Team:EPF Lausanne/Overview

From 2014.igem.org

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<h1 class="cntr">Project</h1>
<h1 class="cntr">Project</h1>
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<h2 class="section-heading" id="title_intro">Introduction</h2>
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<section id="title_intro">
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<h3 class="section-heading">DNA parts submitted by the 2014 EPFL iGEM team</h3>
<p class="lead">
<p class="lead">
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The 2014 EPFL iGEM team has been working on showing that biologically engineered organisms can detect and process signals quickly and efficiently. With this in mind, our team brought forward a novel idea: combining Protein Complementation techniques with biosensors to achieve fast spatiotemporal analysis of bacterial or yeast response to mechanical stimuli.  
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Our team submitted a total of 55 Biobricks (biobrick 51 does not exist).</p>
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</p>
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<br /><br />
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<div class="pull-left img-left">
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Touch_bacteria_EPFL_Ted.png" alt="touch bacteria" width="200" class="img-border" />
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</div>
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<p class="lead">
<p class="lead">
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Our team explored this hypothesis by engineering two stress related pathways in E.Coli and S.Cerevisiae with in mind the development of a BioPad: a biological touchscreen consisting of a microfluidic chip, touch responsive bacteria, and a signal detector. Learn more about <a href="#howitworks">how the BioPad works !</a> </p>
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In addition, 4 microfluidic designs have also been submitted to the registry.</p>
 +
<table class="table table-striped table-hover" id="biobricks_list">
 +
  <tr>
 +
    <th>Biobrick</th>
 +
    <th>What it is</th>
 +
    <th>Function</th>
 +
    <th>Why do we use it?</th>
 +
    <th>Group</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486000</td>
 +
    <td>CpxR coding sequence</td>
 +
    <td>Transcription factor</td>
 +
    <td>To make most of our biobricks!</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486001</td>
 +
    <td>CpxR under arabinose promoter</td>
 +
    <td>Treanscription factor</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486002</td>
 +
    <td>PAra + sfGFP CpxR [Nterm]</td>
 +
    <td>Expresses fused protein</td>
 +
    <td>Test CpxR expression & Ara promoter</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486003</td>
 +
    <td>Flexible linker</td>
 +
    <td>Attaches two proteins together</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486004</td>
 +
    <td>Flexible linker</td>
 +
    <td>Attaches two proteins together</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486005</td>
 +
    <td>PAra + CpxR sfGFP [Cterm]</td>
 +
    <td>Expresses fused protein</td>
 +
    <td>Test CpxR expression & Ara promoter</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486006</td>
 +
    <td>IFP[1]</td>
 +
    <td>N terminus of split IFP</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486007</td>
 +
    <td>IFP[2]</td>
 +
    <td>C terminus of split IFP</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486008</td>
 +
    <td>CxpR & Split IFP1.4 [Cterm + Cterm]</td>
 +
    <td>Two CpxR CDS, each C terminus attached to a moiety of IFP</td>
 +
    <td>Characterize CpxR dimerization</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486009</td>
 +
    <td>CxpR & Split IFP1.4 [Nterm + Nterm]</td>
 +
    <td>Two CpxR CDS, each N terminus attached to a moiety of IFP</td>
 +
    <td>Characterize CpxR dimerization</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486010</td>
 +
    <td>CxpR & Split IFP1.4 [Nterm + Cterm]</td>
 +
    <td>Two CpxR CDS, each attached to a moiety of IFP</td>
 +
    <td>Characterize CpxR dimerization</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486011</td>
 +
    <td>CxpR & Split IFP1.4 [Cterm + Nterm]</td>
 +
    <td>Two CpxR CDS, each attached to a moiety of IFP</td>
 +
    <td>Characterize CpxR dimerization</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486012</td>
 +
    <td>CpxR + IFP[1]</td>
 +
    <td>CpxR with the Nterm moiety of IFP attached at its C terminus</td>
 +
    <td>Intermediate & control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486013</td>
 +
    <td>CpxR + IFP[2]</td>
 +
    <td>CpxR with the Cterm moiety of IFP attached at its C terminus</td>
 +
    <td>Intermediate & control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486014</td>
 +
    <td>IFP[1] + CpxR</td>
 +
    <td>CpxR with the Nterm moiety of IFP attached at its N terminus</td>
 +
    <td>Intermediate & control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486015</td>
 +
    <td>IFP[2] + CpxR</td>
 +
    <td>CpxR with the Cterm moiety of IFP attached at its N terminus</td>
 +
    <td>Intermediate & control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486016</td>
 +
    <td>fLuc[1]</td>
 +
    <td>N terminus moiety of the firefly luciferase</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486017</td>
 +
    <td>fLuc[2]</td>
 +
    <td>C terminus moiety of the firefly luciferase</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486018</td>
 +
    <td>PAra + fLuc[1] + fLuc[2]</td>
 +
    <td>Split firefly luciferase under arabinose promoter</td>
 +
    <td>Control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486019</td>
 +
    <td>rLuc[1]</td>
 +
    <td>C terminus moiety of the renilla luciferase</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486020</td>
 +
    <td>rLuc[2]</td>
 +
    <td>N terminus moiety of the renilla luciferase</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486021</td>
 +
    <td>PAra + rLuc[1] + rLuc[2]</td>
 +
    <td>Split renilla luciferase under arabinose promoter</td>
 +
    <td>Control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486022</td>
 +
    <td>rLuc</td>
 +
    <td>Full renilla luciferase</td>
 +
    <td>Control</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
<tr>
 +
    <td class="biobrick_name">BBa_K1486023</td>
 +
    <td>Yeast sfGFP</td>
 +
    <td>Superfolder GFP for yeast cells</td>
 +
    <td>Reporter</td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486024</td>
 +
    <td>Kan</td>
 +
    <td>Yeast kanamycin resistance gene</td>
 +
    <td>Selection marker</td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486025</td>
 +
    <td>ADH1 terminator</td>
 +
    <td>Terminator</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486026</td>
 +
    <td>Yeast sfGFP + Kan</td>
 +
    <td>Yeast sfGFP attached to the yeast kanamycin resistance gene</td>
 +
    <td>Control the expression of pbs2</td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
<tr>
 +
    <td class="biobrick_name">BBa_K1486027</td>
 +
    <td>rLuc + Kan</td>
 +
    <td>Renilla luciferase attached to the kanamycin resistance gene</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486028</td>
 +
    <td>Yeast sfGFP[1]</td>
 +
    <td>N terminal moiety of split yeast sfGFP</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486029</td>
 +
    <td>sfGFP[1] + kan</td>
 +
    <td>Nterm moiety of split yeast sfGFP attached to yeast kanamycin resistance gene</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486030</td>
 +
    <td>rLuc[1] + kan</td>
 +
    <td>Nterm moiety of split renilla luciferase attached to yeast kanamycin resistance gene</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486031</td>
 +
    <td>Ura</td>
 +
    <td>CDS for Uracil (yeast selective purposes)</td>
 +
    <td>Confer resistance to Uracil-deprived medium</td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
<tr>
 +
    <td class="biobrick_name">BBa_K1486032</td>
 +
    <td>Yeast sfGFP + Ura</td>
 +
    <td>Yeast sfGFP attached to the Uracil CDS</td>
 +
    <td>Control the expression of hog1</td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486033</td>
 +
    <td>rLuc + Ura</td>
 +
    <td>Renilla luciferase attached to the Uracil CDS</td>
 +
    <td>Control the expression of hog1</td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486034</td>
 +
    <td>yeast sfGFP[2]</td>
 +
    <td>C terminal moiety of split the yeast sfGFP</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486035</td>
 +
    <td>yeast sfGFP[2] + Ura</td>
 +
    <td>Cterm moiety of split yeast sfGFP attached to the Uracil CDS</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
<tr>
 +
    <td class="biobrick_name">BBa_K1486036</td>
 +
    <td>rLuc[2] + Ura</td>
 +
    <td>Cterm moiety of split renilla luciferase attached to the Uracil CDS</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486037</td>
 +
    <td>linker</td>
 +
    <td>Attaches two proteins together</td>
 +
    <td> </td>
 +
    <td>Yeast</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486038</td>
 +
    <td>sfGFP[1]</td>
 +
    <td>N terminus moiety of split superfolder GFP</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486039</td>
 +
    <td>sfGFP[2]</td>
 +
    <td>C terminus moiety of split superfolder GFP</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486040</td>
 +
    <td>sfGFP[1] + CpxR</td>
 +
    <td>N terminus moiety of split sfGFP attached to CpxR</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486041</td>
 +
    <td>sfGFP[2] + CpxR</td>
 +
    <td>C terminus moiety of split sfGFP attached to CpxR</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486042</td>
 +
    <td>LZip</td>
 +
    <td>Monomer of leucine zipper TF</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486043</td>
 +
    <td>LZip + split rLuc</td>
 +
    <td>Two Leucine Zipper monomers, each attached to a different split rLuc moiety</td>
 +
    <td>Control for split rLuc assays</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486044</td>
 +
    <td>mut IFP[1]</td>
 +
    <td>Biobrick-compatible IFP[1]</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486045</td>
 +
    <td>mut IFP[2]</td>
 +
    <td>Biobrick-compatible IFP[2]</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486046</td>
 +
    <td>CpxR promoter FW</td>
 +
    <td>CpxR binding-region in forward direction</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486047</td>
 +
    <td>CpxR promoter RV</td>
 +
    <td>CpxR binding-region in reverse direction</td>
 +
    <td> </td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486048</td>
 +
    <td>CpxR reporter</td>
 +
    <td>Calgary's CpxR reporter repaired (sequence was missing)</td>
 +
    <td>To see when CpxR is active</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486049</td>
 +
    <td>CpxR promoter FW + RFP</td>
 +
    <td>Reporter of CpxR</td>
 +
    <td>Test the direction of the complete CpxR promoter</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486050</td>
 +
    <td>CpxR promoter RV + RFP</td>
 +
    <td>Reporter of CpxR</td>
 +
    <td>Test the direction of the complete CpxR promoter</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486052</td>
 +
    <td>Spacer</td>
 +
    <td>40 bases placed between constructs</td>
 +
    <td>Separate two constructs in the same plasmid</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486053</td>
 +
    <td>Linker</td>
 +
    <td>10 amino-acid linker</td>
 +
    <td>Attach CheY/Z to split luciferases</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486054</td>
 +
    <td>CheY/CheZ rLuc</td>
 +
    <td>CheY and CheZ, each attached to a moiety of split renilla luciferase</td>
 +
    <td>Positive control for the split rLuc</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486055</td>
 +
    <td>CheY/CheZ fLuc</td>
 +
    <td>CheY and CheZ, each attached to a moiety of split firefly luciferase</td>
 +
    <td>Positive control for the split fLuc</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="biobrick_name">BBa_K1486056</td>
 +
    <td>CxpR & Split mut IFP1.4 [Cterm + Cterm]</td>
 +
    <td>Two CpxR CDS, each C terminus attached to a moiety of the biobrick-compatible IFP</td>
 +
    <td>Characterize CpxR dimerization</td>
 +
    <td>Bacteria</td>
 +
  </tr>
 +
</table>
 +
</section>
-
<br /><br /><br />
 
-
<div class="pull-right img-right">
 
-
<a href="https://static.igem.org/mediawiki/2014/1/15/Screen_Shot_2014-10-12_at_3.29.30_PM.png" data-lightbox="image-0" data-title="Fluorescence">
 
-
<img src="https://static.igem.org/mediawiki/2014/1/15/Screen_Shot_2014-10-12_at_3.29.30_PM.png" alt="touch bacteria" height="200" class="img-border" /></a>
 
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</div>
 
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<p class="lead">
 
-
The pathway engineered in E.Coli, the Cpx Pathway, is a two-component regulatory system responsive to envelope stress. A full description of the pathway is available <a href="#CpxPathway">here</a>. In S.Cerevisiae we modified the HOG Pathway - a MAPKK pathway responsive to osmotic stress. For more information concerning the HOG Pathway click <a href="#thehogpathway">here.</a></p>
 
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<br /><br /><br /><br /><br />
 
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<div class="pull-left img-left">
 
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<a href="https://static.igem.org/mediawiki/2014/d/d8/EPFLmicrofluidics.JPG" data-lightbox="image-1" data-title="EPFL microfluidic chips"><img src="https://static.igem.org/mediawiki/2014/d/d8/EPFLmicrofluidics.JPG" width="200" class="img-border"></a>
 
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</div>
 
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<p class="lead">
 
-
 
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Our project also includes an extensive microfluidics section. Our self designed chips helped us improve precision, safety, and quantification methods used throughout the project. To learn more about the microfluidic components of our project check out <a href="https://2014.igem.org/Team:EPF_Lausanne/Microfluidics">this link !</a></p>
 
-
 
-
 
-
<br /><br /><br />
 
-
<div class="pull-right img-right">
 
-
<img src="http://www.raspberrypi.org/wp-content/uploads/2011/07/RaspiModelB.png" alt="first" width="200" class="img-border">
 
-
</div>
 
-
<p class="lead">
 
-
<br />
 
-
Last but not least, we designed a novel signal detector ! To make signal detection more practical we developed an automatised cheap tracking system made of a mini-computer (Raspberry Pi) and a mini-HD camera. More details concerning this the BioPad detector can be found <a href="#CpxPathway">here.</a>
 
-
<br /><br /><br /></p>
 
-
 
-
 
-
 
-
 
-
<hr />
 
<br /><br />
<br /><br />
-
 
-
<a id="CpxPathway"></a>
 
-
  <h2>The Cpx Pathway</h2>
 
-
<br />
 
-
 
-
 
-
<!-- CpxA-CpxR PATHWAY DESCRIPTION -->
 
-
 
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<div class="cntr">
 
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<img src="https://static.igem.org/mediawiki/2014/6/62/Cpx_pathway_2_far_2.jpg" alt="Cpx_pathway_description_diagram" class="img-responsive">
 
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</div>
 
 +
<section id="CpxPathway">
 +
<h3 class="section-heading">Microfluidics parts (chips created)</h3>
<p class="lead">
<p class="lead">
-
The natural function of the Cpx two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses. The Cpx two component regulatory system belongs to the class I histidine kinases and includes three main proteins: </p>
+
Our team designed and made 4 microfluidic chips. At the beginning, we also used the <a target="_blank" href="http://link.springer.com/protocol/10.1007%2F978-1-61779-292-2_6">MITOMI chip</a>.</p>
-
<br />
+
<p class="lead">When designing the chips, the team took into account the future users and the current iGEM classification of parts. We considered it best to construct our chips as composite microfluidic parts, so their sub-parts could be used and combined in multiple ways. The flow and control layers can be separated and reused, as well as all the basic structures (chamber + connecting channel), nodes, array parts,...</p>
 +
<!-- send all lines here: https://2014.igem.org/Team:EPF_Lausanne/Microfluidics/Designing -->
 +
<table class="table table-striped table-hover" id="chips_list">
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  <tr>
 +
    <th>Name</th>
 +
    <th>Main Function</th>
 +
  </tr>
 +
  <tr>
 +
    <td>MITOMI modified</td>
 +
    <td>By using the MITOMI chip as a template, we designed our first chip that could squish the cells in the chamber.</td>
 +
  </tr>
 +
  <tr>
 +
    <td>SmashColi</td>
 +
    <td>To be able to separate the chip in 4 different compartments and apply 4 different pressures on each row of chambers.</td>
 +
  </tr>
 +
  <tr>
 +
    <td>BioPad</td>
 +
    <td>A large and simple microfluidic chip containing 6400 chambers in which the cells are contained in. Each chamber acts as a pixel for the BioPad project.</td>
 +
  </tr>
 +
  <tr>
 +
    <td>CleanColi</td>
 +
    <td>As a result of our Safety page, we decided to create a chip that is able to seal the bacteria in the chip, preventing them to leave the chip.</td>
 +
  </tr>
 +
  <tr>
 +
    <td>FilterColi</td>
 +
    <td>To successfully immerse cells in a certain solution, this chip was designed to flow in the new medium in the chambers instead of doing it by diffusion.</td>
 +
  </tr>
 +
</table>
 +
</section>
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<div class="cntr">
 
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<img src="https://static.igem.org/mediawiki/2014/4/41/Cpx_pathway_description_EPFL.jpg" width="70%" alt="Cpx_pathway_description">
 
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</div>
 
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<hr />
 
<br /><br />
<br /><br />
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<a id="howitworks"></a>
 
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<h2 class="section-heading">How the BioPad works in E Coli</h2>
 
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<p class="lead">
 
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Our self-designed PDMS microfluidic chip, the BioPad, is made of hundreds of compartments representing "pixels." Each 30µm x 30µm x 3µm compartment contains a few layers of E. coli. When the surface of the chip is touched, a deformation of the chip - and thus of the compartments - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm.
 
-
<br />
 
-
 
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The aggregated/misfolded proteins are then sensed by the histidine kinase CpxA sensor, which auto-phosphorylates and transfers its phosphate to its corresponding relay protein, CpxR. Upon phosphorylation, CpxR homo-dimerizes.
 
-
 
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<br />
 
-
Our engineered bacteria contain CpxR proteins fused to split fluorescent protein fragments (split IFP1.4) via a 10-amino acid, 2x GGGGS flexible linker. This allows us to detect CpxR dimerization, synonymous periplasmic stress and touch. Moreover, the split protein fragments are reversible. Therefore, when stress is removed, CpxA changes conformation and dephosphorylates CpxR allowing it to dissociate. The signal is shutdown and darkness returns The BioTouch Detector (composed of an inexpensive CMOS called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light emitting source) identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.
 
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</p>
 
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<div class="cntr">
 
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<img src="https://static.igem.org/mediawiki/2014/5/55/Touch4_epfl.jpg" alt="first">
 
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<img src="https://static.igem.org/mediawiki/2014/1/10/Touch5_epfl.jpg" alt="second">
 
</div>
</div>
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<br />
 
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<hr />
 
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<br />
 
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<a id="thehogpathway"></a>
 
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  <h2>The HOG Pathway</h2>
 
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<p class="lead">The HOG (High Osmolarity Glycerol) pathway is a MAPK (Mitogen activated protein kinase) pathway which yeast cells use to coordinate intracellular activities to optimise survival and proliferation in not only hyper-osmotic stress but also heat shock, nitrogen stress and oxidative stress. It is represented below.</p>
 
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<br />
 
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<br />
 
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<img src="https://static.igem.org/mediawiki/2014/6/6d/Hog_pathway_copy.jpg" width="750" alt="HOG_pathway_description">
 
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<br />
 
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The pathway includes five main proteins:
 
-
<ul style="padding-left:80px">
 
-
<li><p class="lead">Sho1/Sln1 – Membrane proteins which are classed as STREs (STress Response Elements) which sense the stress and initiate the pathway</p></li>
 
-
<li><p class="lead">Ste11 – The MAPKKK which phosphorylates PBS2</p></li>
 
-
<li><p class="lead">PBS2 – The MAPKK which phosphorylates HOG1</p></li>
 
-
<li><p class="lead">HOG1 – The MAPK which localizes to the nucleus upon phosphorylation and induces target gene transcription</p></li>
 
-
</ul>
 
-
<br>
 
-
<br>
 
-
 
-
<a id="howweengineered"></a>
 
-
  <h2>How we engineered the HOG pathway to make our BioPad</h2>
 
-
 
-
<p class="lead">
 
-
Our engineered yeasts cells can be loaded into a microfluidic chip made of small compartments able to contain a few layers of cells. When the surface of the chip is touched, it leads to a deformation of the chip - and thus of its compartments. Since the HOG pathway is reactive to turgor pressure, the pressure applied activates it. Upon induction of the pathway, which is a classical MAP kinase pathway, PBS2 – a MAPKK – is phosphorylated and binds HOG1 – a MAPK – and in turn phosphorylates it.
 
-
</p>
 
-
 
-
<p class="lead">
 
-
Therefore, we have fused these two kinases to split fluorescent and luminescent proteins, via a 13-amino acid flexible linker, by homologous recombination.  This allows us to detect the phosphorylation of Hog1 by Pbs2 in response to osmotic pressure or touch. We have used split sfGFP and split Renilla luciferase tags on the C-terminals of both proteins.
 
-
</p>
 
-
 
-
<p class="lead">
 
-
As in the E.Coli, the split sfGFP is irreversible and was made to show the interaction between our two Pbs2 and Hog1 while we use the reversible split luciferase tags to assess the activation and inactivation of the pathway. In fact, when stress is removed, the signal should decline. The BioTouch Detector is programmed to identify and process the light position and can transmit the information to a computer.
 
-
</p>
 
-
 
-
 
-
<hr>
 
-
<br /><br />
 
-
 
-
<h2 class="section-heading">The BioPad Detector</h2>
 
-
<p class="lead">
 
-
<br />
 
-
 
-
<!-- ENGINEERING DETECTOR -->
 
-
 
-
The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.
 
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</p>
 
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<nav id="affix-nav" class="sidebar hidden-sm hidden-xs">
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         <li class="active"><a href="#title_intro">Introduction</a></li>
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         <li class="active"><a href="#title_intro">Introduction</a></li>
         <li><a href="#CpxPathway">The Cpx Pathway</a></li>
         <li><a href="#CpxPathway">The Cpx Pathway</a></li>
         <li><a href="#howitworks">How the BioPad works in E Coli</a></li>
         <li><a href="#howitworks">How the BioPad works in E Coli</a></li>

Revision as of 13:19, 15 October 2014

Project

DNA parts submitted by the 2014 EPFL iGEM team

Our team submitted a total of 55 Biobricks (biobrick 51 does not exist).

In addition, 4 microfluidic designs have also been submitted to the registry.

Biobrick What it is Function Why do we use it? Group
BBa_K1486000 CpxR coding sequence Transcription factor To make most of our biobricks! Bacteria
BBa_K1486001 CpxR under arabinose promoter Treanscription factor Bacteria
BBa_K1486002 PAra + sfGFP CpxR [Nterm] Expresses fused protein Test CpxR expression & Ara promoter Bacteria
BBa_K1486003 Flexible linker Attaches two proteins together Bacteria
BBa_K1486004 Flexible linker Attaches two proteins together Bacteria
BBa_K1486005 PAra + CpxR sfGFP [Cterm] Expresses fused protein Test CpxR expression & Ara promoter Bacteria
BBa_K1486006 IFP[1] N terminus of split IFP Bacteria
BBa_K1486007 IFP[2] C terminus of split IFP Bacteria
BBa_K1486008 CxpR & Split IFP1.4 [Cterm + Cterm] Two CpxR CDS, each C terminus attached to a moiety of IFP Characterize CpxR dimerization Bacteria
BBa_K1486009 CxpR & Split IFP1.4 [Nterm + Nterm] Two CpxR CDS, each N terminus attached to a moiety of IFP Characterize CpxR dimerization Bacteria
BBa_K1486010 CxpR & Split IFP1.4 [Nterm + Cterm] Two CpxR CDS, each attached to a moiety of IFP Characterize CpxR dimerization Bacteria
BBa_K1486011 CxpR & Split IFP1.4 [Cterm + Nterm] Two CpxR CDS, each attached to a moiety of IFP Characterize CpxR dimerization Bacteria
BBa_K1486012 CpxR + IFP[1] CpxR with the Nterm moiety of IFP attached at its C terminus Intermediate & control Bacteria
BBa_K1486013 CpxR + IFP[2] CpxR with the Cterm moiety of IFP attached at its C terminus Intermediate & control Bacteria
BBa_K1486014 IFP[1] + CpxR CpxR with the Nterm moiety of IFP attached at its N terminus Intermediate & control Bacteria
BBa_K1486015 IFP[2] + CpxR CpxR with the Cterm moiety of IFP attached at its N terminus Intermediate & control Bacteria
BBa_K1486016 fLuc[1] N terminus moiety of the firefly luciferase Bacteria
BBa_K1486017 fLuc[2] C terminus moiety of the firefly luciferase Bacteria
BBa_K1486018 PAra + fLuc[1] + fLuc[2] Split firefly luciferase under arabinose promoter Control Bacteria
BBa_K1486019 rLuc[1] C terminus moiety of the renilla luciferase Bacteria
BBa_K1486020 rLuc[2] N terminus moiety of the renilla luciferase Bacteria
BBa_K1486021 PAra + rLuc[1] + rLuc[2] Split renilla luciferase under arabinose promoter Control Bacteria
BBa_K1486022 rLuc Full renilla luciferase Control Bacteria
BBa_K1486023 Yeast sfGFP Superfolder GFP for yeast cells Reporter Yeast
BBa_K1486024 Kan Yeast kanamycin resistance gene Selection marker Yeast
BBa_K1486025 ADH1 terminator Terminator Yeast
BBa_K1486026 Yeast sfGFP + Kan Yeast sfGFP attached to the yeast kanamycin resistance gene Control the expression of pbs2 Yeast
BBa_K1486027 rLuc + Kan Renilla luciferase attached to the kanamycin resistance gene Yeast
BBa_K1486028 Yeast sfGFP[1] N terminal moiety of split yeast sfGFP Yeast
BBa_K1486029 sfGFP[1] + kan Nterm moiety of split yeast sfGFP attached to yeast kanamycin resistance gene Yeast
BBa_K1486030 rLuc[1] + kan Nterm moiety of split renilla luciferase attached to yeast kanamycin resistance gene Yeast
BBa_K1486031 Ura CDS for Uracil (yeast selective purposes) Confer resistance to Uracil-deprived medium Yeast
BBa_K1486032 Yeast sfGFP + Ura Yeast sfGFP attached to the Uracil CDS Control the expression of hog1 Yeast
BBa_K1486033 rLuc + Ura Renilla luciferase attached to the Uracil CDS Control the expression of hog1 Yeast
BBa_K1486034 yeast sfGFP[2] C terminal moiety of split the yeast sfGFP Yeast
BBa_K1486035 yeast sfGFP[2] + Ura Cterm moiety of split yeast sfGFP attached to the Uracil CDS Yeast
BBa_K1486036 rLuc[2] + Ura Cterm moiety of split renilla luciferase attached to the Uracil CDS Yeast
BBa_K1486037 linker Attaches two proteins together Yeast
BBa_K1486038 sfGFP[1] N terminus moiety of split superfolder GFP Bacteria
BBa_K1486039 sfGFP[2] C terminus moiety of split superfolder GFP Bacteria
BBa_K1486040 sfGFP[1] + CpxR N terminus moiety of split sfGFP attached to CpxR Bacteria
BBa_K1486041 sfGFP[2] + CpxR C terminus moiety of split sfGFP attached to CpxR Bacteria
BBa_K1486042 LZip Monomer of leucine zipper TF Bacteria
BBa_K1486043 LZip + split rLuc Two Leucine Zipper monomers, each attached to a different split rLuc moiety Control for split rLuc assays Bacteria
BBa_K1486044 mut IFP[1] Biobrick-compatible IFP[1] Bacteria
BBa_K1486045 mut IFP[2] Biobrick-compatible IFP[2] Bacteria
BBa_K1486046 CpxR promoter FW CpxR binding-region in forward direction Bacteria
BBa_K1486047 CpxR promoter RV CpxR binding-region in reverse direction Bacteria
BBa_K1486048 CpxR reporter Calgary's CpxR reporter repaired (sequence was missing) To see when CpxR is active Bacteria
BBa_K1486049 CpxR promoter FW + RFP Reporter of CpxR Test the direction of the complete CpxR promoter Bacteria
BBa_K1486050 CpxR promoter RV + RFP Reporter of CpxR Test the direction of the complete CpxR promoter Bacteria
BBa_K1486052 Spacer 40 bases placed between constructs Separate two constructs in the same plasmid Bacteria
BBa_K1486053 Linker 10 amino-acid linker Attach CheY/Z to split luciferases Bacteria
BBa_K1486054 CheY/CheZ rLuc CheY and CheZ, each attached to a moiety of split renilla luciferase Positive control for the split rLuc Bacteria
BBa_K1486055 CheY/CheZ fLuc CheY and CheZ, each attached to a moiety of split firefly luciferase Positive control for the split fLuc Bacteria
BBa_K1486056 CxpR & Split mut IFP1.4 [Cterm + Cterm] Two CpxR CDS, each C terminus attached to a moiety of the biobrick-compatible IFP Characterize CpxR dimerization Bacteria


Microfluidics parts (chips created)

Our team designed and made 4 microfluidic chips. At the beginning, we also used the MITOMI chip.

When designing the chips, the team took into account the future users and the current iGEM classification of parts. We considered it best to construct our chips as composite microfluidic parts, so their sub-parts could be used and combined in multiple ways. The flow and control layers can be separated and reused, as well as all the basic structures (chamber + connecting channel), nodes, array parts,...

Name Main Function
MITOMI modified By using the MITOMI chip as a template, we designed our first chip that could squish the cells in the chamber.
SmashColi To be able to separate the chip in 4 different compartments and apply 4 different pressures on each row of chambers.
BioPad A large and simple microfluidic chip containing 6400 chambers in which the cells are contained in. Each chamber acts as a pixel for the BioPad project.
CleanColi As a result of our Safety page, we decided to create a chip that is able to seal the bacteria in the chip, preventing them to leave the chip.
FilterColi To successfully immerse cells in a certain solution, this chip was designed to flow in the new medium in the chambers instead of doing it by diffusion.


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