Team:EPF-Lausanne

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{{:Team:EPF-Lausanne/Templates/Header|title=Transcription factor development}}
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We have developed a pipeline for selection and characterization of new transcription factors (TFs), specifically:
We have developed a pipeline for selection and characterization of new transcription factors (TFs), specifically:
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# ''in vivo'' selection of functional mutants from a large library of variants using a “survival of the weakest” strategy,
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# ''in vivo'' selection of functional mutants from a large library of variants using a “survival of the weakest” strategy;
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# ''in vitro'' characterization of affinity and specificity of mutants with MITOMI,
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# ''in vitro'' characterization of affinity and specificity of mutants with MITOMI;
# ''in vivo'' characterization of selected mutants using reporter plasmids.
# ''in vivo'' characterization of selected mutants using reporter plasmids.
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Our data can be found here : [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Data Data].
Our project can be decomposed into four main parts:
Our project can be decomposed into four main parts:
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<h2><a href="/Team:EPF-Lausanne/Our_Project/TetR_mutants"><i>In vitro</i> TF characterization</a></h2>
<h2><a href="/Team:EPF-Lausanne/Our_Project/TetR_mutants"><i>In vitro</i> TF characterization</a></h2>
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<p>We used a microfluidic based approach for characterizing TF mutants in vitro. The <a href="/Team:EPF-Lausanne/Tools/MITOMI">MITOMI</a> method allows us to measure absolute binding affinities and specificities of transcription factors. We determined the precise binding energy landscape of the wild type TetR transcription factor. We also generated several TetR transcription factor mutants and determined the specificities of a number of the new variants.</p>
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<p>We used a microfluidic based approach for characterizing TF mutants in vitro. The <a href="/Team:EPF-Lausanne/Tools/MITOMI">MITOMI</a> method allows us to measure absolute binding affinities and specificities of transcription factors. We determined the precise <a href="/Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data">binding energy landscape</a> of the wild type TetR transcription factor. We also generated <a href="/Team:EPF-Lausanne/Our_Project/TetR_mutants/muTetRs">several TetR transcription factor mutants</a> and determined the specificities of a number of the new variants.</p>
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<h2><a href="/Team:EPF-Lausanne/Our_Project/Assembly"><i>In vivo</i> TF characterization</a></h2>
<h2><a href="/Team:EPF-Lausanne/Our_Project/Assembly"><i>In vivo</i> TF characterization</a></h2>
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<p>To be able to determine the in vivo activity and specificity of novel transcription factor variants we generated a suite of <a href="/Team:EPF-Lausanne/Our_Project/Assembly">reporter plasmid systems</a>. We successfully characterized a number of our reporter systems for functionality. We created a number of reporter plasmids to measure TetR activity, which lead to improved characterization data for the Partsregistry.</p>
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<p>To be able to determine the in vivo activity and specificity of novel transcription factor variants we generated a suite of <a href="/Team:EPF-Lausanne/Our_Project/Reporter_Systems">reporter plasmid systems</a>. We successfully characterized a number of our reporter systems for functionality. We created a number of reporter plasmids to measure TetR activity, which lead to improved characterization data for the Partsregistry.</p>
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In summary, over the summer we:
In summary, over the summer we:
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* Developed and characterized a new [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants|''in vivo'' selection system]] based on “survival of the weakest”.
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* developed and characterized a new [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants|''in vivo'' selection system]] based on “survival of the weakest”;
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* Created and characterized a set of [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants/t7prom|T7 promoter variants]] that express with different strengths.
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* created and characterized a set of [[Team:EPF-Lausanne/Our_Project/T7_promoter_variants/t7prom|T7 promoter variants]] that express with different strengths;
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* Created several [[Team:EPF-Lausanne/Our_Project/TetR_mutants/muTetRs|TetR variants]].
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* created several [[Team:EPF-Lausanne/Our_Project/TetR_mutants/muTetRs|TetR variants]];
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* Created several [[Team:EPF-Lausanne/Our_Project/Assembly|reporter systems]] for the ''in vivo'' characterization of TetR.
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* created several [[Team:EPF-Lausanne/Our_Project/Assembly|reporter systems]] for the ''in vivo'' characterization of TetR;
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* Determined the [[Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data|binding energy landscape of TetR]] and a number of TetR mutants using MITOMI.
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* determined the [[Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data|binding energy landscape of TetR]] and a number of TetR mutants using MITOMI;
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* Performed experiments with and accordingly updated the [http://partsregistry.org/Part:BBa_K112808 BBaK112808 lysis device].
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* performed experiments with and accordingly updated the [http://partsregistry.org/Part:BBa_K112808 BBaK112808 lysis device];
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* Developed and documented a [[Team:EPF-Lausanne/Tools/Microfluidics/HowTo2|cheap and easy to build a microfluidic setup]] for the iGEM community.
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* developed and documented a [[Team:EPF-Lausanne/Tools/Microfluidics/HowTo2|cheap and easy to build microfluidic control setup]] for the iGEM community.
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Latest revision as of 05:55, 25 October 2011