Team:Cornell/project/wetlab/assembly

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<h2 class="centered">DNA Assembly</h2>
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<h2 class="centered">Summary of DNA Assembly</h2>
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<h3>Genetic Parts Rely on Complementation Strategy To Sense Analyte </h3></div>
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<img class="inline" src="http://2012.igem.org/wiki/images/e/e5/SWITCHOFF.png" width="550" height="600">
<br><b>When MtrB is not present, it is as if the switch is open, and current cannot flow.</b>
<br><b>When MtrB is not present, it is as if the switch is open, and current cannot flow.</b>
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<img class="inline" src="http://2012.igem.org/wiki/images/a/a5/SWITCHON.png" width="550" height="600">
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<br><b>When MtrB is reintroduced into the system, it is as if the switch is closed, allowing current to flow.</b>
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<i>Shewanella oneidensis</i> MR-1’s metal reduction pathway can be likened to a simple switch analogy. Without MtrB, no extracellular transfer of electrons is possible, and therefore no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.
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As described on our <a href=http://2012.igem.org/Team:Cornell/project/wetlab/chassis>chassis</a> page, the protein MtrB is required for
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<i>S. oneidensis</i> to be able to shuttle electrons outside the cell. Therefore, when a strain of  <i>S. oneidensis</i> lacking <i>mtr</i>B is inoculated in a bioelectrochemical system, current cannot be significantly produced.
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We can think of this in terms of a simple switch analogy&#8212;visualized in the figures to the left: Without MtrB, no extracellular transfer of electrons is possible, and no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.  
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To sensitize <i>Shewanella’s</i> metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the <i>Shewanella</i> MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB&#8212;and therefore current&#8212;should only be produced in the presence of analyte.<br>
To sensitize <i>Shewanella’s</i> metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the <i>Shewanella</i> MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB&#8212;and therefore current&#8212;should only be produced in the presence of analyte.<br>
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<br>One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist. <br><br>
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<br><b>When MtrB is reintroduced into the system, it is as if the switch is closed, allowing current to flow.</b>
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<br>One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist. <br><br>
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Because of its essential role in extracellular electron transfer, MtrB can be thought of as an electric switch: When absent, the switch is open, disallowing current production in microbial electrochemical systems; when present, the switch is closed and current may be produced. We took advantage of this in the development of our biosensing strains: Using a dmtrB strain (JG700) as a host, we adopted a complementation strategy wherein the capability for extracellular electron shuttling was reintroduced via the expression of MtrB from a plasmid. Because we designed our engineered plasmids so that mtrB transcription could be induced in response to our analyte of interest, we were able to construct strains that produce more current in response to analyte—as the MtrB ‘switches’ close.    
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Latest revision as of 03:22, 27 October 2012

Summary of DNA Assembly

Genetic Parts Rely on Complementation Strategy To Sense Analyte


When MtrB is not present, it is as if the switch is open, and current cannot flow.
When MtrB is reintroduced into the system, it is as if the switch is closed, allowing current to flow.
As described on our chassis page, the protein MtrB is required for S. oneidensis to be able to shuttle electrons outside the cell. Therefore, when a strain of S. oneidensis lacking mtrB is inoculated in a bioelectrochemical system, current cannot be significantly produced.

We can think of this in terms of a simple switch analogy—visualized in the figures to the left: Without MtrB, no extracellular transfer of electrons is possible, and no current is produced. However, when reintroduced, MtrB closes the switch therefore allowing extracellular reduction and current generation.

To sensitize Shewanella’s metal reduction pathway to our analytes, we decided to use a complementation strategy. By using the Shewanella MtrB knockout strain, JG 700, which was graciously provided by Professor Jeffery Gralnick from the University of Minnesota, we are able to reintroduce MtrB on a plasmid under the control of inducible promoters sensitive to the analytes we want to detect. Thus, MtrB—and therefore current—should only be produced in the presence of analyte.

One of the greatest strengths of this approach is its modularity; by simply switching out the sensing region on the plasmid, we can sensitize MtrB production to any analyte for which genetic parts exist.