Team:Cornell/testing/project/wetlab/1

From 2012.igem.org

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<h3>Summary</h3>
<h3>Summary</h3>
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We began the summer by holding a synthetic biology bootcamp at the DeLisa Lab. The purpose of this bootcamp was both to introduce new members to techniques in molecular biology and to get a running start on the cloning work for our project. During bootcamp, we successfully constructed both versions of our arsenic reporter, and attempted a Gibson assembly of a naphthalene-degrading plasmid.
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According to a report by an oilsand advisory panel to the Canadian Minister of Environment, long-term continuous sampling systems that monitor oilsands effects in rivers “should be pursued aggressively.” This oilsands advisory panel investigated all of the organizations monitoring the oilsands area; an important conclusion by the panel was that many studies and reports of the oilsands area have been unable to conclude the effects oilsands 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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In late June, we transitioned from bootcamp to our permanent bench space in Dr. Archer’s lab in Weill Hall. After spending a few weeks setting up the lab space troubleshooting general issues, we successfully constructed both versions of our salicylate reporter and began an alternative approach to construct a plasmid with a naphthalene-degrading operon. In parallel, we realized that electroporation efficiency for Shewanella transformation is less than optimal—to say the least. However, we were able to conjugate our constructs into Shewanella using a protocol provided by Dr. Gralnick.  
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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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We have developed a novel biosensing platform based on the well-characterized metal reduction pathway of Shewanella oneidensis 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 different 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. However, 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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As we transitioned into the fall semester, wetlab work was divided into ‘task forces’.  
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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 S. oneidensis 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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<li>Site Directed mutagenesis</li>
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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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<li>Western blotting</li>
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<li>qPCR</li>
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<li>Nah operon into Shewanella</li>
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<li>Running reactors</li>
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<li>Artificial River Media</li>
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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.
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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.
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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
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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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Revision as of 01:07, 4 October 2012

Wet Lab Overview

Summary

According to a report by an oilsand advisory panel to the Canadian Minister of Environment, long-term continuous sampling systems that monitor oilsands effects in rivers “should be pursued aggressively.” This oilsands advisory panel investigated all of the organizations monitoring the oilsands area; an important conclusion by the panel was that many studies and reports of the oilsands area have been unable to conclude the effects oilsands 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].

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.

We have developed a novel biosensing platform based on the well-characterized metal reduction pathway of Shewanella oneidensis 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 different 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. However, we plan to build a simple bacterium based biosensor whereby electric current is the direct output. This offers our platforms several advantages.

To construct reporter systems for both arsenic and naphthalene, we will rely on a complementation strategy based on an mtrB deficient strain of S. oneidensis 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].

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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