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(12) Patent Application Publication (10) Pub. No.: US 2014/0059722 A1 Krichevsky (43) Pub US 20140059722A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0059722 A1 Krichevsky (43) Pub. Date: Feb. 27, 2014 (54) BIOSENSORS Publication Classification (71) Applicant: Bioglow LLC, St. Louis, MO (US) (51) Int. Cl. CI2N 5/82 (2006.01) (72) Inventor: Alexander Krichevsky, St. Louis,MO CI2O I/68 (2006.01) (US) (52) U.S. Cl. CPC .......... CI2N 15/8214 (2013.01); C12O 1/6897 (73) Assignee: Bioglow LLC, St. Louis, MO (US) (2013.01) USPC ........ 800/317:435/419; 435/252.3:435/468; (21) Appl. No.: 13/894,043 8OO/298 (57) ABSTRACT (22) Filed: May 14, 2013 Real-time monitoring of plant or environmental conditions is O O solved by Autoluminescent Phytosensor Plants (ALPS) dis Related U.S. Application Data closed herein that emit light in response to a specified stimu (60) Provisional application No. 61/647.323, filed on May lus or condition, which light emission is detected or measured 15, 2012. by a sensor. PASTD Plastic Genone NUCLEUS LUX operon -D T7 RNA polymerase AYMANMemmoothamomomom Inducible sequence 7 promoter Promoter T7 RNA polymerase mRNA CYTOSOL Transit peptide-> 7 RNA polymerase protein Patent Application Publication Feb. 27, 2014 Sheet 1 of 10 US 2014/0059722 A1 sn=TonNL uojadoXnT ?uuou39p??Seld |eun61-I THOSO.LAO Patent Application Publication Feb. 27, 2014 Sheet 2 of 10 US 2014/0059722 A1 STEIT(OTN) yxn-1 quinqns THOSO.LASO Zeun61-I | Patent Application Publication Feb. 27, 2014 Sheet 3 of 10 US 2014/0059722 A1 queIddoÁ??uu?Xoud suosuºspe?oe??e €eun61-I Patent Application Publication Feb. 27, 2014 Sheet 4 of 10 US 2014/0059722 A1 tyeun61 HSDIT XVO Patent Application Publication Feb. 27, 2014 Sheet 5 of 10 US 2014/0059722 A1 Patent Application Publication Feb. 27, 2014 Sheet 6 of 10 US 2014/0059722 A1 SO Xn-1Vuouedo uoluadoXn-1 -9eun61-I--- 8 Patent Application Publication Feb. 27, 2014 Sheet 7 of 10 US 2014/0059722 A1 sque?ue^Áue|duaxeIeuo???ppy Patent Application Publication Feb. 27, 2014 Sheet 8 of 10 US 2014/0059722 A1 8 eun61-I Patent Application Publication Feb. 27, 2014 Sheet 9 of 10 US 2014/0059722 A1 o O O O X o - Patent Application Publication Feb. 27, 2014 Sheet 10 of 10 US 2014/0059722 A1 od-Iqd OLeun61-I US 2014/0059722 A1 Feb. 27, 2014 BOSENSORS genome, nor does it provide Suitable methods or vectors to integrate the LUX operon into plastidal genomes. Further 1.O BACKGROUND OF THE INVENTION more, it does not contemplate indirect luciferase pathway 0001. 1.1 Field of the Invention activation in plants (e.g., genetic relay assay as in PCT/US10/ 0002 Present invention relates to the field of biosensors. 253.66, etc.), thereby describing a different type of transgenic 0003) 1.2. Description of Related Art plants, as well as failing to provide for monitoring or Survey 0004 Commercial agriculture depends on monitoring of methods. Similarly, U.S. Pat. No. 7,049,483 contemplates various plant parameters, such as hydration, disease, ripe introduction of jellyfish luciferase and its substrate, ness, pest invasion, temperature, adequacy of nutrients, and coelenterazine, biosynthesis machinery into a plant to gener other conditions to achieve Successful yields. Since the begin ate bioluminescent plants. However, it does not contemplate ning of agriculture, farmers relied primarily on their intuition expression the jellyfish luciferase pathway from plastid and observation in assessing crop and field conditions. In genomes, does not provide for Suitable methods or vectors to recent decades, growers increasingly utilize various devices, integrate these genes into plastidal genomes, and does not including computerized systems, containing an assortment of contemplate indirect luciferase pathway activation in plants. sensing capabilities to more precisely follow plant, field, and Finally, this reference does not contemplate the use of biolu greenhouse conditions (Wolf, B. (1996) Diagnostic Tech minescent plants as phytosensors. nique for Improving Crop Production, Haworth Press, pp. 185-187). These new developments are continuously leading 0012. Therefore, a solid and robust system comprising an to optimization of agricultural production through improved autoluminescent plant phytosensor and a computerized moni planting, water management, and other practices. toring system is needed. The present invention provides for 0005. However, while these new evolving approaches light emitting plants, having light emission machinery inte have substantially enhanced phytomonitoring, the existing grated within their plastidal genome, and a method of moni methods are still cumbersome, imprecise, require complex toring and Surveying light emission thereof in order to utilize and expensive equipment and, in many cases, do not provide these plants as biosensors or phytosensors in agricultural and real-time monitoring of a crops condition. other settings. 0006 An additional challenge of today’s industrialized Society is environmental contamination. Increasing effects of 0013 The presently disclosed autoluminescent phytosen chemicals in the environment and their toxicity to human and sor (ALPS) plants, also referred as ALPS plants’ or simply animal health necessitate monitoring of pollutant levels. as “ALPS', address this need by providing a simple, inexpen Common pollutants, among others, include heavy metals sive, real-time monitoring alternative, Superior to other bio (cadmium, arsenic, mercury, etc), phenolic compounds, etc. sensor Systems. This technology can be widely used and find Environmental analysis is typically carried out in by sampling application in the monitoring of agricultural and horticultural of the Suspected polluted area and later analyzing the samples crops, including ornamentals, and in environmental protec using Sophisticated methods. Such as atomic absorption spec tion. Finally, ALPS can be used in basic plant research to trometry, ion chromatography, etc. These are time consuming monitor different plant parameters in real time and with high and expensive methods, which are not always available or precision. Currently available monitoring systems frequently feasible in practicality. measure indirect parameters (e.g., CO exchange rate (U.S. 0007. Therefore, there is a clear recognized need for an Pat. No. 6,701,665)), while ALPS produce signals directly in improved and robust phytosensing method capable of provid response to specific stimuli (dehydration, pest invasion, etc.). ing reliable real-time information. Moreover, while other reporter systems based on direct cou 0008 U.S. Pat. No. 6,701,665 teaches monitoring of natu pling of protein expression (e.g., GFP) in response to a given ral plant conditions using computerized systems; however, it stimulus have been contemplated, those have been proven to does not disclose transgenic plants, nor does it contemplate be impractical. For example, GFP could not be detected using measurement of active light emission or luminescence from visualization approaches (see Kooshkietal (2003) “Pathogen plants. inducible reporting in transgenic tobacco using a GFP con 0009 US patent application 2005/0114923 suggests uti struct”. Plant Science 165:213-219). ALPS provide for prac lizing expression of plant pigments, such as anthocyanins, tically feasible, real-time and direct response to very specific generally in response to contaminants. However, the applica stimuli. The technology is non-destructive to plants, does not tion describes phenotypcial changes in plants, such as pig require any external Substrates to be sprayed, and can be mentation, and does not relate to detecting plant autolumi remotely sensed. nescence or monitoring of light-emitting plants. The 0014 ALPS based on plastid-integrated light emission application also does not relate to or disclose genetic engi systems is a radically new concept. In the past, attempts to neering of plastids, nor does it disclose computerized moni incorporate complex metabolic pathways into transgenic toring methods. plants have been hampered by various limitations of genetic 0010 Patent applications PCT/US2008/009310, 60/953, engineering technology. Creation of the world's first autolu 337 and PCT/US10/25366 describe incorporation of minescent plant—a living plant organism capable of emitting luciferase and luciferin-related genes into plastids. PCT/US visible light on its own, without the need for any external 10/25366 contemplates a genetic relay assay for induction of chemical or light sources—has been initially reported by us light emission. These documents do not, however, disclose (Krichevsky et al. (2010) “Autoluminescent Plants”. PLoS the use of autoluminescent plants as phytosensors, or con ONE 5(11):e15461). Here, for the first time, we describe the template monitoring methods. use of autoluminescent plants as phytosensors that can be 0011 WO2007136432 contemplates bioluminescent used to monitor plant health, pathogen invasion, environmen plants containing LUX operon genes. However, it does not tal contamination, and other conditions and stimuli affecting disclose the expression of the LUX operon from the plastid plant growth and development. US 2014/0059722 A1 Feb. 27, 2014 2O SUMMARY OF THE INVENTION 0027 4. The autoluminescentphytosensor (ALPS) plant monitoring system of any one of 1-3, wherein said 0015. In one aspect, the present invention discloses the use environmental or plant physiological condition is of autoluminescent phytosensor plants (ALPS), containing Selected from the group consisting of hydration, disease, genetically engineered plastids expressing a fully functional
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