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>HEY LISTEN</h3>
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<h3>Transcription Upregulated in Response to Salicylate and Arsenic</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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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.
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<h3>Current Increases in Response to Arsenic</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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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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