Team:EPF-Lausanne

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<html>It's on! After some warmup experiments during the semester, our team is now fully operational, and already having trouble getting away from the lab. Follow our adventures on twitter: <a href="http://twitter.com/#!/IGEM_EPFL">IGEM_EPFL</a></html>
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<html>Team, remember the <a href="https://2011.igem.org/Team:EPF-Lausanne/Todo">Todo List</a></html>
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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 vitro'' characterization of affinity and specificity of mutants with MITOMI;
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# ''in vivo'' characterization of selected mutants using reporter plasmids.
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== Project summary ==
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Our data can be found here : [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Data Data].
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Our project goal was to set up a high-throughput method for generating and characterizing new transcription factors (TFs) that would recognize different promoter sequences. We mutated the TetR transcription factor and got about ten interesting mutants. For each of them, we individually characterized  their affinity to the consensus sequence (the Ptet promoter) as well as their position-weight matrices. This characterization was done <i>in vitro</i> on a microfluidic chip, allowing to test a single mutant against hundreds of Ptet sequences.
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Our project can be decomposed into four main parts:
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The next step of the characterization is an <i>in vivo</i> readout system; we created and tested 2 different reporter systems based on RFP expression, with either a positive or negative selection of the TetR-Ptet interaction. Wanting a high-throughput method, we decided also to use a lysis cassette as a reporter gene. The idea is to transform cells with one plasmid containing a TetR mutant and another plasmid containing a Ptet sequence(either the consensus or a mutated one), then to kill the cells in which the TetR mutant recognizes the Ptet sequence, in order to recover their DNA. This can then be coupled to microfluidics chemostat chambers, where hundreds of cell colonies can be grown at the same time. We also created T7 promoter variants, that can be coupled to the RFP or lysis genes, allowing modularity in the readout systems. Lysis ad DNA recovery experiments have been efficiently conducted, demonstrating the feasibility of our project design.
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Results for the TetR mutants, reporter systems, T7 promoter variants and DNA recovery experiments can be found on the data page.
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For more details on the assembly and mutagenesis strategies, please refer to the following pages: TetR mutants, T7 promoter variants, Reporter plasmids.
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<h2><a href="/Team:EPF-Lausanne/Our_Project/T7_promoter_variants"><i>In vivo</i> TF selection system</a></h2>
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<p>As the first step in our pipeline we propose a new in vivo method for the automated selection of mutant transcription factors or transcription factor binding sites from large and diverse libraries containing millions of variants. The novelty of our approach lies in the fact that we use negative selection, or “survival of the weakest” as the selection strategy. In our method, a functional variant activates lysis of the host, leading to release of the plasmid DNA coding for the functional variant. The plasmid DNA can then be amplified, transformed, or directly sequenced to determine which variants were functional. We believe that our negative selection scheme is a potentially powerful approach when coupled to next-generation sequencing. Using a <a href="/Team:EPF-Lausanne/Our_Project/T7_promoter_variants/recovery">proof-of-concept version of the system</a>, consisting of a T7 driven lysis cassette we were able to show that:<p>
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    <li>Cells can be specifically lysed upon induction of the lysis cassette.</li>
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    <li>DNA can be recovered from the lysed cells.</li>
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    <li>We get an enrichment of plasmids originating from lysed cells in a mock selection experiment.</li>
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Our goal is to design transcription factors (TFs) that bind to new sequences, currently unavailable to synthetic biologists. These would allow to build larger genetic circuits than currently possible, with more independently regulated genes. The new transcription factors will be derived from tetR, one of the better characterised TFs out there. To characterise the new ones, we will introduce the tetR mutants and their corresponding mutant promoters into the circuit illustrated below, containing a LacI inverter and a reporter gene. The reporter for ''in vitro'' experiments is a fluorescence gene such as RFP, and a lysis system for ''in vivo'' experiments. The latter lyses cells with effective transcription factors, so that exclusively their DNA can be recovered. -->
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<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 <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>
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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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<h2><a href="/Team:EPF-Lausanne/Tools/Microfluidics">Microfluidics</a></h2>
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<p>We noticed that only a small number of iGEM teams have also made use of microfluidics in the past. Because we think that microfluidics are a useful tool for the iGEM community, we decided to promote the technique by creating the <a href="/Team:EPF-Lausanne/Tools/Microfluidics/Tamagotchip">tamagotchip</a> live online microfluidics game: from any web browser, you can control the setup located in our lab in Lausanne, as detailed <a href="/Team:EPF-Lausanne/Tools/Microfluidics/Tamagotchip">here</a>. In addition, we wrote a <a href="Team:EPF-Lausanne/Tools/Microfluidics">guide on the wiki</a> to help future teams get started with these techniques.</p>
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We aim at engineering transcription factors so that they can recognize new binding sequences. We are making TetR mutants and different TetR recognition sequences, later characterizing each mutant with a MITOMI experiment. We are building a swith with TetR and LacI that will activate either RFP or a lysis cassette.
 
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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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* 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/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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* 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 microfluidic control setup]] for the iGEM community.
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Latest revision as of 05:55, 25 October 2011

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