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Team:EPF-Lausanne
From 2011.igem.org
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| - | <h2><i>In vitro</i> TF characterization</h2> | + | <h2><a href="/Team:EPF-Lausanne/Our_Project/TetR_mutants"><i>In vitro</i> TF characterization</a></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. 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> | <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> | ||
Revision as of 21:43, 21 September 2011
Transcription factor development
We have developed a pipeline for selection and characterization of new transcription factors (TFs), 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. 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 are a useful tool for the iGEM community, we decided to promote the technique by creating the tamagotchiplive 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.
- Performed experiments with and accordingly updated the BBaK112808 lysis device.
- Developed and documented a cheap and easy to build a microfluidic setup for the iGEM community.
