Team:Paris Bettencourt/YonatanTest

From 2013.igem.org

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     <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2>
     <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Detect">Detect</a></h2>
     <div class="overbox">
     <div class="overbox">
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       <div class="bkgr">
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       <div class="subbox1">
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<h2>Background</h2>
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<div class="bkgr">
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<p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing.  </p>
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  <h2>Background</h2>
 +
  <p>CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing.  </p>
 +
</div>
 +
<div class="aims">
 +
  <h2>Aims</h2>
 +
  <p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p>
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</div>
       </div>
       </div>
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       <div class="results">
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       <div class="subbox2">
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<h2>Results</h2>
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<div class="results">
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<ul>
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  <h2>Results</h2>
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          <li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li>
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  <ul>
-
          <li>Testing the new assembly standard for our cloning.</li>
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            <li>Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks. </li>
 +
            <li>Testing the new assembly standard for our cloning.</li>
 +
  </ul>
 +
</div>
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<div class="biocriks">
 +
  <ol>
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            <li><a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank">BBa_K1137012 (gRNA anti-KAN)</a></li>
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            <li><a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank">BBa_K1137013 (crRNA anti-KAN)</a></li>
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            <li><a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank">BBa_K1137014 (tracrRNA-Cas9)</a></li>
 +
            <li><a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank">BBa_K1137015 (pRecA-LacZ)</a></li>
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  </ol>
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</div>
       </div>
       </div>
       <div class="mainfig">
       <div class="mainfig">
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        <center> <img width="90%" src="https://static.igem.org/mediawiki/2013/1/1f/PB_phagesensor10.png"/><br></center>
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        <center> <img width="90%" src="https://static.igem.org/mediawiki/2013/1/1f/PB_phagesensor10.png"/><br></center>
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        <p>After expression of the Cas9 and gRNA, the gRNA guides the Cas9 to the target sequence, the kanamycin resistance. There, the Cas9 generates a double strand break. This activates the SOS response. The reporter LacZ is under the pRECA promoter, which gets activated during the SOS response and we get hence a blue cell, if the resistance gene has successfully been detected.</p>
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        <p>After expression of the Cas9 and gRNA, the gRNA guides the Cas9 to the target sequence, the kanamycin resistance. There, the Cas9 generates a double strand break. This activates the SOS response. The reporter LacZ is under the pRECA promoter, which gets activated during the SOS response and we get hence a blue cell, if the resistance gene has successfully been detected.</p>
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    </div>
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      </div>
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      /*<div style="clear: both;"></div>*/
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      <div class="aims">
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<h2>Aims</h2>
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<p>Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.</p>
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      </div>
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      <div class="biocriks">
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<ol>
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          <li><a href="http://parts.igem.org/Part:BBa_K1137012" target="_blank">BBa_K1137012 (gRNA anti-KAN)</a></li>
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          <li><a href="http://parts.igem.org/Part:BBa_K1137013" target="_blank">BBa_K1137013 (crRNA anti-KAN)</a></li>
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          <li><a href="http://parts.igem.org/Part:BBa_K1137014" target="_blank">BBa_K1137014 (tracrRNA-Cas9)</a></li>
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          <li><a href="http://parts.igem.org/Part:BBa_K1137015" target="_blank">BBa_K1137015 (pRecA-LacZ)</a></li>
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</ol>
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       </div>
       </div>
     </div>
     </div>
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     <div style="clear: both;"></div>
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    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Target">Target</a></h2>
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    <div class="overbox">
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      <div class="bkgr">
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<h2>Background</h2>
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<p>SirA is an essential gene in latent tuberculosis infections</p>
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      </div>
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      <div class="results">
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<h2>Results</h2>
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<ul>
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  <li>Produced an E. coli strain which relies upon mycobacterial sirA, fprA and fdxA genes to survive in M9 minimal media</li>
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  <li>Demonstrated that E. coli can survive with mycobacterial sulfite reduction pathway with Flux Balance Analysis</li>
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  <li>Located drug target sites on sirA as well as identified high structural similarity between cysI and sirA through structural anaylsis</li>
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</ul>
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<p></p>
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      </div>
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      <div class="biocriks">
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<h2>BioBricks</h2>
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<ol>
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  <li><a href="http://parts.igem.org/Part:BBa_K1137000">BBa_K1137000 (SirA)</a></li>
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  <li><a href="http://parts.igem.org/Part:BBa_K1137001">BBa_K1137001 (FprA)</a></li>
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  <li><a href="http://parts.igem.org/Part:BBa_K1137002">BBa_K1137002 (FdxA)</a></li>
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</ol>
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      </div>
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      <div style="clear: both;"></div>
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      <div class="aims">
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<h2>Aims</h2>
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<p>To perform an drug screen targeted at the sirA gene from mycobacteria</p>
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      </div>
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      <div style="clear: both;"></div>
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    </div>
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    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Infiltrate">Infiltrate</a></h2>
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    <div class="overbox">
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-
      <div class="bkgr">
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<h2>Background</h2>
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<p>Latent tuberculosis persists inside macrophages of the lungs, where it is partially protected from both the host immune system and conventional antibiotics.</p>
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-
      </div>
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      <div class="results">
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<h2>Results</h2>
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<ul>
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  <li>We expressed the enzyme Trehalose Dimycolate Hydrolase (TDMH) in <i>E.coli</i> and showed that it is highly toxic to mycobacteria in culture.</li>
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  <li>We expressed the lysteriolyin O (LLO) gene in <i>E. coli</i> and showed that it is capable of entering the macrophage cytosol.</li>
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  <li>We co-infected macrophages with both mycobacteria and our engineered <i>E. coli</i> to characterize the resulting phagocytosis and killing.</li>
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-
</ul>
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<p></p>
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      </div>
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      <div class="biocriks">
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<h2>BioBricks</h2>
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-
<ol>
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  <li><a href="http://parts.igem.org/Part:BBa_K1137008" target="_blank">BBa_K1137008 (TDMH)</a></li>
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</ol>
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      </div>
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      <div style="clear: both;"></div>
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      <div class="aims">
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<h2>Aim</h2>
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<p>To create an <i>E. coli</i> strain capable of entering the macrophage cytosol and delivering a lytic enzyme to kill mycobacteria.</p>
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      </div>
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      <div style="clear: both;"></div>
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    </div>
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    <h2><a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage">Sabotage</a></h2>
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-
    <div class="overbox">
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-
      <div class="bkgr">
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<h2>Background</h2>
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<p>One of the main concern about tuberculosis today is the emergence of antibiotic resistant strain</p>
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-
      </div>
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-
      <div class="results">
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-
<h2>Results</h2>
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-
<ul>
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-
  <li>Construction and characterization of phagemids coding for small RNA targeting antibiotic resistance proteins</li>
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-
  <li>successful conversion of antibiotic resistant population of E. coli to a sensitive state</li>
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-
</ul>
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-
<p></p>
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-
      </div>
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-
      <div class="biocriks">
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<h2>BioBricks</h2>
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-
<ol>
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-
  <li><a href="http://parts.igem.org/Part:BBa_K1137009" target="_blank">BBa_K1137009 (sRNA anti Kan)&nbsp;&nbsp;&nbsp; Characterized </a></li>
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-
  <li><a href="http://parts.igem.org/Part:BBa_K1137010" target="_blank">BBa_K1137010 (sRNA anti Cm)&nbsp;&nbsp;&nbsp; Characterized</a></li>
+
-
  <li><a href="http://parts.igem.org/Part:BBa_K1137011" target="_blank">BBa_K1137011 (sRNA anti Lac)&nbsp;&nbsp;&nbsp;</a></li>
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-
</ol>
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-
      </div>
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      <div style="clear: both;"></div>
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      <div class="aims">
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<h2>Aims</h2>
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<p>Our objective is to make an antibiotic-resistant bacterial population sensitive again to those same antibiotics.</p>
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      </div>
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      <div style="clear: both;"></div>
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    </div>
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   </div>
   </div>
    
    

Revision as of 09:01, 25 October 2013

Detect

Background

CRISPR/Cas systems generate site-specific double strand breaks and have recently been used for genome editing.

Aims

Building a genotype sensor based on CRISPR/Cas that reports existance of an antibiotic resistance gene.

Results

  • Successfully cloned gRNA anti-KAN, crRNA anti-KAN, tracrRNA-Cas9 and pRecA-LacZ into Biobrick backbones and therefore generated four new BioBricks.
  • Testing the new assembly standard for our cloning.
  1. BBa_K1137012 (gRNA anti-KAN)
  2. BBa_K1137013 (crRNA anti-KAN)
  3. BBa_K1137014 (tracrRNA-Cas9)
  4. BBa_K1137015 (pRecA-LacZ)

After expression of the Cas9 and gRNA, the gRNA guides the Cas9 to the target sequence, the kanamycin resistance. There, the Cas9 generates a double strand break. This activates the SOS response. The reporter LacZ is under the pRECA promoter, which gets activated during the SOS response and we get hence a blue cell, if the resistance gene has successfully been detected.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
team2013@igem-paris.org
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