Team:Cornell/Sandbox/project/wetlab

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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results">Testing &amp; Results</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">BioBricks</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/transcription">Transcriptional Characterization</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</a>
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<h2 class="centered">Wet Lab Overview</h2>
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<h3>Summary</h3>
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We have developed a novel biosensing platform to be used for long-term, continuous monitoring of environmental toxins, such as arsenic and naphthalene. Traditional biosensors commonly output fluorescence, pH, or luminescence&#8212;which then need to be interpreted. Our simpler bacterium-based biosensor directly outputs electric current. This platform offers several advantages.
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In order to produce an electrical output in response to analyte, we base our biosensing solution on the well-characterized metal reduction (Mtr) pathway of Shewanella oneidensis MR-1. By shutting electrons through the Mtr pathway, MR-1 is capable of transferring electrons to inorganic solids and generating current at solid-state electrodes. In particular, we choose to utilize MtrB in our biosensing system, as it plays an essential role in localization of components in the Mtr pathway [3].
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We have designed our biosensing platform to upregulate MtrB production in the presence of analyte. To construct reporter systems for both arsenic and naphthalene, we rely on a complementation strategy based on an mtrB deficient strain of S. oneidensis MR-1 [Strain JG700 [&Delta;mtrB], 2]. The endogenous copy of mtrB has been removed in JG700; therefore, by reintroducing mtrB to this knockout strain under the control of an analyte-sensitive regulation system, we restore the functionality of mtrB in proportion to the amount of analyte present in culture media. Because MtrB is essential for electrode reduction in microbial fuel cells, we will observe a current increase in response to analyte when our engineered strains are used to inoculate bioelectrochemical reactors [4].
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Because of their capability to directly transfer electrons to acceptors outside the cell, Shewanella strains are often used in microbial electrochemical systems wherein an electrode serves as a terminal electron acceptor in the Mtr pathway. In general, a microbial electrochemical system is just like any other electrochemical cell, except that a microbe is responsible for catalyzing the oxidation/reduction reaction at either the anode or the cathode. For our purposes, we are interested in half-microbial electrochemical systems with three-electrode setups, since such systems can be easily maintained at constant conditions over extended periods of time by poising the potential of a working electrode—to which the bacteria respire—with respect to a reference.
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<h3>References</h3>
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1. Dowdesw, L., Dillon, P., Miall, A., &amp; Smol, J. P. (2010). A foundation for the future: building an environmental monitoring system for the oil sands, Environment Canada.
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2. Coursolle, D., and Gralnick, J.A. (2012). Reconstruction of extracellular respiratory pathways for iron(III) reduction in Shewanella oneidensis strain MR-1. Frontiers in Microbiology 3(56)
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3. Hartshorne, R. S., Reardon, C. L., Ross, D., Nuester, J., Clarke, T. A., Gates, A. J., Mills, P. C., et al. (2009). Characterization of an electron conduit between bacteria and the extracellular environment . Proceedings of the National Academy of Sciences . doi:10.1073/pnas.0900086106
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4. Coursolle, D., Baron, D.B., Bond, D.R., and Gralnick, J.A. (2010). The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. Journal of Bacteriology 192(2): 467-474
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5. Nivens, D.E., McKnight, T.E., Moser, S.A., Osbourn, S.J., Simpson, M.L., &amp; Sayler, G. S. (2004). Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. Journal of Applied Microbiology, 96(1): 33-46.
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6. Siegfried, K., Endes, C., Bhuiyan, A. F. M. K., Kuppardt, A., Mattusch, J., van der Meer, J. R., Chatzinotas, A., et al. (2012). Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria. Environmental Science &amp; Technology 46(6), 3281-7
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Revision as of 22:55, 14 February 2013

Wet Lab Overview

Summary

We have developed a novel biosensing platform to be used for long-term, continuous monitoring of environmental toxins, such as arsenic and naphthalene. Traditional biosensors commonly output fluorescence, pH, or luminescence—which then need to be interpreted. Our simpler bacterium-based biosensor directly outputs electric current. This platform offers several advantages.

In order to produce an electrical output in response to analyte, we base our biosensing solution on the well-characterized metal reduction (Mtr) pathway of Shewanella oneidensis MR-1. By shutting electrons through the Mtr pathway, MR-1 is capable of transferring electrons to inorganic solids and generating current at solid-state electrodes. In particular, we choose to utilize MtrB in our biosensing system, as it plays an essential role in localization of components in the Mtr pathway [3].

We have designed our biosensing platform to upregulate MtrB production in the presence of analyte. To construct reporter systems for both arsenic and naphthalene, we rely on a complementation strategy based on an mtrB deficient strain of S. oneidensis MR-1 [Strain JG700 [ΔmtrB], 2]. The endogenous copy of mtrB has been removed in JG700; therefore, by reintroducing mtrB to this knockout strain under the control of an analyte-sensitive regulation system, we restore the functionality of mtrB in proportion to the amount of analyte present in culture media. Because MtrB is essential for electrode reduction in microbial fuel cells, we will observe a current increase in response to analyte when our engineered strains are used to inoculate bioelectrochemical reactors [4].

Because of their capability to directly transfer electrons to acceptors outside the cell, Shewanella strains are often used in microbial electrochemical systems wherein an electrode serves as a terminal electron acceptor in the Mtr pathway. In general, a microbial electrochemical system is just like any other electrochemical cell, except that a microbe is responsible for catalyzing the oxidation/reduction reaction at either the anode or the cathode. For our purposes, we are interested in half-microbial electrochemical systems with three-electrode setups, since such systems can be easily maintained at constant conditions over extended periods of time by poising the potential of a working electrode—to which the bacteria respire—with respect to a reference.


References

1. Dowdesw, L., Dillon, P., Miall, A., & Smol, J. P. (2010). A foundation for the future: building an environmental monitoring system for the oil sands, Environment Canada.

2. Coursolle, D., and Gralnick, J.A. (2012). Reconstruction of extracellular respiratory pathways for iron(III) reduction in Shewanella oneidensis strain MR-1. Frontiers in Microbiology 3(56)

3. Hartshorne, R. S., Reardon, C. L., Ross, D., Nuester, J., Clarke, T. A., Gates, A. J., Mills, P. C., et al. (2009). Characterization of an electron conduit between bacteria and the extracellular environment . Proceedings of the National Academy of Sciences . doi:10.1073/pnas.0900086106

4. Coursolle, D., Baron, D.B., Bond, D.R., and Gralnick, J.A. (2010). The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. Journal of Bacteriology 192(2): 467-474

5. Nivens, D.E., McKnight, T.E., Moser, S.A., Osbourn, S.J., Simpson, M.L., & Sayler, G. S. (2004). Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. Journal of Applied Microbiology, 96(1): 33-46.

6. Siegfried, K., Endes, C., Bhuiyan, A. F. M. K., Kuppardt, A., Mattusch, J., van der Meer, J. R., Chatzinotas, A., et al. (2012). Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria. Environmental Science & Technology 46(6), 3281-7