3-glucosidase, preferably less than 1% (w/w) and most preferably 0.1% or Glucan 1.4-3-glucosidase, glucan endo-1,6-B-glucosidase, less. Preferred hydrolases are polypeptides capable of glucan 1.3-O-glucosidase, 6-phospho-B-glucosidase, steryl hydrolyzing the substrates CPRG, PNPG, PAPG and PDPG. B-glucosidase, 3-O-M-strictosidine B-glucosidase, manno Thus in preferred embodiments, the hydrolases are selected Syl-oligosaccharide glucosidase, protein-glucosylgalacto from a B-galactosidase, a B-D-glucuronidase and a B-D- 10 Sylhydroxylysine glucosidase, 2-deoxyglucosidase, glucosidase, and more preferably the hydrolases selected branched-dextran exo-1,2-O-glucosidase, amygdalin B-glu from the B-galactosidase having SEQ.ID. NO:2, the B-D- cosidase, prunasin B-glucosidase, vicianin B-glucosidase, glucoronidase having SEQ.ID. NO.4 and the f-D-glucosi oligoxyloglucan B-glycosidase, maltose-6'-phosphate glu dase, having SEQ.ID NO:6. As illustrated in FIG. 1, the cosidase, raucaffricine B-glucosidase, coniferin B-glucosi hydrolases -galactosidase having SEQ.ID. NO:2, B-D- 15 dase, thioglucosidase, 4-hydroxy-7-methoxy-3-oxo-3,4-di glucuronidase having SEQ.ID. NO:4 and B-D-glucosidase, hydro-2H-1,4-benzoxazin-2-yl glucoside B-D-glucosidase, having SEQ.ID NO:6. are capable of hydrolyzing CPRG, B-D-glucopyranosyl abscisate B-glucosidase, B-apiosyl-3- PNPG and PDGP, respectively. The first and second reporter glucosidase, and hesperidin 6-O-O-L-rhamnosyl-B-D-glu polypeptide, in addition to a first and second hydrolase cosidase; glucuronidases, including 3-glucuronidase, C-glu polypeptide, may comprise additional peptide sequence, for curonidase, hyaluronoglucuronidase, glucuronosyl example, a detection tag sequence, or the first and second disulfoglucosamine glucuronidase, glycyrrhizinate reporter polypeptide may be restricted to and consist of the B-glucuronidase, Xylan C-1,2-glucuronosidase, and baicalin first and second hydrolase. B-D-glucuronidase; glycosylases including DNA-3-methyl Further hydrolases that may be used in accordance here adenine glycosylase I, thymine-DNA glycosylase, DNA with include: agarases including C-agarase, and B-agarase; 25 deoxyinosine glycosylase, DNA-3-methyladenine amylases including C.-amylase, B-amylase, and isoamylase; glycosylase II, rRNA N-glycosylase, DNA-formamidopy arabinosidases including B-L-arabinosidase, and arabinan rimidine glycosylase, uracil-DNA glycosylase, and double endo-1,5-O-L-arabinosidase; facto-N-biosidase; carageen Stranded uracil-DNA glycosylase; heparanase; hexosamini ases including t-carrageenase, K-carrageenase, and W-carra dases, including B-N-hexosaminidase; idanases, including geenase; cellulases; cellobiosidases including, cellulose 1,4- 30 fucoidanase; idurunodases, including L-iduronidase; inu B-cellobiosidase (non-reducing end), and cellulose 1.4-3- lases; lactases; levanase; licheninase; dictyostelium cellobiosidase (reducing end); ceramidases, including lysozyme A: maltosidases, including glucan 1,6-O-isomalto glucosulceramidase, galactosylceramidase galactosylgalac sidase; maltotriosidases including dextran 1,6-O- toyslglucosylceramidase, glycosylceramidase, and endogly isomaltotriosidase, and glucan 1,4-O-maltotriohydrolase; cosylceramidase; chitinases; chitosanase; dextrinases, 35 maltotetraohydrolases, including glucan 1,4-O-maltotetrao including cyclomaltodextrinase, mycodextranase, and limit hydrolase; maltohexaosidases, including glucan 1,4-O- dextrinase; epimerases including UDP-N-acetylglu maltohexaosidase; maltohydrolases including glucan 1,4-O- cosamine 2-epimerase, and UDP-N,N'-diacetylbacil maltohydrolase; mannosidases including mannan 1,4- losamine 2-epimerase; fructofuranosidases, including mannobiosidase, ala endo-1,6-O-mannosidase, B-fructofuranosidase; fucosidases, including B-D-fucosi 40 mannosyl-oligosaccharide 12-O-mannosidase, and glyco dase, C-L-fucosidase, 1.2-O-L-fucosidase, 1.3-O-L-fucosi protein endo-C.-1.2-mannosidase; mannosidases, including dase, and 1,6-O-L-fucosidase; fructosidases, including fruc C-mannosidase, B-mannosidase, mannan 1.2-(1,3)-O-man tan B-fructosidase, fructan B-(2,1)-fructosidase, and fructan nosidase, mannan endo-1,4-B-mannosidase, mannosyl-oli B-(2,6)-fructosidase; furanosidases, including non-reducing gosaccharide 1,3-1,6-O-mannosidase, mannan exo-1,2-1,6- end C-L-arabinofuranosidase, and B-galactofuranosidase, 45 C-mannosidase, mannosylglycoprotein endo-B- non-reducing end B-L-arabinofuranosidase; galactosamini mannosidase, and 1,6-O-D-mannosidase; muramidases, dase, including O-acetyl-galactosamindase, B-acetyl-galac including peptidoglycan B-N-acetylmuramidase; nucleosi to seaminidase, endo-C-N-acetylgalactosaminidase, and dases, including N-methyl nucleosidase, purine nucleosi endogalactosaminidase; galactanases, including arabinoga dase, inosine nucleosidase, uridine nucleosidase, AMP lactan endo-3-1,4-galactanase, and galactan endo-B-1,3-ga 50 nucleosidase, NAD(+) nucleosidase, NAD(P)(+) nucleosi lactanase; galactosidases, including O-galactosidase, f-ga dase, adenosine nucleosidase, ribosylpyririnidine nucleosi lactosidase, 6-phospho-B-galactosidase, capsular dase, deoxyribodipyrimidine endonucleosidase, NMN polysaccharide endo-1,3-O-galactosidase, blood-group nucleosidase, adenosylhomocysteine nucleosidase, pyrimi Substance endo-1,4-B-galactosidase, keratan-sulfate endo-1, dine-5'-nucleotide nucleosidase, inosinate nucleosidase, 4-3-galactosidase, galactan endo-1,6-B-galactosidase, 55 1-methyladenosine nucleosidase, and methylthioadenosine galactan 1.3-3-galactosidase, blood group B linear chain nucleosidase; octulosonidases including 3-deoxy-2-octu C-1,3-galactosidase, and blood group B branched chain losonidase, and 3-deoxyoctulosonase; B-primeverosidase; C-1,3-galactosidase; galacturonases, including polygalactu B-porphyranase; pullulanases, including isopullulanase and ronase; galacturonidases, including galacturan 1,4-O-galac neopullulanase; quercitrinase; rhamnosidases, including turonidase, and exo-poly-O-galacturonosidase; protein 60 C-L-rhamnosidase, and B-L-rhamnosidase; Sialadases, O-GlcNAcase; glucanases, including endo-1.3(4)-3-gluca including exo-O-Sialadase, and endo-O-Sialidase; difructose nase, Xyloglucan-specific endo-B-1,4-glucanase, and Xylog anhydride synthase; trehalases, including C-O-trehalase, and lucan-specific exo-B-1,4-glucanase; glucosaminidases, C-O-phosphotrehalase; Xylanases, including endo-1,4-B-Xy including hyaluronglucosaminidase, Cl-N-acetyl-glucosa lanase, endo-1,3-B-Xylanase, Xylan-1,4-Xylosidase, Xylan mindase, Mannosyl-glycoprotein endo-B-N-acetylglu 65 1,3-B-Xylosidase, glucuronoarabinoxylan endo-1,4-B-Xy cosaminidase, and eXo-1,4-B-D-glucosaminidase, B-aspar lanase, and oligosaccharide reducing-end Xylanase; and still tyl-N-acetylglucosaminidase; glucosidases, including 1,4-O- further include hydrolases selected from mannosylglycerate US 9,689,046 B2 13 14 hydrolase 4-O-D-(1->4)-O-D-glucano)trehalose trehalohy including EC 3.9.1; EC 3.10, including EC 3.10.1; EC 3.11, drolase, rhamnogalacturonan hydrolase, futalosine hydro including EC 3.11.1; and EC 3.12, including EC 3.12.1. lase, poly(ADP-ribose) glycohydrolase, polymannuronate In preferred embodiments hydrolases are selected from hydrolase, oligoxyloglucan reducing-end-specific cellobio the class of enzymes belonging to EC 3.2.1 and 3.2.2, and hydrolase, unsaturated rhamnogalacturonyl hydrolaserham more preferably, EC 3.2.1.1, EC 3.2.1.2, EC 3.2.1.3, EC nogalacturonan galacturonohydrolase, levanbiohydrolases, 3.2.1.4, EC 3.2.1.6, EC 3.2.1.7, EC 3.2.1.8, EC 3.2.1.10, EC 2,6-B-fructan 6-levanbiohydrolase, C.-neoagaro-oligosac 3.2.1.11, EC 3.2.1.14, EC 3.2.1.15, EC 3.2.1.18, EC charide hydrolase, protein ADP-ribosylarginine hydrolase, 3.2.1.20, EC 3.2.1.21, EC 3.2.1.22, EC 3.2.1.23, EC ADP-ribosyl-dinitrogen reductase hydrolase, rhamnogalac 3.2.1.24, EC 3.2.1.25, EC 3.2.1.26, EC 3.2.1.28, EC turonan rhamnohydrolase, C-D-Xyloside Xylohydrolase, 10 3.2.1.3.1, EC 3.2.1.32, EC 3.2.1.33, EC 3.2.1.35, EC 3.2.1.36, EC 3.2.1.37, EC 3.2.1.38, EC 3.2.1.39, EC gellan tetrasaccharide unsaturated glucuronyl hydrolase, and 3.2.1.40, EC 3.2.1.41, EC 3.2.1.42, EC 3.2.1.2, EC 3.2.1.43, unsaturated chondroitin disaccharide hydrolase. EC 3.2.1.44, EC 3.2.1.45, EC 3.2.1.46, EC 3.2.1.47, EC Further hydrolases that may be used in accordance here 3.2.1.48, EC 3.2.1.49, EC 3.2.1.50, EC 3.2.1.51, EC with include carboxylesterase: arylesterase; triacylglycerol 15 3.2.1.52, EC 3.2.1.53, EC 3.2.1.54, EC 3.2.1.55, EC lipase; phospholipase A, lysophospholipase; acetylesterase; 3.2.1.56, EC 3.2.1.57, EC 3.2.1.58, EC 3.2.1.59, EC acetylcholinesterase; actylcholine acetylhydrolase; cholin 3.2.1.60, EC 3.2.1.61, EC 3.2.1.62, EC 3.2.1.63, EC esterase; tropinesterase; pectinesterase; sterol esterase; chlo 3.2.1.64, EC 3.2.1.65, EC 3.2.1.66, EC 3.2.1.67, EC rophyllase; thioester hydrolase including acetyl-CoA hydro 3.2.1.68, EC 3.2.1.70, EC 3.2.1.71, EC 3.2.1.72, EC lase; succinyl-CoA hydrolase; glutathione thiolesterase; 3.2.1.73, EC 3.2.1.74, EC 3.2.1.75, EC 3.2.1.76, EC choloyl-CoA hydrolase; phosphoric-monoester hydrolase; 3.2.1.77, EC 3.2.1.78, EC 3.2.1.80, EC 3.2.1.81, EC alkaline phosphatase; acid phosphatase; phosphoserine 3.2.1.82, EC 3.2.1.83, EC 3.2.1.84, EC 3.2.1.85, EC phosphatase; phosphatidate phosphatase, 5'-nucleotidase, 3.2.1.86, EC 3.2.1.87, EC 3.2.1.88, EC 3.2.1.89, EC 3'-nucleotidase; phytase; phosphatases including glucose 3.2.1.90, EC 3.2.1.91, EC 3.2.1.92, EC 3.2.1.93, EC phosphatase, tre-halose phosphatase, histidinol-phos 25 3.2.1.94, EC 3.2.1.95, EC 3.2.1.96, EC 3.2.1.97, EC phatase, phosphoprotein phosphatase, Sugar-phosphatase, 3.2.1.98, EC 3.2.1.99, EC 3.2.1.100, EC 3.2.1.101, EC inositol-phosphate phosphatase, 4-nitrophenylphosphatase, 3.2.1.102, EC 3.2.1.103, EC 3.2.1.104, EC 3.2.1.105, EC protein-tyrosine-phosphatase, Sorbitol-6-phosphatase; and 3.2.1.106, EC 3.2.1.107, EC 3.2.1.108, EC 3.2.1.109, EC pyridoxal phosphatase; phosphodiesterase I: phospholipase 3.2.1.110, EC 3.2.1.111, EC 3.2.1.112, EC 3.2.1.113, EC D; 3',5'-cyclic-nucleotide phosphodiesterase; dGTPase; sul 30 3.2.1.114, EC 3.2.1.115, EC 3.2.1.116, EC 3.2.1.117, EC fatases, including arylsulfatase, steryl-sulfatase, glycosulfa 3.2.1.118, EC 3.2.1.119, EC 3.2.1.120, EC 3.2.1.121, EC tase, choline-sulfatase, and iduronate-2-sulfatase; prenyl 3.2.1.122, EC 3.2.1.123, EC 3.2.1.124, EC 3.2.1.125, EC diphosphatase; Sclareol cyclase; ether hydrolase; 3.2.1.126, EC 3.2.1.127, EC 3.2.1.128, EC 3.2.1.129, EC adenosylhomocysteinase; cholesterol-5,6-oxide hydrolase; 3.2.1.130, EC 3.2.1.131, EC 3.2.1.132, EC 3.2.1.133, EC peptidases, including aminopeptidase, cystinyl peptidase, 35 3.2.1.134, EC 3.2.1.135, EC 3.2.1.136, EC 3.2.1.137, EC tripeptide aminopeptidase, prolyl aminopeptidase, glutamyl 3.2.1.138, EC 3.2.1.139, EC 3.2.1.140, EC 3.2.1.141, EC peptidase, bacterial leucyl aminopeptidase, clostridial 3.2.1.142, EC 3.2.1.143, EC 3.2.1.144, EC 3.2.1.145, EC aminopeptidase, cytosol alanyl aminopeptidase, tryptopha 3.2.1.146, EC 3.2.1.147, EC 3.2.1.149, EC 3.2.1.150, EC nyl aminopeptidase, methionyl aminopeptidase, asparty1 3.2.1.151, EC 3.2.1.152, EC 3.2.1.153, EC 3.2.1.154, EC aminopeptidase, dipeptidases, serine-type carboxypepti 40 3.2.1.155, EC 3.2.1.156, EC 3.2.1.157, EC 3.2.1.158, EC dases, and metallocarboxypeptidases; cathepsin, ubiquitinyl 3.2.1.159, EC 3.2.1.161, EC 3.2.1.162, EC 3.2.1.163, EC hydrolase; chymotrypsin, trypsin; thrombin; glutaminase; 3.2.1.164, EC 3.2.1.165, EC 3.2.1.166, EC 3.2.1.167, EC asparaginase; amidase; urease: arylformamidase; biotini 3.2.1.168, EC 3.2.1.169, EC 3.2.1.170, EC 3.2.1.171, EC dase; aminoacylase; nicotinamidase; glutaminase; pentana 3.2.1.172, EC 3.2.1.173, EC 3.2.1.174, EC 3.2.1.175, EC midase; barbiturase; lactamase; creatininase; enamidase; 45 3.2.1.176, EC 3.2.1.177, EC 3.2.1.178, EC 3.2.1.179, EC arginase; deaminases, including adenine deaminase, adenos 3.2.1.180, EC 3.2.1.181 EC 3.2.1.182, EC 3.2.1.183, EC ine deaminase, ADP deaminase, ATP deaminase, CMP 3.2.1.184, EC 3.2.1.185, EC 3.2.1n 1, EC 3.2.1n2, EC deaminase, CTP and deaminase guanosine deaminase; nit 3.2.1n2, EC 3.2.1.2; and EC 3.2.2.1, EC 3.2.2.2, EC 3.2.2.3, rilase: arylacetonitrilase; thiocyanate hydrolase; riboflavi EC 3.2.2.4, EC 3.2.2.5, EC 3.2.2.6, EC 3.2.2.7, EC 3.2.2.8, nase; inorganic diphosphatase; apyrase; nucleotide diphos 50 EC 3.2.2.9, EC 3.2.2.10, EC 3.2.2.11, EC 3.2.2.12, EC phatase, FAD diphosphatase; ADP-Sugar diphosphatase; 3.2.2.13, EC 3.2.2.14, EC 3.2.2.15, EC 3.2.2.16, EC thiamine-triphosphatase; UDP-Sugar diphosphatase; adeny 3.2.2.17, EC 3.2.2.18, EC 3.2.2.19, EC 3.2.2.20, EC lylsulfatase; alkylhalidase; haloacetate dehalogenase; atra 3.2.2.21 EC 3.2.2.22, EC 3.2.2.23, EC 3.2.2.24, EC Zine chlorohydrolase; phosphoamidase; cyclamate Sulfohy 3.2.2.25, EC 3.2.2.26, EC 3.2.2.27, EC 3.2.2.28, and EC drolase; phosphonoacetate hydrolase; and trithionate 55 3.22.29. hydrolase, In accordance herewith, cells comprising a chimeric con Still further hydrolases that may be used in accordance struct comprising a promoter and a nucleic acid sequence herewith are any hydrolases within the class of enzymes encoding a reporter polypeptide comprising a hydrolase are belonging to Enzyme Commission numbers 1 and 3, (EC1; prepared. Such cells may be any cells including, without EC3), and preferably those belonging to EC3. In more 60 limitation, any plant cells, animal cells, including human preferred embodiments enzymes belonging to EC 3.1 are cells, or cells obtainable from or obtained from a microor used, including those belonging to EC 3.1.1, EC 3.1.2, EC ganism, such as a bacterial cell or a fungal cell, e.g. 3.1.3, EC 3.1.4, EC 3.1.5, EC 3.1.6, and EC 3.17; EC 3.2: Saccharomyces cerevisiae. Bacterial cells that may be used EC 3.3, including EC 3.3.1, EC 3.4, EC 3.4.11, EC 3.4.13, include Escherichia coli cells, Bacillus cells, Pseudomonas EC 3.4.16, EC 3.4.17, EC 3.4.18; EC 3.5, including EC 65 cells, Clostridium cells, Xanthamonas cells and Staphyllo 3.5.2, EC 3.5.3, EC 3.5.4, EC 3.5.5, 3.5.99: EC 3.6, includ coccus cells. Nucleic acid sequences may be introduced in ing EC 3.6.1, EC 3.6.2: EC 3.8, including EC 3.8.1; EC 3.9, the cell through a host cell vector suitable for expression in US 9,689,046 B2 15 16 a host cell. The term "suitable for expression in a host cell induction by the chemical compound, which may be used to means that the host vector comprises genetic elements isolate the inducible promoter, or colonies may be used required to achieve expression of one or more polypeptides, directly. including the reporter polypeptide comprising a hydrolase, In further embodiments, the expression vector further encoded by the nucleic acid sequences introduced therein. 5 may comprise genetic elements required for the integration Genetic elements that may be included in the host cell vector of the vector or a portion thereof in the host cells genome, in this regard include a promoter, a transcriptional termina for example if a plant host cell is used the T-DNA left and tion region, one or more nucleic acid sequences encoding right border sequences which facilitate the integration into marker genes, one or more origins of replication and the like. the plant's nuclear genome. Pursuant to the present disclo Generally, promoters obtained from bacterial cells are used 10 when a bacterial host is selected in accordance herewith, Sure the expression vector may further contain a marker while a fungal promoter is used when a fungal host is gene. Marker genes that may be used in accordance with the selected, a plant promoter will be used when a plant cell is present disclosure include all genes that allow the distinction selected, and so on. of transformed cells from non-transformed cells, including In accordance herewith, a first and second promoter are 15 all selectable and Screenable marker genes. A marker gene selected, wherein the first and second promoter are inducible may be a resistance marker Such as an antibiotic resistance by the first and second parameter in the liquid medium. A marker against, for example, kanamycin or amplicillin, or an wide variety of existing or novel promoters may be used in auxotrophic marker. Screenable markers that may be this regard. In preferred embodiments, the promoter is employed to identify transformants through visual inspec selected to be inducible by an environmental stimulus, tion include B-glucuronidase (GUS) and green fluorescent including, without limitation: pH, the presence/absence of protein (GFP). One cell that particularly conveniently may an organic compound, presencefabsence of a toxic com be used is Escherichia coli. The preparation of the E. coli pound, pressure, light, Sound, and the like. In a preferred vectors may be accomplished using commonly known tech embodiment, a nucleic acid sequence comprising the lac7. niques such as restriction digestion, ligation, gel electropho promoter is used, and more preferably the nucleic acid 25 resis, DNA sequencing, the Polymerase Chain Reaction sequence comprising SEQ.ID. NO:7 is used. The lacZ (PCR) and other methodologies well known to those of skill promoter is induced by isopropyl-B-D-thiogalactopyrano in the art. A wide variety of cloning vectors is available to side (IPTG) present in the liquid medium. Other promoters perform the necessary steps required to prepare a recombi that may be used in accordance herewith include, any nant expression vector. Among the vectors with a replication synthetically produced or naturally occurring promoter 30 system functional in E. coli, are vectors such as pBR322, the including: lac promoter (also known as P), P. promoter pUC series of vectors, the M13 mp series of vectors, (rhamnose inducible, glucose repressible), tet promoter (P. pBluescript etc. Typically, these cloning vectors contain a tetracycline inducible), lambda cl promoter, P. promoter, marker allowing selection of transformed cells. Nucleic acid lux promoter (P.), leX promoter (P), P., promoter, sequences may be introduced in these vectors, and the omp promoter (P), cinL/cinR promoter, P (pH sensi 35 vectors may be introduced in E. coli by preparing competent tive promoter) and a UV sensitive promoter; Further pro cells, electroporation, transfection or using other well known moters include compound inducible promoters such as a methodologies to a person of skill in the art. E. coli may be copper sensitive promoter, including the cusR promoter grown in an appropriate medium, Such as Luria-Broth (P), and an iron sensitive promoter, including the feca medium and harvested. Recombinant expression vectors promoter (P.); further promoters that may be used include 40 may readily be recovered from cells upon harvesting and constitutive promoters a T7 promoter, O24 promoter, O28 lysing of the cells. Further, general guidance with respect to promoter, O32 promoter, O38 promoter, and O70 promoter the preparation of recombinant vectors and growth of (any sigma collection of promoter). Specific nucleic acid recombinant cells, including, without limitation, E. coli, sequences of promoters that may be used in accordance may be found in, for example: Sambrook et al., Molecular herewith include SEQ.ID NO:8, SEQ.ID NO:9, SEQ.ID 45 Cloning, a Laboratory Manual, Cold Spring Harbor Labo NO:10, SEQ.ID NO:11, SEQ.ID NO:12, SEQ.ID NO:13, ratory Press, 2012, Fourth Ed. SEQ.ID NO:14, SEQ.ID NO:15, SEQ.ID NO:16, SEQ.ID In another aspect, the present disclosure provides a cell NO:17, SEQ.ID NO:18 SEQID NO:19 SEQ.I.D. NO: 20 comprising a chimeric nucleic acid sequence comprising in and SEQ.ID NO: 21. the 5' to 3' direction of translation as operably linked Additional novel inducible promoters may be identified, 50 components a first nucleic acid construct comprising: for example by Screening a genetic library for Such promot (a) a first promoter operable in the cell; and ers. Thus, by way of example, in order to identify novel (b) a nucleic acid sequence encoding a first reporter promoters that respond to a chemical compound (e.g. a toxic polypeptide comprising a first hydrolase capable of chemical compound) a library (e.g. a transposon library or a hydrolyzing a first Substrate comprising a first electro phage library) may be created in an organism (e.g. bacterial 55 active analyte and a first carrier and releasing the first cells) known to metabolize the chemical compound. The electroactive analyte from the carrier; and transposon may be constructed to be a mobile genetic a second nucleic acid construct comprising: element containing a reporter gene (e.g. lac7), a selectable (c) a second promoter operable in the cell; and marker (e.g. antibiotic resistance marker Such as a tetracy (d) a nucleic acid sequence encoding a second reporter cline or ampicillin resistance marker), and the transposase 60 polypeptide comprising a second hydrolase capable of enzyme responsible for gene insertion. By inserting the hydrolyzing a second Substrate comprising a second transposon randomly into the organism's genome a genetic electroactive analyte and a second carrier and releasing library can be tested for inducible promoters. Isolated colo the second electroactive analyte from the carrier. nies, selected via the selectable marker cassette in the The first and second promoter may be a constitutive transposon, may be grown in the presence of the reporter 65 promoter or an inducible promoter. In a preferred embodi gene and the chemical compound. Colonies in which the ment, the first promoter is an inducible promoter. In a further reporter gene is expressed comprise a promoter capable of preferred embodiment the first and the second promoter are US 9,689,046 B2 17 18 inducible promoters. The cell may be an isolated or more or (ii) detecting the electrical signal facilitated by the first less pure cell or a cell cultured or Suspended in a medium, and second electroactive analyte in the assay mixture, Solution, or growth medium. wherein detecting the electrical signal facilitated by the In yet a further aspect, the present disclosure provides a first and second electroactive analyte detects the first mixture comprising two cells, the first cell comprising a first 5 and second parameters in the sample. chimeric nucleic acid sequence comprising in the 5' to 3' In order to detect the first and second parameter in the direction of translation as operably linked components a first sample, the electrical signal facilitated by the first and nucleic acid construct comprising: second electroactive analyte is detected and related to the (a) a first promoter operable in the first cell; and presence of the first and second parameter. By the term (b) a nucleic acid sequence encoding a first reporter 10 “facilitated it is meant that an electroactive analyte consti polypeptide comprising a first hydrolase capable of tutes a conduit element of an electrical circuit, Thus the flow or conduct of an electrical current in a sample, to which a hydrolyzing a first Substrate comprising a first carrier Voltage is applied, is attained via an electroactive analyte, and a first electroactive analyte and releasing the first released from a carrier, resulting in the reduction or oxida electroactive analyte from the carrier; and 15 tion of the electroactive analyte. the second cell comprising a second chimeric nucleic acid The electrical signal may be detected in accordance sequence comprising in the 5' to 3' direction of translation as herewith using any methodology involving the application operably linked components a first nucleic acid construct of a Voltage to the assay medium. The Voltage may be comprising: applied potentiostatically (i.e. at one Voltage) or in cyclic (c) a second promoter operable in the second cell; and Voltammetrical fashion (i.e. across a range of defined Volt (d) a nucleic acid sequence encoding a second reporter ages). This further includes any Voltamperometric method polypeptide comprising a second hydrolase capable of ology, including, without limitation, pulse Voltammetry, hydrolyzing a second Substrate comprising a second linear Sweep voltammetry, chromoamerometry, staircase carrier and a second electroactive analyte and releasing Voltammetry, and cyclical Voltammetry, and variations or the second electroactive analyte from the carrier. 25 adaptations thereof Such as differential pulse Voltammetry, The first and second cells may be similar or even identi or wave based Voltammetry with chronoamperometric steps cal, but for the presence of the chimeric nucleic acid included in the Sweeps. In accordance herewith, the appli sequences therein, thus the first and second cells may each cation of Voltage to the assay medium results in the oxida be E. coli cells, from the same or different strains. The cells tion or reduction of the electroactive analyte and gain or also may be a mixture of two cell cultures comprising two 30 release of electrons by the electroactive analyte or the different biological species. It will be clear to those of skill electrode, which can be measured amperometrically in the in the art that the mixture may comprise a plurality of cells form of a current. Voltages may be applied to a solution genetically identical to the first and second cell. versus any stable reference electrode, including, without Growth of the cells leads to production of the first and limitation, Ag/AgCl, saturated calomel electrode (SCE), second hydrolase. In one embodiment, the hydrolase poly 35 saturated sodium chloride calomel electrode (SSCE), and peptides may be recovered, isolated and separated from the reduction of hydrogen electrode (RHE), and the voltage other components of the cell by a variety of different protein is between the production of oxygen from water at the purification techniques including, e.g. ion-exchange chro positive end of the spectrum, and the production of hydrogen matography, size exclusion chromatography, affinity chro from water at the negative end. Voltages may be applied, for matography, hydrophobic interaction chromatography, 40 example, in the range from 0-2.0 V versus a reduction reverse phase chromatography, gel filtration, etc. Further hydrogen electrode (RHE electrode) reference electrode, or general guidance with respect to protein purification may for -1 Volt to +1 Volt against a pseudo Ag/AgCl electrode. example be found in: Cutler, P. Protein Purification Proto Amperages detected may range, for example, from 1 nA to cols, Humana Press, 2004, Second Ed. Thus a substantially 1 LA or more when a potentiostatic Voltage is applied, or pure protein may be obtained. By “substantially pure' it is 45 from about 1 nA to 10 JLA or more when using cyclic meant that the protein is separated from other host cell Voltammetry. Detection of the electrical signal in accordance components. In accordance here with the protein is at least with the herein disclosed methodologies involves the use of 95% pure, and more preferably at least 96%, 97%, 98% or a single electrochemical cell, which is introduced in a single 99% pure. Such substantially pure hydrolase protein may be vessel, e.g. a single flask, tube, beaker, well plate or any used to detect the presence of two or more electroactive 50 other receptacle of any geometry or shape, comprising the analytes. Accordingly, the present disclosure in a further assay mixture. The electrical cell generally comprises one or aspect includes an embodiment of a method of detecting a more working electrodes, a reference electrode, and a coun first and second electroactive analyte in a liquid medium, the ter electrode. Further generally included is a potentiostat to method comprising: control the electric cell. The composition of each of the (i) contacting in a vessel the liquid medium and (a) a first 55 electrodes in the electrochemical cell may vary, and depend Substrate comprising a first electroactive analyte ing on the composition of the reference electrode, the chemically linked to a first carrier; (b) a second sub Voltage measurements may change. The reference electrode strate comprising a second electroactive analyte chemi may be any electrode that holds a consistent Voltage when cally linked to a second carrier; (c) a first reporter placed into the electrochemical cell, and is suitably a polypeptide comprising a first hydrolase; and (d) sec 60 Ag/AgCl, saturated calomel electrode (SCE), or a saturated ond reporter polypeptide comprising a second hydro sodium chloride calomel electrode (SSCE). The counter lase, to form an assay mixture, wherein the first hydro electrode may, for example, be a gold or platinum electrode, lase is capable of hydrolyzing the first substrate and or a carbon electrode, e.g. a printed carbon, glassy carbon or releasing the first electroactive analyte from the carrier Vulcan carbon electrode. The composition of the working and the second hydrolase is capable of hydrolyzing the 65 electrode may vary in composition, and, for example, be second Substrate and releasing the second electroactive gold, platinum or carbon. Examples of preferred combina analyte from the carrier, and tions of electrodes include: gold working electrode, reduc US 9,689,046 B2 19 20 tion of hydrogen reference electrode, and a platinum counter a second nucleic acid construct comprising: electrode; glassy carbon working electrode, carbon counter (c) a second promoter operable in the cell; and electrode, and Ag/AgCl reference electrode; and platinum (d) a nucleic acid sequence encoding a second reporter working electrode, gold counter electrode, SCE reference polypeptide comprising a second hydrolase capable electrode. It is further advantageous that the electrode is of hydrolyzing a second Substrate comprising a sec coated e.g. by thiolate self-assembled monolayers on a metal ond electroactive analyte and a second carrier and Surface (e.g. gold), and/or protected, for example, a carbon releasing the second electroactive analyte from the electrode may be protected by applying phthalocyanine carrier; layer, by application of certain ions or metals, e.g. nickel, (ii) a first and second Substrate; and 10 (iii) instructions for use or storage of the kit. which may be dried on the electrode surface or platinum The kit may be provided in Such a manner that it com which may be plated on the surface. Combinations of the prises one or more vessels, e.g. flasks, beakers, tubes, well foregoing may also be applied. plates, or microfluidic based device. The kit further may also The electrochemical cell may be calibrated prior to initi be constructed in Such a manner that it contains two cells: (I) ating detection. This may conveniently be done using solu 15 a first cell comprising a chimeric nucleic acid sequence tions comprising the first and second electroactive analyte. comprising in the 5' to 3’ direction of translation as operably Voltage can be applied through various techniques, such as linked components a first nucleic acid construct comprising: cyclic Voltammetry, pulse Voltammetry, square wave Volta a first promoter operable in the cell; and a nucleic acid mmetry, potentiostatically or any other form of Voltampero sequence encoding a first reporter polypeptide comprising a metric methodologies and techniques. Amperages may be first hydrolase capable of hydrolyzing a first substrate and detected and obtained as desired, for example in the form of producing a first electroactive analyte; and (II) a chimeric a voltammogram, or chronoamperometrically. Thus, it will nucleic acid sequence comprising in the 5' to 3' direction of be clear to those of skill in the art, that in accordance with translation as operably linked components a second nucleic the present disclosure, the first and second parameter in the acid construct comprising a second promoter operable in the assay medium are detected by relating the electrical signal to 25 cell; and a nucleic acid sequence encoding a second reporter the presence of the first and second parameter in the assay polypeptide comprising a second hydrolase capable of mixture. As hereinbefore mentioned, voltages are preferably hydrolyzing a second Substrate and producing a second selected in accordance with the distinct redox properties of electroactive analyte. The kit further may further be fabri the electroactive analytes. In certain embodiments, an elec cated to comprise an electrochemical cell and/or a poten trical signal is only detected once per assay mixture. In other 30 tiostat. embodiments hereof, a plurality of electrical signals is Use of the Assays detected either at regular or irregular time intervals, or The liquid medium as used herein can be any liquid detection of the electrical signals may be conducted more or medium including any aqueous Solution, for example any less continuously. For example, where the method of the buffer or salt Solution, or any organic Solution. In certain present disclosure is used to detect a first and second 35 embodiments the medium may be an LB medium or more nutritional compound in a growth medium in order to preferably an M9 minimal medium which are commonly determine depletion thereof, repeated or continuous detec used to grow E. coli bacterial cells. The first and second tion may be desirable. In yet another embodiment, Samples parameters in the liquid medium are two distinct parameters, are drawn from a liquid medium to form the assay mixture. for example, light and temperature, or ionic strength and It is further noted that detection using the methods of the 40 temperature. In particularly preferred embodiments, the two present disclosure may be automated, and the electrochemi parameters are two distinct chemical compounds. These cal cell may be linked to a computer system to control the compounds may be any chemical compounds and may be electrochemical cell and to record and analyze the electrical obtainable from any source, including from any liquid, gas signal. or a Solid, as desired. In embodiments where the compounds Flow of an electrical current and detection thereof, upon 45 of interest are present in a liquid assay sample the com application of a Voltage to the assay medium, thus signals pounds may be detected directly in the liquid sample, the presence of the first and second parameter in the assay however where the compounds of interest are present in a sample. Conversely, the absence of an electrical signal, upon gaseous sample or Solid sample, the compounds are dis the application of a Voltage to the assay medium, is indica Solved in a liquid medium prior to initiating detection tive of the absence of the first and second parameter in the 50 thereof. As hereinbefore mentioned, the methods of the assay sample. In this manner, the detection of the electrical present disclosure permit the simultaneous detection of the signal, in accordance with the present disclosure, correlates presence of two parameters. It is noted however that the with and detects the first and second parameter. methodologies further permit the detection of a plurality of In yet another aspect, the present disclosure also relates to parameters, e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more parameters. a diagnostic kit and provides at least one embodiment of a 55 Thus when reference is made herein to a method involving diagnostic kit for simultaneously detecting two parameters the detection of a first and second parameter, it is intended comprising: that the methodology disclosed herein involves the detection (i) a cell comprising a chimeric nucleic acid sequence of at least two parameters. comprising in the 5' to 3’ direction of translation as In further preferred embodiments, a first and second toxic operably linked components a first nucleic acid con 60 compound found in the environment are detected, for struct comprising: example toxic compounds that may be present in e.g. in (a) a first promoter operable in the cell; and Surface water or sediments, including for example com (b) a nucleic acid sequence encoding a first reporter pounds found in tailings ponds associated with mining or polypeptide comprising a first hydrolase capable of fossil fuel production, or toxic compounds associated with hydrolyzing a first Substrate comprising a first elec 65 petroleum extraction, Such as hydrocarbons and naphthenic troactive analyte and a first carrier and releasing the acid, which may be released in the environment, for first electroactive analyte from the carrier; and example, as a result of a pipeline or fracking well leak. In US 9,689,046 B2 21 22 other embodiments, the chemical compounds are nutritional the current allowed to stabilize for 1200 seconds. After compounds used, e.g. nutritional compounds used by a stabilization the substrate PNPG was added to the solution microorganism culture, or toxins which accumulate in a (time Zero in FIG. 4) and the current produced at the working microorganism’s production system. It will be clear to those electrode was recorded. FIG. 4 provides the results obtained. of skill in the art that the methodologies herein provided are independent of the exact parameters, including chemical Example 3 Detection of PDP Using compounds, to be detected. The first and second parameter, Inducibly Expressed f-Glucosidase or the chemical formula of the first and second chemical compound does not matter and any chemical compound, or For the potentiostatic detection of PDP as a product of the any parameter for which the presence of a compound serves 10 cleavage of PDPG by B-glucosidase, a solution of 0.1M pH as a proxy, may be detected in accordance with the methods 7 sodium phosphate buffer was used. 20 mL of this solution provided by the instant disclosure. was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working EXAMPLES electrode, a platinum mesh as the counter electrode, and a 15 reduction of hydrogen reference electrode (RHE) immersed Hereinafter are provided examples of specific embodi in an identical Solution with constant hydrogen gas bubbling ments for performing the methods of the present disclosure, and connected via a luggin capillary. The buffer was bubbled as well as embodiments representing the compositions of the with nitrogen for 15 minutes prior to use. 1 mL of an present disclosure. The examples are provided for illustra overnight culture of TOP10 E. coli expressing native levels tive purposes only, and are not intended to limit the scope of of bglX when grown in rich media was pelleted and resus the present disclosure in any way. pended in 0.1M pH 7 phosphate buffer and then added to the electrochemical cell. The working electrode was then held at Example 1-Detection of CPR Using 0.825 V vs RHE and the current allowed to Stabilize for 1200 Constitutively Expressed B-Galactosidase seconds. After stabilization the substrate PDPG was added 25 to the solution (time Zero in FIG. 3) and the current produced For the potentiostatic detection of CPR as a product of the at the working electrode was recorded. FIG. 3 shows the cleavage of CPRG by B-galactosidase, a solution of 0.1M results obtained. pH 7 sodium phosphate buffer was used. 20 mL of this Solution was placed into an electrochemical cell with a Example 4-Simultaneous screen printed carbon electrode (Pine Instruments, USA) as 30 Detection of PDP and CPR the working electrode, a platinum mesh as the counter electrode, and a reduction of hydrogen reference electrode For the simultaneous voltammetric detection of PDP and (RHE) immersed in an identical solution with constant CPR at once as a product of the cleavage of PDPG and hydrogen gas bubbling and connected via a luggin capillary. CPRG by B-glucosidase and B-galactosidase respectively, a The buffer was bubbled with nitrogen for 15 minutes prior 35 solution of 0.1M pH 7 sodium phosphate buffer was used. 15 to use. 1 mL of an overnight culture of Pseudomonas mL of this solution was placed into an electrochemical cell fluorescens PFS constitutively expressing lacZ via a trans with a screen printed carbon electrode (Pine Instruments, poson insertion was pelleted and resuspended in 0.1M pH 7 USA) as the working electrode, counter electrode, and a phosphate buffer and then added to the electrochemical cell. screen printed Ag/AgCl pseudo-reference electrode The working electrode was then held at 1.325V vs RHE and 40 immersed in the same solution. The buffer was bubbled with the current allowed to stabilize for 1200 seconds. After nitrogen for 15 minutes prior to use. 2 mL of an overnight stabilization the substrate CPRG was added to the solution culture of BL-21 E. coli was pelleted and resuspended in (time Zero in FIG. 2) and the current produced at the working 0.1M pH 7 phosphate buffer and then added to the electro electrode was recorded. FIG. 2 provides the results obtained. chemical cell. PDPG and CPRG were added to the Solution 45 and the working electrode was then swept between -1 Vand Example 2 Detection of PNP Using 1 V vs the Ag/AgCl reference at 100 mV/s for 100 cycles. Constitutively Expressed B-Glucuronidase Six cycles 800 seconds apart, from the initial start of the Sweep considering it to be the baseline (0 seconds), are For the potentiostatic detection of PNP as a product of the reported in FIG. 5. Oxidation of PDP and CPR are noted cleavage of PNPG by B-glucuronidase, a solution of 0.1M 50 (-0.2 and 0.4V respectively) as shown by the increase of pH 7 sodium phosphate buffer was used. 20 mL of this peak heights after each Voltammetric Sweep. Solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as Example 5. Simultaneous the working electrode, a platinum mesh as the counter Detection of PDP and PNP electrode, and a reduction of hydrogen reference electrode 55 (RHE) immersed in an identical solution with constant For the simultaneous in vivo voltammetric detection of hydrogen gas bubbling and connected via a luggin capillary. PDP and PNP at once as a product of the cleavage of PDPG The buffer was bubbled with nitrogen for 15 minutes prior and PNPG by B-glucosidase and B-glucuronidase respec to use. 1 mL of an overnight culture of TOP10 E. coli tively, a solution of 0.1M pH 7 sodium phosphate buffer was expressing uidA under the control of the Lacl repressible 60 used. 15 mL of this solution was placed into an electro promoter on a high copy number plasmid (PSB1C3) was chemical cell with a screen printed carbon electrode (Pine pelleted and resuspended in 0.1M pH 7 phosphate buffer and Instruments, USA) as the working electrode, counter elec then added to the electrochemical cell. One sample was trode, and a screen printed Ag/AgCl pseudo-reference elec treated with 500 uM IPTG during the overnight culture, trode immersed in the same solution. The buffer was while the second was grown without induction to assess 65 bubbled with nitrogen for 15 minutes prior to use. 2 mL of basal (leaky) expression in the genetic circuit. The working an overnight culture of BL-21 E. coli with a plasmid electrode was then held at 1.6 V vs RHE for each sample and harbouring the uidA gene under the control of an uninduced US 9,689,046 B2 23 24 LacI responsive promoter was pelleted and resuspended in reference electrode at 100 mV/s for 3 cycles and the third 0.1M pH 7 phosphate buffer and then added to the electro cycle was plotted (the baseline was extended to 0.6 V to chemical cell. PDPG and PNPG were added to the Solution check for additional Voltammogram features). A voltammo and the working electrode was then swept between -1 Vand gram showing the results is provided in FIG. 9. 1 V vs the Ag/AgCl reference at 100 mV/s for 100 cycles. Six cycles 800 seconds apart, from the initial start of the Example 9. Simultaneous Detection of PDP and Sweep considering it to be the baseline (0 seconds), are PAP Using Inducibly Expressed f-galactosidase reported in FIG. 6. Oxidation of PDP and PNP are noted and Inducibly Expressed B-glucuronidase (-0.2 and 0.7V respectively) as shown by the increase of peak heights after each Sweep. 10 For the observation of both PDP and PAP produced from two inducible promoters translating the Lacz and UidA Example 6. Simultaneous Detection of proteins from E. coli (Pr and P. promoters, respec PDP, CPR and PNP tively), a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a For the simultaneous in vivo voltammetric detection of 15 glassy carbon electrode with a Pt mesh counter electrode and PDP, CPR, and PNP at once, a solution of 0.1M pH 7 sodium a Ag/AgCl reference. 2 mL of transformed cells were grown phosphate buffer was used. 15 mL of this solution was overnight (as previously described), induced with a genetic placed into an electrochemical cell with a screen printed circuit activating component specific for each transformed carbon electrode (Pine Instruments, USA) as the working gene (1 mM IPTG for the P promoter and 1% (w/v) electrode, counter electrode, and a screen printed Ag/AgCl rhamnose for the P promoter), and then pelleted and pseudo-reference electrode immersed in the same solution. rinsed in the phosphate buffer. The cells were subsequently The buffer was bubbled with nitrogen for 15 minutes prior added to a tube containing 5 mM PAPG and 5 mM PDPG, to use. 0.2 mL of 2 mM PDP and PNP was added to the and 50 uL was added every ten minutes to the electrochemi solution as well as 0.5 mL of 0.001% (w/v) CPR. After cal cell for thirty minutes. At each concentration, the work mixing the working electrode was then swept between -1 V 25 ing electrode was then swept from -0.3 V to 0.7 V vs the and 1 V vs the Ag/AgCl reference at 100 mV/s for 3 cycles. Ag/AgCl reference electrode at 100 mV/s for 3 cycles and Results are shown in FIG. 7. the third cycle was plotted. A voltammogram showing the results is provided in FIG. 10. Example 7 Detection of PDP Using Inducibly Expressed f-Glucuronidase 30 Example 10 Simultaneous Detection of PDP and PAP Using Inducibly Expressed f-galactosidase For the observation of PDP produced from an inducible and Inducibly Expressed B-glucuronidase by promoter translating the Lacz protein from E. coli (JM109 Differential Pulse Voltammetry K-12), a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a 35 For an alternate observation of PDP and PAP produced glassy carbon electrode with a Pt mesh counter electrode and from two inducible promoters translating the Lac Z and a Ag/AgCl reference. 2 mL of transformed cells (with UidA proteins from E. coli (P, and P. promoters, respec rhamnose promoter P, inducible uidA protein) were tively), a solution of 0.1 M pH 7 sodium phosphate buffer grown for 4 hours at 37 degrees, induced with a genetic was used. 10 mL of this solution was used to immerse a circuit activating component (1% w/v rhamnose) for 6 40 glassy carbon electrode with a Pt mesh counter electrode and hours, and then pelleted and rinsed in the phosphate buffer a Ag/AgCl reference. 2 mL of transformed cells were grown pH 7.0 three times. The cells were subsequently added to a overnight, induced with a genetic circuit activating compo tube containing 5 mM PDPG and 50 uL was added every ten nent (1 mM IPTG for the P promoter and 1% (w/v) minutes to the electrochemical cell for thirty minutes. At rhamnose for the P promoter) specific for each trans each concentration, the working electrode was then swept 45 formed gene, and then pelleted and rinsed in the phosphate from -0.3 V to 0.7 V vs the Ag/AgCl reference electrode at buffer. The cells were subsequently added to a tube contain 100 mV/s for 3 cycles and the third cycle was plotted. A ing 5 mM PAPG and 5 mM PDPG, and 50 uL was added voltammogram showing the results is provided in FIG. 8. every ten minutes to the electrochemical cell for thirty minutes. At each concentration, the working electrode was Example 8 Detection of PAP Using 50 held at 0.2 V vs Ag/AgCl for ten seconds and then pulsed Inducibly Expressed ?-Galactosidase negatively to -0.3 V vs Ag/AgCl using the differential pulse voltammetry method. The pulse height used was 5 mV for For the observation of PAP produced from an inducible 50 ms, with a step width of 10 mV for 100 ms. A differential promoter translating the Lacz protein from E. coli (DH5 C. pulse Voltammogram showing the results is provided in FIG. K-12) a solution of 0.1 M pH 7 sodium phosphate buffer was 55 11. used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Example 11—Comparison of Induced Ag/AgCl reference. 2 mL of transformed cells (with an Versus Non-Induced 3-galactosidase IPTG inducible promoter P inducible lacZ protein) were grown for 4 hours at 37 degrees, induced with a genetic 60 To compare the production of PAP from an inducible circuit activating component (1 mM final concentration bacteria translating the Lacz protein from E. coli under the IPTG) for 6 hours, and then pelleted and rinsed in the control of an inducible promoter, P., a solution of 0.1 M phosphate buffer pH 7.0 three times. The cells were subse pH 7 sodium phosphate buffer was used. 10 mL of this quently added to a tube containing 5 mM PAPG and 50 uL Solution was used to immerse a glassy carbon electrode with was added every ten minutes to the electrochemical cell for 65 a Pt mesh counter electrode and a Ag/AgCl reference, 2 mL thirty minutes. At each concentration, the working electrode of transformed cells were grown overnight, induced with a was then swept from -0.3 V to 0.5 V vs the Ag/AgCl genetic circuit activating component (1 mM IPTG) specific US 9,689,046 B2 25 26 for the transformed gene or left non-induced, and then SUMMARY OF SEQUENCES pelleted and rinsed in the phosphate buffer. The cells were subsequently added to a tube containing 5 mM PAPG, and SEQ.ID NO:1 and 2 set forth the nucleotide sequence and 50 uL was added every ten minutes to the electrochemical deduced amino acid sequence of 3-galactosidase (LacZ). SEQ.ID NO:3 and 4 set forth the nucleotide sequence and cell for thirty minutes. At each concentration the working deduced amino acid sequence off-D-glucuronidase (UidA). electrode was swept from -0.3 V to 0.7 V vs Ag/AgCl for SEQ.ID NO.4 and 6 set forth the nucleotide sequence and three cycles. The maximum current in the oxidation peak of deduced amino acid sequence of B-D-glucosidase (BglX). PAP was calculated for each sample and plotted as a function SEQ.ID. NO:7 sets forth the nucleotide sequence of the of time (occurring at approximately 0.1 V). The results are lacZ promoter. shown in FIG. 12. 10 SEQ. ID. NO:8 sets forth the nucleotide sequence of the prha promoter. SEQ. ID. NO:9 sets forth the nucleotide sequence of the Example 12 Baseline Production of PAP from E. tet promoter. coli Capable of Inducibly Producing SEQ. ID. NO:10 sets forth the nucleotide sequence of the B-galactosidase 15 lambda cl promoter. SEQ. ID. NO:11 sets forth the nucleotide sequence of the lasR promoter. For the observation of the baseline production of PAP SEQ. ID. NO: 12 sets forth the nucleotide sequence of the from an inducible promoter capable of translating the Lac Z luxR promoter. protein from E. coli, under non-induced conditions, a solu SEQ. ID. NO:13 sets forth the nucleotide sequence of the tion of 0.1 M pH 7 sodium phosphate buffer was used. 10 leXA promoter. mL of this solution was used to immerse a glassy carbon SEQ. ID. NO:14 sets forth the nucleotide sequence of the electrode with a Pt mesh counter electrode and a Ag/AgCl pBad/araC promoter. reference. 2 mL of transformed cells were grown overnight SEQ. ID. NO:15 sets forth the nucleotide sequence of the and then pelleted and rinsed in the phosphate buffer. The 25 feca promoter. cells were subsequently added to a tube containing 5 mM SEQ. ID. NO:16 sets forth the nucleotide sequence of the PAPG and 50 uL was added every ten minutes to the cusR promoter. electrochemical cell for thirty minutes. At each concentra SEQ. ID. NO:17 sets forth the nucleotide sequence of the tion, the working electrode was then swept from -0.3 V to T7 promoter. 0.7 V vs the Ag/AgCl reference electrode at 100 mV/s for 3 30 SEQ. ID. NO:18 sets forth the nucleotide sequence of the cycles and the third cycle was plotted. The results are shown O32 promoter. in FIG. 13. SEQ. ID. NO:19 sets forth the nucleotide sequence of the All publications, patents and patent applications are O70 promoter. herein incorporated by reference in their entirety to the same SEQ. ID. NO:20 sets forth the nucleotide sequence of the extent as if each individual publication, patent or patent 35 E. coli pRha promoter application was specifically and individually indicated to be SEQ. ID. NO: 21 sets forth the nucleotide sequence of a incorporated by reference in its entirety. Thiobacillus ferrooxidans UV inducible promoter:

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 21

<21 Os SEQ ID NO 1 &211s LENGTH: 3075 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs SEQUENCE: 1 atgac catga ttacggattic actggc.cgt.c gttt tacaac git cqtgactg ggaaaacc ct 60

gg.cgttaccc aacttaatcg ccttgcago a catcc.ccctt togc.ca.gct g gogtaatago 12O galagaggc.cc gCaccgatcg cc ctitcc caa cagttgcgca gcctgaatgg caatggcgc 18O

tttgcctggit tt CC9gcacc agaag.cggtg ccggaaagct ggctggagtg Catct tcct 24 O

gaggc.cgata ctgtcgt.cgt cc cct caaac togcagatgc acggittacga tigcgcc catc 3 OO

tacac caacg tdaccitatic c cattacggit c aatcc.gc.cgt ttgttcc.cac ggagaatc.cg 360 acgggttgtt acticgct cac atttaatgtt gatgaaagct ggct acagga aggc.ca.gacg 42O

cga attattt ttgatggcgt talacticgcg titt Catctgt ggtgcaacgg gCdCtgggtc 48O

ggittacggcc aggacagtcg tttgc.cgt.ct gaatttgacc tagcgcatt tttacgc.gc.c 54 O

ggagaaaac C gcctcgcggit gatggtgctg cgctggagtg acggcagtta totggalagat 6 OO

Caggatatgt gg.cggatgag cqgcatttt C. c9tgacgt.ct cqttgctgca taalaccgact 660

US 9,689,046 B2 29 30 - Continued tggtgtcaaa aataa 3 Ofs

<210s, SEQ ID NO 2 &211s LENGTH: 1024 212. TYPE : PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2

Met Thir Met Ile Thir Asp Ser Luell Ala Wall Wall Lell Glin Arg Arg Asp 1. 5 15

Trp Glu Asn Pro Gly Wall Thir Glin Luell Asn Arg Lell Ala Ala His Pro 25

Pro Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thir Asp Arg Pro 35 4 O 45

Ser Glin Glin Luell Arg Ser Lell Asn Gly Glu Trp Arg Phe Ala Trp Phe SO 55 6 O

Pro Ala Pro Glu Ala Wall Pro Glu Ser Trp Luell Glu Asp Luell Pro 65 70

Glu Ala Asp Thir Wall Wall Wall Pro Ser Asn Trp Glin Met His Gly Tyr 85 90 95

Asp Ala Pro Ile Thir Asn Wall Thir Pro Ile Thir Wall Asn Pro 105 11 O

Pro Phe Wall Pro Thir Glu Asn Pro Thir Gly Cys Tyr Ser Luell Thir Phe 115 12 O 125

Asn Wall Asp Glu Ser Trp Lell Glin Glu Gly Glin Thir Arg Ile Ile Phe 13 O 135 14 O

Asp Gly Wall Asn Ser Ala Phe His Luell Trp Cys Asn Gly Arg Trp Wall 145 150 155 160

Gly Tyr Gly Glin Asp Ser Arg Luell Pro Ser Glu Phe Asp Luell Ser Ala 1.65 17O 17s

Phe Luell Arg Ala Gly Glu Asn Arg Luell Ala Wall Met Wall Luell Arg Trp 18O 185 19 O

Ser Asp Gly Ser Lell Glu Asp Glin Asp Met Trp Arg Met Ser Gly 195

Ile Phe Arg Asp Wall Ser Lell Luell His Pro Thir Thir Glin Ile Ser 21 O 215

Asp Phe His Wall Ala Thir Arg Phe Asn Asp Asp Phe Ser Arg Ala Wall 225 23 O 235 24 O

Lell Glu Ala Glu Wall Glin Met Gly Glu Luell Arg Asp Luell Arg 245 250 255

Wall Thir Wall Ser Lell Trp Glin Gly Glu Thir Glin Wall Ala Ser Gly Thir 26 O 265 27 O

Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp 27s 28O 285

Arg Wall Thir Luell Arg Lell Asn Wall Glu Asn Pro Lys Lell Trp Ser Ala 29 O 295 3 OO

Glu Ile Pro Asn Lell Tyr Arg Ala Wall Wall Glu Lell His Thir Ala Asp 3. OS 310 315

Gly Thir Luell Ile Glu Ala Glu Ala Asp Wall Gly Phe Arg Glu Wall 3.25 330 335

Arg Ile Glu Asn Gly Lell Lell Luell Luell Asn Gly Pro Luell Luell Ile 34 O 345 35. O

Arg Gly Wall Asn Arg His Glu His His Pro Luell His Gly Glin Wall Met 355 360 365 US 9,689,046 B2 31 32 - Continued

Asp Glu Glin Thir Met Wall Glin Asp Ile Luell Luell Met Lys Glin Asn Asn 37 O 375

Phe Asn Ala Wall Arg Cys Ser His Pro ASn His Pro Luell Trp Tyr 385 390 395 4 OO

Thir Luell Asp Arg Gly Luell Wall Wall Asp Glu Ala Asn Ile 4 OS 415

Glu Thir His Gly Met Wall Pro Met Asn Arg Luell Thir Asp Asp Pro Arg 425 43 O

Trp Luell Pro Ala Met Ser Glu Arg Wall Thir Arg Met Wall Glin Arg Asp 435 44 O 445

Arg Asn His Pro Ser Wall Ile Ile Trp Ser Luell Gly Asn Glu Ser Gly 450 45.5 460

His Gly Ala Asn His Asp Ala Luell Arg Trp Ile Ser Wall Asp 465 470

Pro Ser Arg Pro Wall Glin Glu Gly Gly Gly Ala Asp Thir Thir Ala 485 490 495

Thir Asp Ile Ile Pro Met Ala Arg Wall Asp Glu Asp Glin Pro SOO 505

Phe Pro Ala Wall Pro Trp Ser Ile Trp Lell Ser Luell Pro 515 52O 525

Gly Glu Thir Arg Pro Lell Ile Luell Glu Ala His Ala Met Gly 53 O 535 54 O

Asn Ser Luell Gly Gly Phe Ala Trp Glin Ala Phe Arg Glin Tyr 5.45 550 555 560

Pro Arg Luell Glin Gly Gly Phe Wall Trp Asp Trp Wall Asp Glin Ser Luell 565 st O sts

Ile Asp Glu Asn Gly Asn Pro Trp Ser Ala Gly Gly Asp 585 59 O

Phe Asp Thir Pro Asn Asp Arg Glin Phe Met Asn Gly Luell Wall 595 605

Phe Asp Arg Thir Pro His Pro Ala Luell Thir Glu Ala His Glin 615

Glin Phe Phe Glin Phe Arg Luell Ser Gly Glin Thir Ile Glu Wall Thir 625 630 635 64 O

Ser Luell Phe Arg His Ser Asp Asn Glu Lell Lell His Trp Met 645 650 655

Wall Luell Asp Gly Pro Luell Ala Ser Gly Glu Wall Pro Luell Asp 660 665 67 O

Wall Pro Glin Gly Glin Luell Ile Glu Luell Pro Glu Luell Pro Glin 675 685

Pro Ser Ala Gly Glin Lell Trp Luell Thir Wall Arg Wall Wall Glin Pro 695 7 OO

Asn Thir Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Glin Glin 7 Os 71O

Trp Arg Luell Ala Glu Asn Lell Ser Wall Thir Luell Pro Ala Ala Ser His 72 73 O 73

Ala Ile Pro His Lell Thir Thir Ser Glu Met Asp Phe Ile Glu Luell 740 74. 7 O

Gly Asn Lys Arg Trp Glin Phe Asn Arg Glin Ser Gly Phe Luell Ser Glin 760 765

Met Trp Ile Gly Asp Lys Glin Luell Luell Thir Pro Lell Arg Asp Glin 770 775 78O US 9,689,046 B2 33 34 - Continued

Phe Thr Arg Ala Pro Lieu. Asp Asn Asp Ile Gly Wall Ser Glu Ala Thr 78s 79 O 79. 8OO Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His 805 810 815

Tyr Glin Ala Glu Ala Ala Lieu. Lieu Gln Cys Thr Ala Asp Thr Lieu Ala 825 83 O

Asp Ala Val Lieu. Ile Thir Thir Ala His Ala Trp Gln His Glin Gly Lys 835 84 O 845

Thir Lieu. Phe Ile Ser Arg Llys Thr Tyr Arg Ile Asp Gly Ser Gly Glin 850 855 860

Met Ala Ile Thr Val Asp Val Glu Wall Ala Ser Asp Thr Pro His Pro 865 87O 88O

Ala Arg Ile Gly Lieu. Asn. Cys Glin Lieu Ala Glin Wall Ala Glu Arg Val 885 890 895

Asn Trp Lieu. Gly Lieu. Gly Pro Glin Glu Asn Tyr Pro Asp Arg Lieu. Thir 9 OO 905 91 O

Ala Ala Cys Phe Asp Arg Trp Asp Luell Pro Luell Ser Asp Met Tyr Thr 915 92 O 925

Pro Tyr Val Phe Pro Ser Glu Asn Gly Lieu. Arg Cys Gly Thr Arg Glu 93 O 935 94 O

Lieu. Asn Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Glin Phe ASn Ile 945 950 955 96.O

Ser Arg Tyr Ser Glin Glin Gln Lieu. Met Glu. Thir Ser His Arg His Lieu. 965 97O 97.

Lieu. His Ala Glu Glu Gly. Thir Trp Lieu. ASn Ile Asp Gly Phe His Met 98O 985 99 O

Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Wal Ser Ala Glu Phe 995 1005 Glin Lieu. Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Glin 1010 1015 Lys

<210s, SEQ I D NO 3 &211s LENGT H: 1812 212. TYPE : DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 3 atgttacgt.c Ctgtagaaac cc caa.ccc.gt gaaatcaaaa aacticgacgg Cctgtgggca 6 O ttcagt ctgg atcgcgaaaa Ctgtggaatt gat cagogtt ggtgggaaag cgcgttacaa 12 O gaaag.ccggg caattgctgt gcc aggcagt tittaacgatc agttcgc.cga tgcagatatt 18O cgtaattatg cgggcaacgt. Ctggitat cag cgcgaagttct ttataccgaa aggttgggca 24 O ggc.cagcgta titt catgcg gtcact catt acggcaaagt gtgggtcaat 3OO aatcaggaag tgatggagca tCagggcggc tatacgc cat tgtcacgc.cg 360 tatgttattg ccgggaaaag tgitacgitatic accgtttgttg tgaacaacga actgaactgg cagacitat co cgc.cgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagt cittac 48O titccatgatt totttalacta tgc.cgggatc catcgcagcg taatgcticta Caccacgc.cg 54 O alacacctggg tggacgat at Caccgtggtg acgcatgtcg cgcaagactg talaccacgc.g tctgttgact ggCaggtggt ggccalatggit gatgtcagcg ttgaactg.cg tgatgcggat 660

Caac aggtgg ttgcaactgg acaaggcact agcgggactt tgcaa.gtggit gaatc.cgcac 72 O

US 9,689,046 B2 37 38 - Continued Tyr Thir Thr Pro Asn. Thir Trp Val Asp Asp Ile Thr Val Val Thr His 18O 185 19 O Val Ala Glin Asp Cys Asn His Ala Ser Val Asp Trp Glin Val Val Ala 195 2OO 2O5 Asn Gly Asp Val Ser Val Glu Lieu. Arg Asp Ala Asp Glin Glin Val Val 21 O 215 22O Ala Thr Gly Glin Gly Thr Ser Gly Thr Lieu. Glin Val Val Asn Pro His 225 23 O 235 24 O Lieu. Trp Gln Pro Gly Glu Gly Tyr Lieu. Tyr Glu Lieu. Cys Val Thr Ala 245 250 255 Llys Ser Glin Thr Glu. Cys Asp Ile Tyr Pro Lieu. Arg Val Gly Ile Arg 26 O 265 27 O Ser Val Ala Val Lys Gly Glu Glin Phe Lieu. Ile Asn His Llys Pro Phe 27s 28O 285 Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Lieu. Arg Gly Lys 29 O 295 3 OO Gly Phe Asp Asin Val Lieu Met Val His Asp His Ala Lieu Met Asp Trp 3. OS 310 315 32O Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu 3.25 330 335 Met Lieu. Asp Trp Ala Asp Glu. His Gly Ile Val Val Ile Asp Glu Thir 34 O 345 35. O Ala Ala Val Gly Phe Asn Lieu. Ser Lieu. Gly Ile Gly Phe Glu Ala Gly 355 360 365 ASn Llys Pro Llys Glu Lieu. Tyr Ser Glu Glu Ala Val ASn Gly Glu Thir 37 O 375 38O Glin Glin Ala His Lieu. Glin Ala Ile Lys Glu Lieu. Ile Ala Arg Asp Llys 385 390 395 4 OO

Asn His Pro Ser Val Val Met Trp Ser I e Ala Asn. Glu Pro Asp Thr 4 OS 4. O 415 Arg Pro Glin Gly Ala Arg Glu Tyr Phe Ala Pro Lieu Ala Glu Ala Thr 42O 425 43 O Arg Llys Lieu. Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe 435 44 O 445 Cys Asp Ala His Thr Asp Thir Ile Ser Asp Lieu. Phe Asp Val Lieu. Cys 450 45.5 460 Lieu. Asn Arg Tyr Tyr Gly Trp Tyr Val Glin Ser Gly Asp Lieu. Glu Thir 465 470 47s 48O Ala Glu Lys Val Lieu. Glu Lys Glu Lieu. Lieu Ala Trp Glin Glu Lys Lieu 485 490 495 His Glin Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Lieu Ala Gly SOO 505 51O Lieu. His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Glin Cys Ala 515 52O 525 Trp Lieu. Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val 53 O 535 54 O

Gly Glu Glin Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Glin Gly Ile 5.45 550 555 560

Lieu. Arg Val Gly Gly Asn Llys Lys Gly Ile Phe Thr Arg Asp Arg Llys 565 st O sts

Pro Llys Ser Ala Ala Phe Lieu. Lieu. Glin Lys Arg Trp Thr Gly Met Asn 58O 585 59 O

Phe Gly Glu Lys Pro Glin Glin Gly Gly Lys Glin

US 9,689,046 B2 41 - Continued

Cctgttgaaac agctgaaagg Ctttgagaaa at Caccctga aaccgggcga aacticagact 216 O gtcagott Co catcgat at taggcgctgaagttctgga atcaiacagat gaaatatgac 222 O gcc.gagcctg gcaagttcaa ttctittatc ggcactgatt CCGC acgcgt taagaaaggc 228O gagtttgagt totgtaa 2298

<210s, SEQ ID NO 6 &211s LENGTH: 765 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 6 Met Lys Trp Lieu. Cys Ser Val Gly Ile Ala Val Ser Lieu Ala Lieu. Glin 1. 5 1O 15 Pro Ala Lieu Ala Asp Asp Lieu. Phe Gly Asn His Pro Lieu. Thr Pro Glu 2O 25 3O Ala Arg Asp Ala Phe Val Thr Glu Lieu. Lieu Lys Llys Met Thr Val Asp 35 4 O 45 Glu Lys Ile Gly Glin Lieu. Arg Lieu. Ile Ser Val Gly Pro Asp Asn Pro SO 55 6 O Lys Glu Ala Ile Arg Glu Met Ile Lys Asp Gly Glin Val Gly Ala Ile 65 70 7s 8O Phe Asn Thr Val Thr Arg Glin Asp Ile Arg Ala Met Glin Asp Glin Val 85 90 95 Met Glu Lieu. Ser Arg Lieu Lys Ile Pro Lieu. Phe Phe Ala Tyr Asp Wall 1OO 105 11 O Lieu. His Gly Glin Arg Thr Val Phe Pro Ile Ser Lieu. Gly Leu Ala Ser 115 12 O 125 Ser Phe Asn Lieu. Asp Ala Wall Lys Thr Val Gly Arg Val Ser Ala Tyr 13 O 135 14 O Glu Ala Ala Asp Asp Gly Lieu. Asn Met Thir Trp Ala Pro Met Val Asp 145 150 155 160 Val Ser Arg Asp Pro Arg Trp Gly Arg Ala Ser Glu Gly Phe Gly Glu 1.65 17O 17s Asp Thr Tyr Lieu. Thir Ser Thr Met Gly Llys Thr Met Val Glu Ala Met 18O 185 19 O Gln Gly Lys Ser Pro Ala Asp Arg Tyr Ser Val Met Thr Ser Val Lys 195 2OO 2O5 His Phe Ala Ala Tyr Gly Ala Val Glu Gly Gly Lys Glu Tyr Asn. Thir 21 O 215 22O Val Asp Met Ser Pro Glin Arg Lieu Phe Asn Asp Tyr Met Pro Pro Tyr 225 23 O 235 24 O Lys Ala Gly Lieu. Asp Ala Gly Ser Gly Ala Wal Met Val Ala Lieu. Asn 245 250 255

Ser Lieu. Asn Gly Thr Pro Ala Thir Ser Asp Ser Trp Lieu. Lieu Lys Asp 26 O 265 27 O Val Lieu. Arg Asp Gln Trp Gly Phe Lys Gly Ile Thr Val Ser Asp His 27s 28O 285 Gly Ala Ile Lys Glu Lieu. Ile Llys His Gly Thr Ala Ala Asp Pro Glu 29 O 295 3 OO

Asp Ala Val Arg Val Ala Lieu Lys Ser Gly Ile Asn Met Ser Met Ser 3. OS 310 315 32O

Asp Glu Tyr Tyr Ser Llys Tyr Lieu Pro Gly Lieu. Ile Llys Ser Gly Lys 3.25 330 335 US 9,689,046 B2 43 44 - Continued

Wall Thir Met Ala Glu Lell Asp Asp Ala Ala Arg His Wall Luell Asn Wall 34 O 345 35. O

Asp Met Gly Lell Phe Asn Asp Pro Ser His Luell Gly Pro 355 360 365

Glu Ser Asp Pro Wall Asp Thir Asn Ala Glu Ser Arg Luell His Arg 37 O 375

Lys Glu Ala Arg Glu Wall Ala Arg Glu Ser Luell Wall Lell Luell Asn 385 390 395 4 OO

Arg Luell Glu Thir Lell Pro Lell Ser Ala Thir Ile Ala Wall Wall 4 OS 415

Gly Pro Luell Ala Asp Ser Arg Asp Wall Met Gly Ser Trp Ser Ala 425 43 O

Ala Gly Wall Ala Asp Glin Ser Wall Thir Wall Luell Thir Gly Ile Asn 435 44 O 445

Ala Wall Gly Glu Asn Gly Lys Wall Luell Ala Lys Gly Ala Asn Wall 450 45.5 460

Thir Ser Asp Gly Ile Ile Asp Phe Luell ASn Glin Glu Glu Ala 465 470

Wall Wall Asp Pro Arg Ser Pro Glin Glu Met Ile Asp Glu Ala Wall 485 490 495

Glin Thir Ala Lys Glin Ser Asp Wall Wall Wall Ala Wall Wall Gly Glu Ala SOO 505

Glin Gly Met Ala His Glu Ala Ser Ser Arg Thir Asp Ile Thir Ile Pro 515 525

Glin Ser Glin Arg Asp Lell Ile Ala Ala Luell Ala Thir Gly Pro 53 O 535 54 O

Lell Wall Luell Wall Lell Met Asn Gly Arg Pro Luell Ala Lell Wall Glu 5.45 550 555 560

Asp Glin Glin Ala Asp Ala Ile Luell Glu Thir Trp Phe Ala Gly Thir Glu 565 st O sts

Gly Gly Asn Ala Ile Ala Asp Wall Luell Phe Gly Asp Asn Pro Ser 585 59 O

Gly Luell Pro Met Ser Phe Pro Arg Ser Wall Gly Glin Ile Pro Wall 595 605

Tyr Ser His Lell Asn Thir Gly Arg Pro Asn Ala Asp Pro 610 615

Asn Thir Ser Arg Phe Asp Glu Ala Asn Gly Ala Luell Tyr 625 630 635 64 O

Pro Phe Gly Gly Lell Ser Thir Thir Phe Thir Wall Ser Asp Wall 645 650 655

Luell Ser Ala Pro Thir Met Arg Asp Gly Wall Thir Ala Ser 660 665 67 O

Wall Glin Wall Thir Asn Thir Gly Lys Arg Glu Gly Ala Thir Wall Wall Glin 675 685

Met Tyr Luell Glin Asp Wall Thir Ala Ser Met Ser Arg Pro Wall Glin 69 O. 695 7 OO

Lell Gly Phe Glu Lys Ile Thir Luell Pro Gly Glu Thir Glin Thir 7 Os 71s

Wall Ser Phe Pro Ile Asp Ile Glu Ala Luell Phe Trp Asn Glin Glin 72 73 O 73

Met Asp Ala Glu Pro Gly Lys Phe ASn Wall Phe Ile Gly Thir 740 74. 7 O

US 9,689,046 B2 49 50 - Continued

212. TYPE : DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 16 aaaatgacaa ttttgt catt tt 22

<210s, SEQ ID NO 17 &211s LENGTH: 23 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: T7 Bacteriophage

<4 OOs, SEQUENCE: 17 taatacgact Cactataggg aga 23

<210s, SEQ ID NO 18 &211s LENGTH: 405 212. TYPE : DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 18

Cactgaagtg atcct cqc.ca C Caac CocaC ggttgaaggt gaagctaccg CtalactaCat 6 O tgcc.gagctt tgcgc.gcaat atgacgtgga agc.ca.gc.cga atcgct catg gcgttc.cggit 12 O tggcgg.cgag Ctggaaatgg tcgacggcac cacgttgtca cactic ccttg ccgggcgt.ca 18O taagatt.cgt. ttittaa.gcaa acgaga.gcag gat cacctgc t ct cqcttga aattatt Ctc. 24 O ccttgtc.ccc atctot CC Ca catcc tdttt ttaac Cttaa. aatggcatta ttgaggtaga 3OO cctacatgaa aggacaagaa act cqtggitt ttcagt caga agtgaaa.ca.g cittctgcacc 360 tgatgatcca ttctgttct at tccaataaag aaatctt CCt 405

<210s, SEQ ID NO 19 &211s LENGTH: 35 212. TYPE : DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 19 ttgacggcta gct cagtic ct agg tacagtg ctago 35

<210s, SEQ ID NO 2 O &211s LENGTH: 24 O 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: E. coli rhamnose promoter (pRha)

<4 OOs, SEQUENCE: 2O cggcgaaata gtaatcacga ggt caggttc ttacct taala tttitcgacgg aaaac cacgt. 6 O aaaaaacgt.c gatttittcaa gatacagcgt. gaattitt cag gaaatgcggit gag catcaca 12 O toac Cacaat t cagoaaatt gtgaacat Ca t cacgttcat ctitt ccctgg ttgccaatgg 18O cc cattitt co tgtcagtaac gagaaggtog cgagttcagg cgctttittag actggtcgta 24 O

<210s, SEQ ID NO 21 &211s LENGTH: 76 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Thiobacillus ferrioxidans

<4 OOs, SEQUENCE: 21 US 9,689,046 B2 51 52 - Continued ggcaggaaga ccgggcgcat gagcg tattt ttitt at Cta atatgcctga aag.cgcatac 6 O cgctatggag ggggtt 76

We claim as our invention: 2. The method according to claim 1 wherein the B-D- 1. A method for simultaneously detecting two parameters galactosidase comprises SEQID NO: 2, the B-D glucuroni in a liquid medium comprising: 10 dase comprises SEQ ID NO: 4, and the B-D-glucosidase (i) providing a liquid medium comprising a first and comprises SEQ ID NO: 6. second parameter; (ii) contacting in a vessel the liquid medium with cells and 3. A method of simultaneously detecting a first and second a first and second Substrate to form an assay mixture, parameter in a liquid medium, the method comprising: wherein the first substrate comprises a first electroac (i) contacting in a vessel the liquid medium and (a) a first tive analyte chemically linked to a first carrier and the 15 Substrate comprising a first electroactive analyte second Substrate comprises a second electroactive ana chemically linked to a first carrier; (b) a second sub lyte chemically linked to a second carrier, and wherein strate comprising a second electroactive analyte chemi the cells comprise a chimeric nucleic acid construct cally linked to a second carrier; (c) a first reporter comprising in the 5' to 3’ direction of translation as polypeptide comprising a first hydrolase; and (d) a operably linked components a first nucleic acid con second reporter polypeptide comprising a second struct comprising: (a) a first promoter inducible by the first parameter; and hydrolase, to form an assay mixture, wherein the first (b) a nucleic acid sequence encoding a first reporter hydrolase is capable of hydrolyzing the first substrate polypeptide comprising a first hydrolase capable of and releasing the first electroactive analyte from the hydrolyzing the first substrate and releasing the first 25 carrier and the second hydrolase is capable of hydro electroactive analyte from the carrier; and lyzing the second Substrate and releasing the second a second nucleic acid construct comprising: electroactive analyte from the carrier; and (c) a second promoter inducible by the second param (ii) detecting an electrical signal facilitated by the first and eter, and second electroactive analyte in the assay mixture, (d) a nucleic acid sequence encoding a second reporter 30 wherein detecting the electrical signal is facilitated by polypeptide comprising a second hydrolase capable the first and second electroactive analyte detects the first and second parameters in the liquid medium; of hydrolyzing the second substrate and releasing the wherein the first and second hydrolase are independently second electroactive analyte from the carrier; Selected from the group of hydrolases consisting of (iii) detecting an electrical signal facilitated by the first B-D-glucosidase, 3-D galactosidase, and 3-D-glu and second electroactive analyte in the assay mixture, 35 wherein detecting the electrical signal facilitated by the curonidase; first and second electroactive analyte detects the first wherein the first and second substrate are independently and second parameters in the liquid medium; Selected from the group of Substrates consisting of wherein the first and second hydrolase are independently chlorophenol red-f-D-galactopyranoside (CPRG), Selected from the group of hydrolases consisting of 40 para-nitrophenol-f-D-glucuronide (PNPG), para B-D-glucosidase, 3-D galactosidase, and B-D-glu aminophenol-3-D-galactopyranoside (PAPG) and para curonidase; di-phenol-f-D-glucopyranoside (PDPG); wherein the first and second substrate are independently wherein detecting the electrical signal is performed by Selected from the group of Substrates consisting of applying a Voltage to the assay mixture by a Voltam chlorophenol red-f-D-galactopyranoside (CPRG), 45 metric Sweep, and wherein the first and second reporter para-nitrophenol-f-D-glucuronide (PNPG), para polypeptides are different. aminophenol-3-D-galactopyranoside (PAPG) and para 4. The method according to claim 3 wherein the B-D- di-phenol-f-D-glucopyranoside (PDPG); galactosidase comprises SEQID NO: 2, the B-D glucuroni wherein the Voltage in step (iii) is applied to the assay dase comprises SEQ ID NO: 4, and the B-D-glucosidase mixture by a voltammetric sweep, and wherein the first 50 comprises SEQ ID NO: 6. and second nucleic acid constructs are different. k k k k k