Team:Cornell/project/wetlab/results

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<h2 class="centered">Results</h2>
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<h2 class="centered">Summary of Results</h2>
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<h3>Transcriptional Characterization</h3>
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<h3>Transcription Upregulated in Response to Salicylate and Arsenic</h3>
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.  
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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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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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. As seen in the figures to the left, 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.
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>Current Response</h3>
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<h3>Current Increases in Response to Arsenic</h3>
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.
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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<a href="http://2012.igem.org/wiki/images/b/bf/Currentresponse_600.png" rel="lightbox[project-page]"><img src="http://2012.igem.org/wiki/images/2/2e/Currentresponse_300.png">Click to Enlarge</a>
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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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Latest revision as of 03:55, 27 October 2012

Summary of Results

Transcription Upregulated in Response to Salicylate and Arsenic

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. As seen in the figures to the left, 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 Increases in Response to Arsenic

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.

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