Team:EPF Lausanne/Data

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            <!-- RESULTS -->
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<h1><b>DATA</b></h1>
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<h2>Engineering stress-related pathways to create a BioPad</h2>
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</div>
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<br />
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<h3><b>Bacterial Biosensors </b> </h3>
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<div  class="cntr">
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<a href="https://static.igem.org/mediawiki/2014/f/fd/CpxR-HOG.jpg" data-lightbox="cpxr" data-title="Touch response in yeast and bacteria">
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<img src="https://static.igem.org/mediawiki/2014/f/fd/CpxR-HOG.jpg" alt="touch response" class="img-responsive img-border" /></a>
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<figcaption class="cntr">Touch response in yeast and bacteria</figcaption>
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<br/><br/>
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<h3 id="characterization">Our BioBricks</h3>
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<h4 id="doc_and_sub"><b>New Parts</b> </h4>
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<h1><b>DATA</b></h1>
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<li>Submitted a BioBrick consisting of CpxR under Arabinose Promoter (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486001">BBa_K1486001</a>). Reporter sfGFP was fused to CpxR's N terminus (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486002">BBa_K1486002</a>) and C terminus (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486005">BBa_K1486005</a>), to evaluate the expression of our construct and characterize the Arabinose promoter in <i>E. coli</i>.</li><br/>
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<h3><b>Bacterial Biosensors </b> </h3>
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<li>Submitted four BioBricks coding for CpxR fused to one of the two fragments of the split reporter Infrared Fluorescent Protein (IFP1.4). Fragments IFP1 and IFP2 (corresponding to the N and C terminal of the IFP respectively) were fused at the N or C terminal of the CpxR, leading two four different constructs: CpxR-IFP1 (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486012">BBa_K1486012</a>), CpxR-IFP2 (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486013">BBa_K1486013</a>), IFP1-CpxR (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486014">BBa_K1486014</a>) and IFP2-CpxR (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486015">BBa_K1486015</a>). Sequences are under arabinose promoter. These Biobricks are construction intermediates to build our four final constructs below. </li><br/>
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<li>Submitted four BioBricks, which are the final constructs of the IFP subproject. Each BioBrick codes for two proteins: CpxR fused to IFP1, and CpxR fused to IFP2. The different combinations with the split at the C or N terminal of CpxR lead to four constructs: CpxR-IFP1 & CpxR-IFP2 (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486056">BBa_K1486056</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486008">BBa_K1486008</a>), IFP1-CpxR & IFP2-CpxR (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486009">BBa_K1486009</a>), IFP1-CpxR & Cpxr-IFP2 (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486010">BBa_K1486010</a>), CpxR-IFP1 & IFP2-CpxR (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486011">BBa_K1486011</a>). Sequences are under arabinose promoter.
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These BioBricks aimed at first to study the orientation of CpxR's homodimerization by observing the IFP signal measured from the four strains under stress. We discovered that only the orientations with both parts of the split are at the C terminal of the CpxR lead to an IFP signal when cells are stressed. Since all of these Biobricks are incompatible with RFC[10], we removed illegal restriction sites from <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486008">BBa_K1486008</a> and created <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486056">BBa_K1486056</a> (version without PstI illegal restriction enzyme sites). The experiment mentioned above was repeated with <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486056">BBa_K1486056</a> and identical results were reproduced. All further experiments were done with <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486056">BBa_K1486056</a>.  </li><br/>
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<li>CpxR reporters were made with the promoter in forward and reverse direction, respectively <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>. These Biobricks are improvements of <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007">BBa_K339007</a> </li><br/>
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<li>Submitted the two parts of the split yeast optimized superfolder GFP  (N-terminal part (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486029">BBa_K1486029</a>) and C-terminal part (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486035">BBa_K1486035</a>)) created from plasmids pFA6a-link-yoSuperfolderGFP-Kan (44901) and pFA6a-link-yoSuperfolderGFP-Ura (44873) ordered from Addgene. We attached them to the ADH1 terminator, regulating the transcripion of our fusion proteins and to the selection markers Kan and Ura3. We stressed our cells under various conditions to trigger the HOG pathway and were able to show that interaction of Hog1 and Pbs2 in response to osmotic stress allowed the re-assembly of the full GFP protein.
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</li><br />
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<h3><b>Split Luciferase Complementation Assay </b> </h3></br>
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<li>Submitted a BioBrick (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486043">BBa_K1486043</a>) containing two leucine zipper sequences, each fused with one moiety of renilla Luciferase, to test the efficiency of the split renilla luciferase in order to use it for a complementation assay.</li>
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Advantages of luciferase for our project :
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</ul><br/>
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<ul>
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<li>Fast and reversible signal (quasi-immediate) </li>
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<li> Light excitation not needed (minimal autofluorescence) => simplification of the signal detection and processing mechanisms</li>
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<li> Signal highly correlated with concentration of substrate => very modulable </li>
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<li> Small concentrations of substrate are needed</li>
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</ul>
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</br>
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<p>Our constructs :</br>
 
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N terminal part of Luciferase fused to CheZ and C terminal part of Luciferase fused to CheY. </p>
 
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<div class = "cntr">
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<br />
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<img src="https://static.igem.org/mediawiki/2014/e/ed/CheYCheZscheme.png" alt="Exp_IFP" width="625">
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<figcaption></figcaption>
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<p>In absence of chemoattractant, CheZ and CheY are interacting and the two parts of the split reassemble to oxidize luciferin and thus luminescence is triggered. After addition of a chemoattractant (e.g. Glucose), CheZ and CheY don't interact anymore and luminescence is shut down.</p>
 
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<p>We created two different constructs, one with split Firefly luciferase and one with split Renilla luciferase, to test different possibilities with different substrates (Firefly luciferase oxidizes d-luciferin and Renilla luciferase oxidizes coelenterazine)</p>
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<h4 id="improvement"><b>Further Characterization and Improvement of Parts Already in the Registry</b> </h4>
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<p>
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</br>
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<ul>
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<li>After several experiments with stress induction we realised that the biobrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007"> BBa_K339007 </a> was missing its CpxR responsive promoter. So we repaired it and sent it as <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486048">BBa_K1486048</a>. The biobrick was further engineered by testing the native CpxR target sequence that is found in front of CpxA in the E.coli genome (as Calgary's <a target="_blank" href="http://parts.igem.org/Part:BBa_K339007"> BBa_K339007 </a> did not include the whole sequence). These are the biobricks <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486049">BBa_K1486049</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486050">BBa_K1486050</a>, with the promoter in forward and reverse direction respectively.</li><br/>
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Data For Our Favorite New Parts</br></br>
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<li>Submitted the two parts of the split of <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108">EPIC Firefly luciferase</a> (N-terminal part (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486016">BBa_K1486016</a>) and C-terminal part (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486017">BBa_K1486017</a>)) from Cambridge 2010. The plasmid (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486018">BBa_K1486018</a>) containing the two parts of the split separated by a spacer can be very useful as a negative control or to establish a background noise for a complementation assay experiment.</li><br/>
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</br>
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<li>Compared the <a target="_blank" href="http://parts.igem.org/Part:BBa_K325108">EPIC Firefly luciferase</a> from Cambridge 2010 team to the renilla luciferase (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486022">BBa_K1486022</a>) in the same conditions, to determine which one is best suited for a complementation assay experiment. The full and split luciferases have been compared. Renilla luciferase (full and splits(<a target="_blank" href="http://parts.igem.org/Part:BBa_K1486021">BBa_K1486021</a>)) have been submitted.</li>
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</ul></p><br/>
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<h3><b>Characterization of New Parts Submitted to the Registry</b> </h3>
 
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<p>Documented & submitted:
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<h3 id="title_microfluidics"><b>Microfluidics</b> </h3>
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<ul>
+
-
<li>BBa_K1486001 (Ara CpxR http://parts.igem.org/Part:BBa_K1486001 ) </li>
+
-
<li>BBa_K1486002 (Ara GFPCpxR http://parts.igem.org/Part:BBa_K1486002 )</li>
+
-
<li>BBa_K1486005 (Ara CpxrGFP http://parts.igem.org/Part:BBa_K1486005 )</li>
+
-
<li>BBa_K1486043 (LeuZ + rLuc http://parts.igem.org/Part:BBa_K1486043 ) </li>
+
-
<li>BBa_K1486049 (CpxR reporter promoter FW http://parts.igem.org/Part:BBa_K1486049 )</li>
+
-
<li>BBa_K1486050 (CpxR reporter promoter RV http://parts.igem.org/Part:BBa_K1486050 )</li>
+
-
<li>BBa_K1486056 (Ga1Mut http:/parts.igem.org/Part:BBa_K1486056 ) </li>
+
-
</ul>
+
-
</p>
+
-
<p>Works as expected:
+
-
<ul>
+
-
<li>BBa_K1486002 (Ara GFPCpxR http://parts.igem.org/Part:BBa_K1486002 )</li>
+
-
<li>BBa_K1486005 (Ara CpxRGFP http://parts.igem.org/Part:BBa_K1486005 )</li>
+
-
<li>BBa_K1486008 (GA1 TedCécile http://parts.igem.org/Part:BBa_K1486008 )</li>
+
-
<li>BBa_K1486012 (CpxR IFP1 http://parts.igem.org/Part:BBa_K1486012 )</li>
+
-
<li>BBa_K1486013 (cpxR IFP2 http://parts.igem.org/Part:BBa_K1486013 )</li>
+
-
<li>BBa_K1486014 (IFP1 CpxR rLuc http://parts.igem.org/Part:BBa_K1486014 )</li>
+
-
<li>BBa_K1486015 (IFP2 CpxR rLuc http://parts.igem.org/Part:BBa_K1486015 )</li>
+
-
<li>BBa_K1486018 (Ara split fLuc http://parts.igem.org/Part:BBa_K1486018 )</li>
+
-
<li>BBa_K1486021 (Ara split rLuc http://parts.igem.org/Part:BBa_K1486021 )</li>
+
-
</ul></p>
+
-
<p>Works as expected & submitted:
+
-
<ul>
+
-
<li>BBa_K1486002 (Ara GFPCpxR http://parts.igem.org/Part:BBa_K1486002 )</li>
+
-
<li>BBa_K1486005 (Ara CpxrGFP http://parts.igem.org/Part:BBa_K1486005 )</li>
+
-
<li>BBa_K1486018 (Ara split fLuc http://parts.igem.org/Part:BBa_K1486018 )</li>
+
-
<li>BBa_K1486021 (Ara split rLuc http://parts.igem.org/Part:BBa_K1486021 )</li>
+
-
<li>BBa_K1486049 or BBaK1486050 (CpxR reporter promoter FW or RV http://parts.igem.org/Part:BBa_K1486050 )</li>
+
-
</ul></p>
+
-
</br>
+
-
 
+
-
<h3><b>Further Characterization and Improvement of Parts Already in the Registry</b> </h3>
+
<p>
<p>
<ul>
<ul>
-
<li>Documented & submitted: BBa_K1486056 (CpxR reporter (Calgary) http://parts.igem.org/Part:BBa_K1486056 ) We repaired it</li>
+
<li>Design of <a target="_blank" href="https://static.igem.org/mediawiki/2014/b/b7/SmashColi_iGEM_EPFL_2014.zip">SmashColi</a> - a testing chip to analyse the effects of different mechanical stresses on cells. This chip was used to characterize <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486056">BBa_K1486056</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1486013">BBa_K1486013</a>. </li>
-
</ul></p>
+
<li>Design of <a target="_blank" href="https://static.igem.org/mediawiki/2014/7/79/FilterColi_iGEM_EPFL_2014.zip">FilterColi</a> - a testing chip to analyse the effects of different osmotic stresses on cells</li>
-
 
+
<li>Design of <a target="_blank" href="https://static.igem.org/mediawiki/2014/3/33/TheBioPad_iGEM_EPFL_2014.zip">The BioPad</a> - a large-scaled chip implemented to be the touch-senstive interface of our final trackpad</li>
-
 
+
<li>Design of <a target="_blank" href="https://static.igem.org/mediawiki/2014/b/b3/CleanColi_iGEM_EPFL_2014.zip">CleanColi</a> - an "on-chip waste treatment" unit that can be integrated at the end of any chip to decontaminate GMOs or pathogens</li>
-
<h3><b>Microfluidics</b> </h3>
+
<br /><p>To find out more about what we did for each chip, click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results#Micro_exp1">here</a></p>
-
<h3><b>Modelling</b> </h3>
+
</ul></p><br/>
-
<h3><b>Human Practices</b> </h3>
+
<h3 id="title_human_practices"><b>Human Practices</b> </h3><br/>
<ul>
<ul>
<li>Met with a journalist from the biggest newspaper of our region (Le Temps) and got an article about our project.</li>
<li>Met with a journalist from the biggest newspaper of our region (Le Temps) and got an article about our project.</li>
-
<li>Our work was commented by Bent Stumpe, inventor of the touchscreen, as well as Rolf Heuer, the current director of the CERN, in Geneva</li>
+
<li>Our work was commented by Bent Stumpe, inventor of the touchscreen, as well as Rolf Heuer, the current director of the CERN, in Geneva.</li>
-
<li>Organized an outreach event with 80 highschool students at EPFL, teaching them about synthetic biology as well as laboratory techniques and made them participate in a game called « mini iGEM ».</li>
+
<li>Organized an outreach event with 80 highschool students at EPFL, teaching them about synthetic biology as well as laboratory techniques and made them participate in a game called « <a target="_blank" href="https://static.igem.org/mediawiki/2014/7/76/Mini_iGEM_projects.pdf">mini iGEM</a> ».</li>
-
<li>Presented our work at the Hackuarium in Renens</li>
+
<li>Presented iGEM and our work at the Hackuarium, the new BioHackerspace in Lausanne.</li>
-
</ul></p>
+
</ul>
-
 
+
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</br></br></br>
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+
 +
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 +
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          <nav id="affix-nav" class="sidebar hidden-sm hidden-xs">
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            <ul class="nav sidenav box" data-spy="affix" data-offset-top="200" data-offset-bottom="600">
 +
              <li><a href="#characterization">Characterization</a>
 +
                <ul class="nav">
 +
                  <li><a href="#doc_and_sub">New Parts</a></li>
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                  <li><a href="#improvement">Existing Parts</a></li>
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                </ul>
 +
            </li>
 +
            <li><a href="#title_microfluidics">Microfluidics</a></li>
 +
            <li><a href="#title_human_practices">Human Practices</a></li>
 +
        </ul>
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      </nav>
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Latest revision as of 03:21, 18 October 2014

DATA

Engineering stress-related pathways to create a BioPad


touch response
Touch response in yeast and bacteria


Our BioBricks

New Parts

  • Submitted a BioBrick consisting of CpxR under Arabinose Promoter (BBa_K1486001). Reporter sfGFP was fused to CpxR's N terminus (BBa_K1486002) and C terminus (BBa_K1486005), to evaluate the expression of our construct and characterize the Arabinose promoter in E. coli.

  • Submitted four BioBricks coding for CpxR fused to one of the two fragments of the split reporter Infrared Fluorescent Protein (IFP1.4). Fragments IFP1 and IFP2 (corresponding to the N and C terminal of the IFP respectively) were fused at the N or C terminal of the CpxR, leading two four different constructs: CpxR-IFP1 (BBa_K1486012), CpxR-IFP2 (BBa_K1486013), IFP1-CpxR (BBa_K1486014) and IFP2-CpxR (BBa_K1486015). Sequences are under arabinose promoter. These Biobricks are construction intermediates to build our four final constructs below.

  • Submitted four BioBricks, which are the final constructs of the IFP subproject. Each BioBrick codes for two proteins: CpxR fused to IFP1, and CpxR fused to IFP2. The different combinations with the split at the C or N terminal of CpxR lead to four constructs: CpxR-IFP1 & CpxR-IFP2 (BBa_K1486056 and BBa_K1486008), IFP1-CpxR & IFP2-CpxR (BBa_K1486009), IFP1-CpxR & Cpxr-IFP2 (BBa_K1486010), CpxR-IFP1 & IFP2-CpxR (BBa_K1486011). Sequences are under arabinose promoter. These BioBricks aimed at first to study the orientation of CpxR's homodimerization by observing the IFP signal measured from the four strains under stress. We discovered that only the orientations with both parts of the split are at the C terminal of the CpxR lead to an IFP signal when cells are stressed. Since all of these Biobricks are incompatible with RFC[10], we removed illegal restriction sites from BBa_K1486008 and created BBa_K1486056 (version without PstI illegal restriction enzyme sites). The experiment mentioned above was repeated with BBa_K1486056 and identical results were reproduced. All further experiments were done with BBa_K1486056.

  • CpxR reporters were made with the promoter in forward and reverse direction, respectively BBa_K1486049 and BBa_K1486050. These Biobricks are improvements of BBa_K339007

  • Submitted the two parts of the split yeast optimized superfolder GFP (N-terminal part (BBa_K1486029) and C-terminal part (BBa_K1486035)) created from plasmids pFA6a-link-yoSuperfolderGFP-Kan (44901) and pFA6a-link-yoSuperfolderGFP-Ura (44873) ordered from Addgene. We attached them to the ADH1 terminator, regulating the transcripion of our fusion proteins and to the selection markers Kan and Ura3. We stressed our cells under various conditions to trigger the HOG pathway and were able to show that interaction of Hog1 and Pbs2 in response to osmotic stress allowed the re-assembly of the full GFP protein.

  • Submitted a BioBrick (BBa_K1486043) containing two leucine zipper sequences, each fused with one moiety of renilla Luciferase, to test the efficiency of the split renilla luciferase in order to use it for a complementation assay.


Further Characterization and Improvement of Parts Already in the Registry

  • After several experiments with stress induction we realised that the biobrick BBa_K339007 was missing its CpxR responsive promoter. So we repaired it and sent it as BBa_K1486048. The biobrick was further engineered by testing the native CpxR target sequence that is found in front of CpxA in the E.coli genome (as Calgary's BBa_K339007 did not include the whole sequence). These are the biobricks BBa_K1486049 and BBa_K1486050, with the promoter in forward and reverse direction respectively.

  • Submitted the two parts of the split of EPIC Firefly luciferase (N-terminal part (BBa_K1486016) and C-terminal part (BBa_K1486017)) from Cambridge 2010. The plasmid (BBa_K1486018) containing the two parts of the split separated by a spacer can be very useful as a negative control or to establish a background noise for a complementation assay experiment.

  • Compared the EPIC Firefly luciferase from Cambridge 2010 team to the renilla luciferase (BBa_K1486022) in the same conditions, to determine which one is best suited for a complementation assay experiment. The full and split luciferases have been compared. Renilla luciferase (full and splits(BBa_K1486021)) have been submitted.


Microfluidics

  • Design of SmashColi - a testing chip to analyse the effects of different mechanical stresses on cells. This chip was used to characterize BBa_K1486056 and BBa_K1486013.
  • Design of FilterColi - a testing chip to analyse the effects of different osmotic stresses on cells
  • Design of The BioPad - a large-scaled chip implemented to be the touch-senstive interface of our final trackpad
  • Design of CleanColi - an "on-chip waste treatment" unit that can be integrated at the end of any chip to decontaminate GMOs or pathogens

  • To find out more about what we did for each chip, click here


Human Practices


  • Met with a journalist from the biggest newspaper of our region (Le Temps) and got an article about our project.
  • Our work was commented by Bent Stumpe, inventor of the touchscreen, as well as Rolf Heuer, the current director of the CERN, in Geneva.
  • Organized an outreach event with 80 highschool students at EPFL, teaching them about synthetic biology as well as laboratory techniques and made them participate in a game called « mini iGEM ».
  • Presented iGEM and our work at the Hackuarium, the new BioHackerspace in Lausanne.

Sponsors