Team:INSA Toulouse/contenu/project/e calculus design

From 2013.igem.org

(Difference between revisions)
(Created page with "{{:Team:INSA_Toulouse/template/header}} {{:Team:INSA_Toulouse/template/sidebar}} <!--Contenu*/ /**********/--> <html> <!--- open Sans : fonnt Google: --> <link href='htt...")
 
(21 intermediate revisions not shown)
Line 68: Line 68:
  <!--/* HTML Contenu*/-->
  <!--/* HTML Contenu*/-->
-
<div class="maincontent" style="width: 720px; margin: 25px 0 25px 0; float: right;">
+
<div class="maincontent" style="width: 700px; margin: 25px 0 25px 0; float: right;">
-
   <h1 class="title1">Parts</h1>
+
   <h1 class="title1"><i>E. calculus</i> design</h1>
-
   <h2 class="title2">Used Parts</h2>
+
   <h2 class="title2">General idea</h2>
-
   <p class="texte">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut et dolor turpis. Suspendisse interdum, dolor eu  ultricies dignissim, arcu sapien placerat quam, ut bibendum lorem nibh id magna. Nunc condimentum lectus at diam tempus sodales. Nullam scelerisque auctor tellus, nec auctor ante elementum sit amet. Aenean venenatis velit eget porttitor laoreet. Aliquam aliquam libero in ante imperdiet vehicula. Donec nibh urna, tincidunt quis condimentum at, tempus sed sapien. Nunc elit dui, dignissim tincidunt mi non, ultrices ultricies erat.</p>
+
   <p class="texte">The general idea of the project was to have distinct colonies of bacteria, each being a one bit calculator. The <i>E. calculus</i> strain can then be streaked several times, in the form of a thin line or circle on an agar plate, at regular intervals. The input signal (blue or red light) illuminates only one group of <i>E. calculus</i> at a time, allowing the full one bit calculation and the synthesis of the carry (AHL) if needed. After a certain period of time (long enough for the carry to diffuse to the next colony), illumination of the second colony can start. As the system is genetically stable, a relatively small amount of cells can be used, leading to amplification of the signal during growth of the switched bacteria. The output signal (RFP) would then be produced, stating a clear difference between the 0 or 1 state. </p>
-
 
+
 
-
  <img src="https://static.igem.org/mediawiki/2013/b/b7/Insa-toulouse2013-phototestpage.jpg" class="imgcontent" />
+
-
  <p class="texteleft"><span class="spantitle">lorem ipsum</span></br>
+
  <img style="width:650px; float:left;" src="https://static.igem.org/mediawiki/2013/6/60/E.caclulusdesign.GeneralIdea.png" class="imgcontent" />
-
<br>Mauris ac suscipit erat, sit amet blandit lectus. Fusce placerat, lectus at suscipit viverra, lorem lorem mattis turpis, id pharetra velit nunc vitae velit. Etiam ultrices aliquam ligula, sed ultrices nibh vulputate non. Ut dapibus arcu luctus, suscipit urna et, imperdiet magna. Curabitur tristique sed diam non elementum. Sed lectus urna, consequat quis porta eu, ultrices in nunc. Aenean vitae elit neque.<p>
+
<div class="clear"></div>
-
  <p class="texteright"><span class="spantitle">lorem ipsum</span></br>
+
      <p class="texte">We also imagined that the <i>E. calculus</i> strain should have two different states, one for living happy state, feeding on the agar plate and dividing, not bothering about blue or red lights and a second state, dedicated to calculation. We therefore implemented a general inducer based on TetR repression and activation that would switch the bacteria to the "calculation ready" state. TetR, constitutively expressed would repress the transcription of the three receptors (blue light, red light and AHL receptors). In the presence of aTc (anhydrotetracycline) TetR would not repress anymore the promoter controlling the receptors genes. Upon illumination or when AHL is bound, each receptor will activate the transcription of the different recombinases, PhiC31 for the carry, Bxb1 for the blue light and Tp901.1 for the red light. When the recombinases are produced, they will switch their specific gates. Depending on the switch performed, the output will (or will not!) be produced.</p>
-
<br>Mauris ac suscipit erat, sit amet blandit lectus. Fusce placerat, lectus at suscipit viverra, lorem lorem mattis turpis, id pharetra velit nunc vitae velit. Etiam ultrices aliquam ligula, sed ultrices nibh vulputate non. Ut dapibus arcu luctus, suscipit urna et, imperdiet magna. Curabitur tristique sed diam non elementum. Sed lectus urna, consequat quis porta eu, ultrices in nunc. Aenean vitae elit neque.</p>
+
 +
  <img style="width:650px; float:left;" src="https://static.igem.org/mediawiki/2013/0/06/Ecalculus_design.png" class="imgcontent" />
<div class="clear"></div>
<div class="clear"></div>
-
  <div class="list">
 
-
   
 
-
    <ul class="arrow">
 
-
      <li>Quisque at massa ipsum</li>
 
-
      <li>Maecenas a lorem augue, egestas </li>
 
-
      <li>Cras vitae felis at lacus ele </li>
 
-
      <li>Etiam auctor diam pellentesque </li>
 
-
      <li>Nulla ac massa at dolor </li>
 
-
    </ul>
 
-
  </div>
+
  <h2 class="title2">Implementing the whole calculator in one strain </h2>
-
  <div class="list">
+
  <p class="texte">The full DNA construct, general inducer, input system, logic gates, output and carry was clearly too large to fit on a single plasmid (22 kb). Furthermore, we also imagined that switching DNA elements on a multicopy plasmid may not be completely wise, especially when our riboregulators were supposed to exert a strong control over the recombinases expression (i.e. maintaining a very low level of recombinases). Integration of some modules in the <i>E. calculus</i> genome was clearly the solution. We imagined that two different integration sites, one bearing AND1 and AND2, the second bearing XOR1 and XOR2 would be ideal. With this design, recombination events between XOR1 and AND1 or XOR2 and AND2 would lead to chromosomal DNA defects and cell death. The system would then be forced to recombine only the two sites belonging to the same gate. </p>
-
   
+
    <img style="width:650px; float:left;" src="https://static.igem.org/mediawiki/2013/2/2f/E.caclulusdesign.final.png" class="imgcontent" />
-
    <ul class="circlearrow">
+
-
      <li>Quisque at massa ipsum</li>
+
-
      <li>Maecenas a lorem augue, egestas </li>
+
-
      <li>Cras vitae felis at lacus ele </li>
+
-
      <li>Etiam auctor diam pellentesque </li>
+
-
      <li>Nulla ac massa at dolor </li>
+
-
    </ul>
+
-
  </div>
+
<div class="clear"></div>
-
  <div class="list">
+
  <p class="texte">
-
   
+
  The general inducer, input and output system as well as the carry can then be assembled on a single plasmid. The general strain would then carry the logic gates and would be stable (no recombinases produced). Upon transformation with the plasmid containing the general inducer, input, output and carry modules, the <i>E. calculus</i> strain would be generated, but still not able to switch before the general inducer put in the medium.
-
    <ul class="arrow">
+
 
-
      <li>Quisque at massa ipsum</li>
+
<br>
-
      <li>Maecenas a lorem augue, egestas </li>
+
<br>
-
      <li>Cras vitae felis at lacus ele </li>
+
For the final design, a great help was provided by François Cornet and Caroline Schiavon from the <a href="https://www-lmgm.biotoul.fr/" target="_blank">LMGM laboratory</a>. They gave us the MG1655 strain that contains special integration sites with two plasmids that would allow these specific integrations.
-
      <li>Etiam auctor diam pellentesque </li>
+
</p>
-
      <li>Nulla ac massa at dolor </li>
+
-
    </ul>
+
-
  </div>
 
-
 
 
-
<div class="clear"></div>
 
-
  <h3 class="title3">TITRE 3 lorem ipsum</h3>
 
-
  <p class="texte">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Ut et dolor turpis. Suspendisse interdum, dolor eu  ultricies dignissim, arcu sapien placerat quam, ut bibendum lorem nibh id magna. Nunc condimentum lectus at diam tempus sodales. Nullam scelerisque auctor tellus, nec auctor ante elementum sit amet. Aenean venenatis velit eget porttitor laoreet. Aliquam aliquam libero in ante imperdiet vehicula. Donec nibh urna, tincidunt quis condimentum at, tempus sed sapien. Nunc elit dui, dignissim tincidunt mi non, ultrices ultricies erat.</p>
+
<br>
 +
<br>
 +
<br><br>  
-
   <table class="tablecontent">
+
    
 +
-
<tr style="background-color:#20a8da; height:50px; color:#ffffff;" >
 
-
<td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;">TR  Title</td>
 
-
<td style="border-bottom:4px solid #e5e6e6;">TR  Title</td>
 
-
<td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;">TR  Title</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="border-right:1px solid #e5e6e6;">Quisque</td>
 
-
<td style="border-right:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
<td>Maecenas a lorem augue</td>
 
-
</tr>
 
-
<tr style="border-top:1px solid #e5e6e6">
 
-
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Quisque</td>
 
-
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
<td style="border-top:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Quisque</td>
 
-
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
<td style="border-top:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
</tr>
 
-
<tr>
 
-
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Quisque</td>
 
-
<td style="border-right:1px solid #e5e6e6; border-top:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
<td style="border-top:1px solid #e5e6e6;">Maecenas a lorem augue</td>
 
-
</tr>
 
-
</table>
 

Latest revision as of 20:40, 4 October 2013

logo


E. calculus design

General idea

The general idea of the project was to have distinct colonies of bacteria, each being a one bit calculator. The E. calculus strain can then be streaked several times, in the form of a thin line or circle on an agar plate, at regular intervals. The input signal (blue or red light) illuminates only one group of E. calculus at a time, allowing the full one bit calculation and the synthesis of the carry (AHL) if needed. After a certain period of time (long enough for the carry to diffuse to the next colony), illumination of the second colony can start. As the system is genetically stable, a relatively small amount of cells can be used, leading to amplification of the signal during growth of the switched bacteria. The output signal (RFP) would then be produced, stating a clear difference between the 0 or 1 state.

We also imagined that the E. calculus strain should have two different states, one for living happy state, feeding on the agar plate and dividing, not bothering about blue or red lights and a second state, dedicated to calculation. We therefore implemented a general inducer based on TetR repression and activation that would switch the bacteria to the "calculation ready" state. TetR, constitutively expressed would repress the transcription of the three receptors (blue light, red light and AHL receptors). In the presence of aTc (anhydrotetracycline) TetR would not repress anymore the promoter controlling the receptors genes. Upon illumination or when AHL is bound, each receptor will activate the transcription of the different recombinases, PhiC31 for the carry, Bxb1 for the blue light and Tp901.1 for the red light. When the recombinases are produced, they will switch their specific gates. Depending on the switch performed, the output will (or will not!) be produced.

Implementing the whole calculator in one strain

The full DNA construct, general inducer, input system, logic gates, output and carry was clearly too large to fit on a single plasmid (22 kb). Furthermore, we also imagined that switching DNA elements on a multicopy plasmid may not be completely wise, especially when our riboregulators were supposed to exert a strong control over the recombinases expression (i.e. maintaining a very low level of recombinases). Integration of some modules in the E. calculus genome was clearly the solution. We imagined that two different integration sites, one bearing AND1 and AND2, the second bearing XOR1 and XOR2 would be ideal. With this design, recombination events between XOR1 and AND1 or XOR2 and AND2 would lead to chromosomal DNA defects and cell death. The system would then be forced to recombine only the two sites belonging to the same gate.

The general inducer, input and output system as well as the carry can then be assembled on a single plasmid. The general strain would then carry the logic gates and would be stable (no recombinases produced). Upon transformation with the plasmid containing the general inducer, input, output and carry modules, the E. calculus strain would be generated, but still not able to switch before the general inducer put in the medium.

For the final design, a great help was provided by François Cornet and Caroline Schiavon from the LMGM laboratory. They gave us the MG1655 strain that contains special integration sites with two plasmids that would allow these specific integrations.