Revision as of 14:23, 21 September 2011

Introduction
Our Project
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Acknowledgements
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Transcription factor development

iGEM Logo

We have developed a pipeline for selection and characterization of new transcription factors, specifically:

in vivo selection of functional mutants from a large library of variants using a “survival of the weakest” strategy,
in vitro characterization of affinity and specificity of mutants with MITOMI,
in vivo characterization of selected mutants using reporter plasmids.

Our project can be decomposed into four main parts:

In vivo TF selection system

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 proof-of-concept version of the system, consisting of a T7 driven lysis cassette we were able to show that:

Cells can be specifically lysed upon induction of the lysis cassette.
DNA can be recovered from the lysed cells.
We get an enrichment of plasmids originating from lysed cells in a mock selection experiment.

In vitro TF characterization

We used a microfluidic based approach for characterizing TF mutants in vitro. The MITOMI method allows us to measure absolute binding affinities and specificities of transcription factors (Cite the science paper). 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.

In vivo TF characterization

To be able to determine the in vivo activity and specificity of novel transcription factor variants we generated a suite of reporter plasmid systems. 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.

Microfluidics

We noticed that only a small number of iGEM teams have also made use of microfluidics in the past. Because we think that microfluidics is a useful tool for the iGEM community, we decided to promote the technique by creating the [http://tamagotchip.epfl.ch tamagotchip] live online microfluidics game: from any web browser, you can control the setup located in our lab in Lausanne, as detailed here. In addition, we wrote a guide on the wiki to help future teams get started with these techniques.

In summary, over the summer we:

developed and characterized a new in vivo selection system based on “survival of the weakest”.
created and characterized a set of T7 promoter variants that express with different strengths.
created several TetR variants.
created several reporter systems for the in vivo characterization of TetR.
determined the binding energy landscape of TetR and a number of TetR mutants using MITOMI.
developed a cheap and easy to build a microfluidic setup for the iGEM community

@@ Line 1: / Line 1: @@
-{{:Team:EPF-Lausanne/Templates/Header|title=Welcome}}
+{{:Team:EPF-Lausanne/Templates/Header|title=Transcription factor development}}
-<html><img src="https://static.igem.org/mediawiki/2010/b/b0/UCL-Igem.png" alt="iGEM Logo" width="200px" style="float:right"/><html>
+<html><img src="https://static.igem.org/mediawiki/2010/b/b0/UCL-Igem.png" alt="iGEM Logo" width="200px" style="float:right"/></html>
-<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>
+We have developed a pipeline for selection and characterization of new transcription factors, specifically:
-<html>Team, remember the <a href="https://2011.igem.org/Team:EPF-Lausanne/Todo">Todo List</a></html>
+# ''in vivo'' selection of functional mutants from a large library of variants using a “survival of the weakest” strategy,
+# ''in vitro'' characterization of affinity and specificity of mutants with MITOMI,
+# ''in vivo'' characterization of selected mutants using reporter plasmids.
+Our project can be decomposed into four main parts:
-== Project summary ==
+<html>
+<div class="frontpagebox odd">
+<h2><i>In vivo</i> TF selection system</h2>
-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 obtained ten interesting new mutants. For each of these mutants, we 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 using a process called MITOMI, which allowed us to test a single mutant against hundreds of Ptet sequences.
+<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 proof-of-concept version of the system, consisting of a T7 driven lysis cassette we were able to show that:<p>
+<ol>
+    <li>Cells can be specifically lysed upon induction of the lysis cassette.</li>
+    <li>DNA can be recovered from the lysed cells.</li>
+    <li>We get an enrichment of plasmids originating from lysed cells in a mock selection experiment.</li>
+</ol>
+</div>
-As a complement to the <i> in vitro </i> approach, the next step in characterizing these TF mutants was to produce an <i>in vivo</i> readout system. To that end, we created and tested two different reporter systems based on RFP expression, with one producing a positive and the other a negative selection of the TetR-Ptet interaction. Because we also wanted a high-throughput method, we decided to use a lysis cassette as a reporter gene. The idea was to transform cells with one plasmid containing a TetR mutant and another plasmid containing a Ptet sequence (either the consensus or a mutated one), and then to have automatic lysis of the cells in which the TetR mutant recognizes the Ptet sequence, the goal being to recover their DNA. This approach could then be coupled to microfluidic chemostat in which hundreds of cell colonies can be grown all at the same time. To enhance the modularity of this readout system, we also created T7 promoter variants which can be coupled to the RFP or lysis genes. Lysis and DNA recovery experiments using this readout system have been efficiently conducted, demonstrating the feasibility of our project design.
+<div class="frontpagebox even">
+<h2><i>In vitro</i> TF characterization</h2>
+<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 (Cite the science paper). 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>
+</div>
-Results of the different experiments can be found on the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Data data] page.
+<div class="frontpagebox odd">
-For more details on the assembly and mutagenesis strategies, please refer to the following pages: [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants TetR mutants], [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/T7_promoter_variants T7 promoter variants], [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly Reporter plasmids].
+<h2><i>In vivo</i> TF characterization</h2>
+<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>
+</div>
-<!--
+<div class="frontpagebox even">
-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. -->
+<h2>Microfluidics</h2>
+<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 is a useful tool for the iGEM community, we decided to promote the technique by creating the [http://tamagotchip.epfl.ch tamagotchip] 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>
+</div>
-<html> <!--
-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.
--->
 </html>
+In summary, over the summer we:
+* developed and characterized a new in vivo selection system based on “survival of the weakest”.
+* created and characterized a set of T7 promoter variants that express with different strengths.
+* created several TetR variants.
+* created several reporter systems for the in vivo characterization of TetR.
+* determined the binding energy landscape of TetR and a number of TetR mutants using MITOMI.
+* developed a cheap and easy to build a microfluidic setup for the iGEM community
 <html>