Team:Cornell/project/wetlab/results

From 2012.igem.org

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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/reactors">Reactors</a>
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<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/currentresponse">Current Response</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/protein">MtrB Protein Expression</a>
<a href="http://2012.igem.org/Team:Cornell/project/wetlab/results/protein">MtrB Protein Expression</a>
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<h3>HEY LISTEN</h3>
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<h3>Transcriptional Characterization</h3>
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Can the fate of Hyrule really depend on such a lazy boy?
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After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in <i>S. oneidensis</i>. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR.
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<br><br>
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In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB.
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We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.
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<h3>HEY LISTEN</h3>
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<h3>Current Response</h3>
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Do you want to talk to Saria? Do you want to talk to me?
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Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.
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As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.
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<h3>MtrB Protein Expression</h3>
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In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots.
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<h3><i>nah</i> Operon Expression</h3>
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To test the functionality of our third major construct, the <i>nah</i> operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the <i>nah</i> operon.
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<h3>Artificial River Media</h3>
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As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution. 
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Revision as of 00:37, 27 October 2012

Results

Transcriptional Characterization

After constructing our reporters, we first wanted to verify that they operated on a transcriptional level in S. oneidensis. Our transcriptional characterization consisted of fluorescence testing and RT-qPCR.

In order to conduct fluorescence characterization, we appended mRFP downstream of mtrB in our arsenic and salicylate reporters. We then measured the relative fluorescence of the reporters when induced with different concentrations of arsenic and salicylate, respectively. Both showed increased fluorescence in response to increased analyte, strongly suggesting transcriptional upregulation of mRFP and therefore of upstream mtrB.

We are also in the process of conducting real-time quantitative PCR, as a more direct method of demonstrating transcriptional upregulation of mtrB in response to analyte.

Current Response

Encouraged by our positive results from fluorescence characterization, we moved forward with testing our reporter strains in bioreactors. We demonstrated that our arsenic reporter strain functions at the system level: current is upregulated in response to increasing arsenite concentration. However, leaky expression in our salicylate reporter strain saturated current response at basal levels, an issue we plan to address in the future.

As a proxy to current production, we are also conducting ferrozine assays, which measure how much iron (III) is reduced to iron (II) by extracellular electron shuttling. This is a high-throughput method of assessing the relative functionality of our reporter strains at a system level.

MtrB Protein Expression

In addition to characterization at the transcriptional and whole-system levels, we also want to verify that MtrB, our protein of interest, is being expressed at higher levels when the reporters are induced with analyte. To this end, we are in the process of conducting Western blots.

nah Operon Expression

To test the functionality of our third major construct, the nah operon, we are currently conducting tests on the biosynthesis of indigo by naphthalene dioxygenase, an enzyme encoded in the nah operon.

Artificial River Media

As a practical consideration for the deployment of our device, we attempted to develop a minimal growth medium that could be highly concentrated in the food tanks of our final device, and fed to the cells at a low flow rate. Our results suggest that our strains could be sustained on a steady flow of 2% (w/v) sodium lactate solution.