[Catalyst 2017]

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(a) (b) (c) (d) Fig 1: Schematics for the genetic circuits to produce iRFP of the three different inducers: (a) aribanose, (b) nitric oxide, (c and d) hypoxia presence of arabinose sugar, allowing for the activation of the pBAD promoter. Fluorescence levels emitted by the iRFPs increased significantly when placed in wells containing increasing concentrations of arabinose (Figure 2). This correlation suggests that our selected iRFPs fluoresce sufficiently when promoters are induced by environmental signals. The results of the arabinose assays showed that we successfully produced iRFPs; the next steps were to engineer bacteria to produce the same iRFPs under nitric oxide and hypoxia. Nitric Oxide The next step was to test the nitric oxide induction of iRFP fluorescence. We used a genetic circuit consisting of a constitutive promoter and the soxR gene, which in turn expresses the soxR protein (Figure 1b). In the presence of nitric oxide, soxR changes form to activate the promoter soxS, which activates the expression of the desired gene. The source of nitric oxide added to our engineered bacteria samples was diethylenetriamine/nitric oxide adduct (DETA/ NO). Figure 3 shows no significant difference of fluorescence/OD600 between DETA/NO concentrations. This finding implies that our engineered bacteria were unable to detect the nitric oxide biomarker and produce iRFP; future troubleshooting includes verifying promoter strength and correct sample conditions. Furthermore, nitric oxide has an extremely short half-life of a few seconds, which may not be enough time for most of the engineered bacteria to sense the nitric oxide, limiting iRFP production and fluorescence. 58 | CATALYST Hypoxia We also tested the induction of iRFP fluorescence with the hypoxia-inducible promoters narK and fdhf. We expected iRFP production and fluorescence to increase when using the narK and fdhf promoters in anaerobic conditions (Figure 1c and d). However, we observed the opposite result. A decreased fluorescence for both iRFP constructs in both promoters was measured when exposed to hypoxia (Figure 4). This finding suggests that our engineered bacteria were unable to detect the hypoxia biomarker and produce iRFP; future troubleshooting includes verifying promoter strength and correct sample conditions. Future Directions Further studies include testing the engineered bacteria co-cultured with colon cancer cells and developing other constructs that will enable bacteria to sense carcinogenic tumors and make them fluoresce for imaging and treatment purposes. Violacein has anti-cancer therapy potential Violacein is a fluorescent pigment for in vivo photoacoustic imaging in the near-infrared range and shows anti-tumoral activity. 6 It has high potential for future work in bacterial tumor targeting. We have succeeded in constructing violacein using Golden Gate shuffling and intend to use it in experiments such as the nitric oxide and hypoxia assays we used for iRFP670 and 713. 7 Invasin can allow for targeted cell therapy Using a beta integrin called invasin, certain bacteria are able to invade mammalian cells. 8-9 If we engineer E. coli that have the beta integrin invasion as well as the genetic circuits capable of sensing nitric oxide and/or hypoxia, we can potentially allow the E. coli to invade colon cells and release contrast agents for photoacoustic imaging or therapeutic agents such as violacein only in the presence of specific biomarkers. 10 Additionally, if we engineer the bacteria that exhibit invasin to invade colon cancer cells only and not normal cells, then this approach would potentially allow for a localized targeting and treatment of cancerous tumors. This design allows us to create scenarios with parameters more similar to the conditions observed in the human gut as we will be unable to test our engineered bacteria in an actual human gut.
Fig 2: Arabinose induces iRFP production by pBAD promoter Fig 3: iRFP production by Sox R/S promoter is not induced by nitric oxide analog Fig 4: iRFP production by Fdhf or NarK is not induced by hypoxic conditions ACKNOWLEDGEMENTS The International Genetically Engineered Machine (iGEM) Foundation (igem.org) is an independent, non-profit organization dedicated to education and competition, the advancement of synthetic biology, and the development of an open community and collaboration. This project would not have been possible without the patient instruction and generous encouragement of our Principal Investigators (Dr. Beth Beason-Abmayr and Dr. Jonathan Silberg, BioSciences at Rice), our graduate student advisors and our undergraduate team. I would also like to thank our iGEM collaborators. This work was supported by the Wiess School of Natural Sciences and the George R. Brown School of Engineering and the Departments of BioSciences, Bioengineering, and Chemical and Biomolecular Engineering at Rice University; Dr. Rebecca Richards-Kortum, HHMI Pre-College and Undergraduate Science Education Program Grant #52008107; and Dr. George N. Phillips, Jr., Looney Endowment Fund. If you would like to know more information about our project and our team, please visit our iGEM wiki at: http://2016.igem.org/Team:Rice. WORKS CITED [1] Ntziachristos, V. Nat Methods. 2010, 7, 603- 614. [2] Weber, J. et al. Nat Methods. 2016, 13, 639- 650. [3] Archer, E. J. et al. ACS Synth. Biol. 2012, 1, 451–457. [4] Hӧckel, M.; Vaupel, P. JNCI J Natl Cancer Inst. 2001, 93, 266−276. [5] Guzman, L. M. et al. J of Bacteriology. 1995, 177, 4121-4130. [6] Shcherbakova, D. M.; Verkhusha, V. V. Nat Methods. 2013, 10, 751-754. [7] Engler, C. et al. PLOS One. 2009, 4, 1-9. [8] Anderson, J. et al. Sci Direct. 2006, 355, 619–627 [9] Arao, S. et al. Pancreas. 2000, 20, 619-627. [10] Jiang, Y. et al. Sci Rep. 2015, 19, 1-9. Icons from Alice Noir via The Noun Project DESIGN BY Vidya Giri, Katrina Cherk EDITED BY Billy Jeon CATALYST | 59
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Page 15 and 16: A Gift From the Pliocene of our pla
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Page 19 and 20: the doubleedged sword at the forefr
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Page 23 and 24: The stress in the everyday job of a
Page 25 and 26: the University of Texas M.D. Anders
Page 27 and 28: INCarceration by Rishi Suresh Healt
Page 29 and 30: CALCIUM AND GLUTAMATE: Facilitating
Page 31 and 32: PILOT SIGNAL CONVERTER MICROWAVE BE
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Page 35 and 36: how some of the world’s deadliest
Page 37 and 38: ENGINEERING EDEN TERRAFORMING A SEC
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Page 41 and 42: Touching lives by Jack Trouvé Ever
Page 43 and 44: depression is still an enigma to ph
Page 45 and 46: The subjects were given tasks to de
Page 47 and 48: PARTNERS IN CANCER RESEARCH SAMANTH
Page 49 and 50: A Glimpse of the Future by Joshua H
Page 51 and 52: WASTE PRODUCT LYSOSOME MALFUNCTION
Page 53 and 54: she said. This inspired Dr. Ostherr
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Page 57: DETECTION OF GUT INFLAMMATION + TUM
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[Catalyst 2017]

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