Team:Cornell/project/wetlab

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DNA Assembly
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/assembly/salicylate">Salicylate Reporter</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/assembly/naphthalene">Naphthalene Reporter</a>
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Testing &amp; Results
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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/fluorescence">Fluorescence</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/qPCR">qPCR</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a>
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<h3>Summary</h3>
<h3>Summary</h3>
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According to a report by an oil sand advisory panel to the Canadian Minister of Environment, long-term continuous sampling systems that monitor oil sands effects in rivers “should be pursued aggressively.” This oil sands advisory panel investigated all of the organizations monitoring the oil sands area; they concluded that many studies and reports of this area have been unable to conclude what effects oil sands have on the environment and human health due to an inadequate monitoring system. This inadequacy can be attributed to a lack of consistent sampling methods in both time and location [1].
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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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High concentrations of arsenic and naphthalene have been detected in tailing ponds. While the extent and effects of tailing pond seepage into local watersheds have not been well documented, the potential environmental and health effects--combined with the effects of arsenic and naphthalene on biodiversity and ecosystem balance--serve as the main motivation for our project.
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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 developed a novel biosensing platform based on the well-characterized metal reduction pathway of <i>Shewanella oneidensis</i> MR-1. MR-1 is capable of transferring electrons to inorganic solids and generating current at solid-state electrodes via an unconventional electron shuttling pathway. Two essential proteins in this pathway are MtrC, an outer-membrane cytochrome responsible for electron transfer to metal oxides and electrodes and MtrB, which aids MtrC. Several MtrB paralogs exist in the MR-1 genome, but MtrB promotes the highest Fe(III) citrate reduction activity when combined with functional MtrA and MtrC [2]. Furthermore, MtrB plays an essential role in the localization of components of the Mtr pathway [3]. For these reasons, we are designing our biosensing platform to upregulate MtrB production in the presence of analyte. Previous iGEM biosensing platforms have relied on the detection of fluorescence or luminescence. We plan to build a simple bacterium-based biosensor whereby electric current is the direct output. This offers our platforms several advantages.
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To construct reporter systems for both arsenic and naphthalene, we will rely on a complementation strategy based on an mtrB deficient strain of <i>S. oneidensis</i> MR-1 [Strain JG700 [ΔmtrB], 1]. 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 will 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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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, <i>Shewanella</i> 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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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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Latest revision as of 02:53, 20 September 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