Difference between revisions of "Team:Toulouse/Parts"

 
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<div class="wrapper row0 bgded" style="background-image:url('https://static.igem.org/mediawiki/2015/0/03/TLSE_bakcground.png')">
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<div class="wrapper row0 bgded" style="background-image:url('https://static.igem.org/mediawiki/2015/8/8f/TLSE_bg_9.jpg')">
 
    
 
    
  
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         <li><a href="#1">- Attraction (butyrate pathway)</a></li>
 
         <li><a href="#1">- Attraction (butyrate pathway)</a></li>
 
         <li><a href="#2">- Eradication (formate pathway)</a></li>
 
         <li><a href="#2">- Eradication (formate pathway)</a></li>
−
         <li><a href="#3">- Circadian swich</a></li>
+
         <li><a href="#3">- Circadian switch</a></li>
  
 
       </ul>
 
       </ul>
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</center>
 
</center>
 
   <!-- ################################ ICI CONTENEUR OU TU PEUX T'AMUSER A METTRE LES DIVS ############################### -->
 
   <!-- ################################ ICI CONTENEUR OU TU PEUX T'AMUSER A METTRE LES DIVS ############################### -->
 +
 
 +
  <!---- TEST BACKGROUND-COULEUR --->
 
    
 
    
 
  <br><br>  
 
  <br><br>  
−
<div class="group center">
+
<div class="group center" id="biobricks">
−
<p class="text">
+
<p class="text" style="padding:10px;">
 
In the list below you will find an overview over the BioBrick parts created by the iGEM Toulouse 2015 team and added to the IGEM registry.  
 
In the list below you will find an overview over the BioBrick parts created by the iGEM Toulouse 2015 team and added to the IGEM registry.  
 
<br>
 
<br>
 
<br>
 
<br>
−
For our project, we worked on three main biological modules : Attract, Eradicate, and Regulation to alternatively produce the two molecules of interest (butyrate by day, formate by night).  
+
For our project, we worked on three main biological modules : Attract, Eradicate, and Regulate to alternatively produce the two molecules of interest (butyrate by day, formate by night).  
 
</p>
 
</p>
 
</div>
 
</div>
Line 87: Line 89:
 
       <th>Type</th>
 
       <th>Type</th>
 
       <th>Genic construction</th>
 
       <th>Genic construction</th>
−
       <th>Lenghth (bp)</th>
+
       <th>Module</th>
 +
  <th>Length (bp)</th>
 +
  <th>Sequencing</th>
 
       <th>References</th>
 
       <th>References</th>
 
     </tr>
 
     </tr>
Line 94: Line 98:
 
       <td>Composite (with RBS)</td>
 
       <td>Composite (with RBS)</td>
 
       <td>RBS-<i>ho1</i></td>
 
       <td>RBS-<i>ho1</i></td>
 +
  <td>Regulate</td>
 
       <td>744</td>
 
       <td>744</td>
−
       <td>[13]</td>
+
  <td>Ok</td>
 +
       <td>[13] [14]</td>
 
     </tr>
 
     </tr>
 
   <tr>
 
   <tr>
Line 101: Line 107:
 
       <td>Basic part</td>
 
       <td>Basic part</td>
 
       <td><i>tesB</i></td>
 
       <td><i>tesB</i></td>
 +
  <td>Attract</td>
 
       <td>861</td>
 
       <td>861</td>
 +
  <td>Ok</td>
 
       <td>[3][4][5]</td>
 
       <td>[3][4][5]</td>
 
     </tr>  
 
     </tr>  
Line 108: Line 116:
 
       <td>Composite (with RBS)</td>
 
       <td>Composite (with RBS)</td>
 
       <td>RBS-<i>pcyA</i></td>
 
       <td>RBS-<i>pcyA</i></td>
 +
  <td>Regulate</td>
 
       <td>796</td>
 
       <td>796</td>
−
       <td>[13]</td>
+
  <td>Ok</td>
 +
       <td>[13][14]</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
Line 115: Line 125:
 
       <td>Basic part</td>
 
       <td>Basic part</td>
 
       <td><i>crt</i></td>
 
       <td><i>crt</i></td>
 +
  <td>Attract</td>
 
       <td>786</td>
 
       <td>786</td>
−
       <td>[1] [2] [3] [4] [7]</td>
+
  <td>Sequenced until 736 pb : ok </td>
 +
       <td>[1][2][3][4][7]</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
 
       <td><a href="http://parts.igem.org/Part:BBa_K1587004"target=_blank">BBa_K1587004</a></td>
 
       <td><a href="http://parts.igem.org/Part:BBa_K1587004"target=_blank">BBa_K1587004</a></td>
 
       <td>Device</td>
 
       <td>Device</td>
−
       <td>pBla-RBS-<i>ccr</i>-RBS-<i>hbd</i>-RBS-<i>crt</i>-RBS-
+
       <td>P(Bla)-RBS-<i>ccr</i>-RBS-<i>hbd</i>-RBS-<i>crt</i>-RBS-
 
<i>tesB</i>-RBS-<i>atoB</i>-Terminator
 
<i>tesB</i>-RBS-<i>atoB</i>-Terminator
 
</td>
 
</td>
−
       <td>5175</td>
+
<td>Attract</td>
−
       <td>[1] [2] [3] [4] [6]</td>
+
       <td>5192</td>
 +
  <td>Ok</td>
 +
       <td>[1][2][3][4][6]</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
 
       <td><a href="http://parts.igem.org/Part:BBa_K1587005"target=_blank">BBa_K1587005</a></td>
 
       <td><a href="http://parts.igem.org/Part:BBa_K1587005"target=_blank">BBa_K1587005</a></td>
 
       <td>Device</td>
 
       <td>Device</td>
−
       <td>pBla-RBS-<i>hbd</i>-RBS-<i>crt</i>-RBS-
+
       <td>P(Bla)-RBS-<i>hbd</i>-RBS-<i>crt</i>-RBS-
 
<i>tesB</i>-RBS-<i>atoB</i>-Terminator</td>
 
<i>tesB</i>-RBS-<i>atoB</i>-Terminator</td>
 +
<td>Attract</td>
 
       <td>3827</td>
 
       <td>3827</td>
−
       <td>[1] [2] [3] [4] [6]</td>
+
  <td>Ok</td>
 +
       <td>[1][2][3][4][6]</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
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       <td>Device</td>
 
       <td>Device</td>
 
       <td>P<sub>OmpC</sub>-<i>LacI</i>box-RBS-<i>cI</i></td>
 
       <td>P<sub>OmpC</sub>-<i>LacI</i>box-RBS-<i>cI</i></td>
 +
  <td>Regulate</td>
 
       <td>905</td>
 
       <td>905</td>
−
       <td>[12][13]</td>
+
  <td>Ok</td>
 +
       <td>[12][13][14]</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
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       <td>Device</td>
 
       <td>Device</td>
 
       <td>RBS-<i>pflB</i>-RBS-<i>pflA</i>-Terminator</td>
 
       <td>RBS-<i>pflB</i>-RBS-<i>pflA</i>-Terminator</td>
 +
  <td>Eradicate</td>
 
       <td>3093</td>
 
       <td>3093</td>
−
       <td>[7] [8] [9] [10] [11] </td>
+
  <td>Ok</td>
 +
       <td>[7][8][9][10] </td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
 
<td><a href="http://parts.igem.org/Part:BBa_K1587008"target=_blank">BBa_K1587008</a></td>
 
<td><a href="http://parts.igem.org/Part:BBa_K1587008"target=_blank">BBa_K1587008</a></td>
 
<td>Device</td>
 
<td>Device</td>
−
<td><i>cph8</i></td>
+
<td>Strong Promotor-RBS-<i>cph8</i></td>
 +
<td>Regulate</td>
 
<td>2288</td>
 
<td>2288</td>
−
<td>REF A METTRE !!!</td>
+
<td>Ok</td>
 +
<td>[11][13][14]</td>
 
</tr>
 
</tr>
 
 
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<td>Composite (with RBS)</td>
 
<td>Composite (with RBS)</td>
 
<td>P<sub>OmpC</sub>-<i>LacI</i>box-RBS-<i>cI</i>-RBS-<i>pflB</i>-RBS-<i>pflA</i>-Terminator</td>
 
<td>P<sub>OmpC</sub>-<i>LacI</i>box-RBS-<i>cI</i>-RBS-<i>pflB</i>-RBS-<i>pflA</i>-Terminator</td>
 +
<td>Eradicate</td>
 
<td>4006</td>
 
<td>4006</td>
−
<td>[7] [8] [9] [10] [11] [12] [13]</td>
+
<td>-</td>
 +
<td>[7][8][9][10][11][12][13][14]</td>
 
</tr>
 
</tr>
 
 
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<p class="text">
 
<p class="text">
  
−
This BioBrick construction is composed of a constitutive promoter p(Bla)
+
This BioBrick construction is composed of a constitutive promoter P(Bla)
−
  (<a href="http://parts.igem.org/Part:BBa_I14018"target=_blank">BBa_I14018)</a>) and 5 genes from three different micro-organisms :  
+
  (<a href="http://parts.igem.org/Part:BBa_I14018"target=_blank">BBa_I14018</a>) and 5 genes from three different micro-organisms :  
 
  in yellow are the <i>E. coli</i> genes, in blue those from <i>Clostridium  
 
  in yellow are the <i>E. coli</i> genes, in blue those from <i>Clostridium  
 
  acetobutylicum</i> and finally, the purple gene is from <i>Streptomyces  
 
  acetobutylicum</i> and finally, the purple gene is from <i>Streptomyces  
−
  collinus</i>. A Ribosome Binding Site (RBS; represented by a green circle;  
+
  collinus</i>. A Ribosome Binding Site (RBS; represented by a green circle; <a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>), is added between each gene in order to improve the proteic
−
(<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>), is added between each gene in order to improve the proteic
+
 
  synthesis.  Finally, a strong terminator <a href="http://parts.igem.org/Part:BBa_B1006"target=_blank">BBa_B1006)</a> represents the end of the sequence.   
 
  synthesis.  Finally, a strong terminator <a href="http://parts.igem.org/Part:BBa_B1006"target=_blank">BBa_B1006)</a> represents the end of the sequence.   
 
<br><br>
 
<br><br>
−
<a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10995"target="_blank"><i>tesB</i></a> and <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2688"target="_blank"><i>crt</i></a> have been described above. <a href ="http://www.uniprot.org/uniprot/Q53865"target="_blank"><i>ccr</i></a> encodes crotonyl  
+
<a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10995"target="_blank"><i>tesB</i></a> and <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2688"target="_blank"><i>crt</i></a> have been described above. <a href ="http://www.uniprot.org/uniprot/Q53865"target="_blank"><i>ccr</i></a> encodes a crotonyl-CoA reductase (an oxidoreductase which acts on the double bond CH=CH). <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2684"target="_blank"><i>hbd</i></a> from <i>Clostridium acetobutylicum</i> encodes a 3-hydroxybutyryl-CoA  
−
CoA reductase, an oxidoreductase which acts on the double bond  
+
dehydrogenase (an oxidoreductase which catalyses the formation of alcohol function). <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11672"target="_blank"><i>atoB</i></a>, from <i>E. coli</i>, encodes an acetyl-CoA acetyltransferase which catalyses the condensation of two acetyl-CoA.  
−
CH=CH. <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2684"target="_blank"><i>hbd</i></a> from <i>Clostridium acetobutylicum</i> encodes 3-hydroxybutyryl-CoA  
+
−
dehydrogenase, an oxidoreductase which catalyses the formation of
+
−
alcohol function. <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11672"target="_blank"><i>atoB</i></a>, from <i>E. coli</i>, encodes an acetyl-CoA acetyltransferase which catalyses the condensation of two acetyl-CoA.  
+
  
 
</p>
 
</p>
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<div class="group center">
 
<div class="group center">
 
<p class="text">   
 
<p class="text">   
−
This BioBrick construction is the same as previously, but does not  
+
This BioBrick construction is the same as the previous one, but for the fact that it does not  
 
contain the <i>ccr</i> gene from <i>Streptomyces collinus</i>.  
 
contain the <i>ccr</i> gene from <i>Streptomyces collinus</i>.  
−
It is composed of a constitutive promoter p(Bla) (BBa_I14018) and of 4  
+
It is composed of a constitutive promoter P(Bla) (<a href="http://parts.igem.org/Part:BBa_I14018"target=_blank">BBa_I14018</a>) and of 4 of the 5 genes present in the previous construction. Genes are issued from two micro-organisms : in yellow from  
−
genes from two different micro-organisms : in yellow are the genes from  
+
<i>E. coli</i>, and in blue from <i>Clostridium acetobutylicum</i>. The green  
−
<i>E. coli</i>, and in blue those from <i>Clostridium acetobutylicum</i>. The green  
+
circles correspond to the strong RBS sequence (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>) based on Ron Weiss thesis and the red one is the terminator (<a href="http://parts.igem.org/Part:BBa_B1006"target=_blank">BBa_B1006</a>).   
−
circles correspond to the strong RBS (BBa_B0030) sequences based on  
+
−
Ron Weiss thesis and the red one is the terminator (BBa_B1006).   
+
 
</p>
 
</p>
 
</div>
 
</div>
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  <div class="group center">
 
  <div class="group center">
 
  <p class="text">
 
  <p class="text">
−
To obtain the second module, we decided to produce formic acid.
+
The second module is dedicated to formate production and composed of two key genes :  
−
Indeed, this molecule has two benefits. The first is that the acaricide
+
  <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10028"target="_blank"><i>pflA</i></a> and <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10701"target="_blank"><i>pflB</i></a>.   
−
effect has been demonstrated, and the second is the natural production
+
−
of the compound by <i>E. coli</i>, the bacterium we chose as chassis.  Glucose
+
−
is the initial substrate and it is degraded into pyruvate during
+
−
glycolysis. Finally, formate is synthesized thanks to two key genes :  
+
−
  <i>pflA</i> and <i>pflB</i>.   
+
 
   </p>
 
   </p>
 
   </div>
 
   </div>
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<div class="subtitle">
 
<div class="subtitle">
−
<h3>Formate pathway (BBa_K1587007)</h3>
+
<h3>Formate pathway (<a href="http://parts.igem.org/Part:BBa_K1587007"target=_blank">BBa_K1587007</a>)</h3>
 
</div>
 
</div>
  
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<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
<i>pflB</i> encodes the pyruvate formate lyase, an enzyme which catalyses
+
<i>pflB</i> encodes a pyruvate formate lyase, an enzyme which catalyses
 
  the cutting between C1 and C2 carbons of pyruvate. This enzyme  
 
  the cutting between C1 and C2 carbons of pyruvate. This enzyme  
 
  is oxygen-sensitive and is only active in microaerobic or anaerobic  
 
  is oxygen-sensitive and is only active in microaerobic or anaerobic  
Line 301: Line 314:
 
   
 
   
 
The formate compound is naturally produced by <i>E. coli</i>, that is why we  
 
The formate compound is naturally produced by <i>E. coli</i>, that is why we  
−
decided to overexpress the two essential genes.
+
decided to overexpress these two genes. For this, <i>pflB</i> and <i>pflA</i> are put  
−
<br><br>
+
together with two RBS (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>) in front of them to allow the proteic synthesis. A strong terminator (<a href="http://parts.igem.org/Part:BBa_B1006"target=_blank">BBa_B1006</a>) ends up the sequence.
−
To test the formate production, <i>pflB</i> and <i>pflA</i> are put  
+
−
together with two RBS (BBa_B0030) in front of them to improve the
+
−
proteic synthesis. A strong terminator (BBa_B1006) ends the sequence.
+
 
</p>
 
</p>
 
</div>
 
</div>
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  <center>
 
  <center>
 
<div class="title" id="3">
 
<div class="title" id="3">
−
<h3>Circadian swich</h3>
+
<h3>Circadian switch</h3>
 
</div>
 
</div>
  
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<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
As said previously, we decided to produce butyric acid in our trap during
+
We aimed to produce butyric acid in our trap during
−
  the day and formic acid to kill the mite during the night.  
+
  the day and formic acid during the night.  
 
  We designed a light response system which is improved to obtain an  
 
  We designed a light response system which is improved to obtain an  
−
  on/off switch of genic expression. We adapted this system because we
+
  on/off switch of genic expression.
−
did not want to obtain only an on/off switch but a switch of genic
+
−
expression between two different polycistronic genes following the
+
−
presence or the absence of light.  
+
 
  <br><br>
 
  <br><br>
 
The center of the light sensor is composed of membrane proteins PCB  
 
The center of the light sensor is composed of membrane proteins PCB  
−
(chromophore phycocyanobilin) and the hybrid protein Cph8 (EnvZ and Cph1).
+
(chromophore phycocyanobilin) and the hybrid protein Cph8 (EnvZ and Cph1)[11].
 
<br><br>
 
<br><br>
−
PCB protein comes from a cyanobacterium <i>Synechocystis</i> sp PCC 6803  
+
PCB protein comes from a cyanobacterium <i>Synechocystis sp.</i> PCC 6803  
 
and to be synthesized, it needs the expression of two genes:  
 
and to be synthesized, it needs the expression of two genes:  
−
heme oxygenase (<i>Ho1</i>) and biliverdin reductase (<i>PcyA</i>).  
+
heme oxygenase (<i>Ho1</i>) and biliverdin reductase (<i>PcyA</i>)[11].  
 
</p>
 
</p>
 
</div>
 
</div>
−
<br><br>
 
−
<center><img src="https://static.igem.org/mediawiki/2015/4/40/TLSE_Parts_image6.PNG" style="width:60%;"/>
 
−
<p class="legend">
 
−
Figure: Phycobilin biosynthetic pathway in cyanobacteria showing
 
−
the formation of PCB from heme. The first step comprises <i>Ho1</i>-
 
−
catalyzed heme degradation (Okada et al 2009).
 
−
</p>
 
  
 
</center>
 
</center>
  
 
  <div class="subtitle">
 
  <div class="subtitle">
−
  <h3><i>Ho1</i> with RBS (BBa_K1587000)</h3>
+
  <h3><i>Ho1</i> with RBS (<a href="http://parts.igem.org/Part:BBa_K1587000"target=_blank">BBa_K1587000</a>)</h3>
 
  </div>
 
  </div>
 
   
 
   
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<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
Gene required for chromophore synthesis in photosynthetic  
+
A gene required for chromophore synthesis in photosynthetic  
 
light-harvesting complexes, photoreceptors, and circadian clocks.  
 
light-harvesting complexes, photoreceptors, and circadian clocks.  
 
<i>ho1</i>, along with <i>pcyA</i>, converts heme into the chromophore  
 
<i>ho1</i>, along with <i>pcyA</i>, converts heme into the chromophore  
−
phycocyanobillin (PCB).
+
phycocyanobillin (PCB). The gene comes from the cyanobacterium <i>Synechocystis sp. </i>PCC 6803.
 
<br><br>
 
<br><br>
−
The biobrick is composed of a strong RBS (BBa_B0030) and <i>Ho1</i> coding region (BBa_K566022).  
+
The biobrick is composed of a strong RBS (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>) and <i>Ho1</i> coding region (<a href="http://parts.igem.org/Part:BBa_K566022"target=_blank">BBa_K566022</a>).  
 
</p>
 
</p>
 
</div>
 
</div>
Line 364: Line 364:
 
    
 
    
 
  <div class="subtitle">
 
  <div class="subtitle">
−
  <h3><i>PcyA</i> with RBS (BBa_K1587002)</h3>
+
  <h3><i>PcyA</i> with RBS (<a href="http://parts.igem.org/Part:BBa_K1587002"target=_blank">BBa_K1587002</a>)</h3>
 
  </div>
 
  </div>
 
   
 
   
Line 372: Line 372:
 
<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
Gene required for chromophore synthesis in photosynthetic  
+
A gene required for chromophore synthesis in photosynthetic  
−
light-harvesting complexes, photoreceptors, and circadian clocks.
+
light-harvesting complexes, photoreceptors, and circadian clocks. The gene comes from the cyanobacterium <i>Synechocystis sp.</i> PCC 6803.
 
<br><br>
 
<br><br>
−
Biobrick composed of a strong RBS (BBa_B0030) and <i>pcyA</i>  
+
The biobrick is composed of a strong RBS (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>) and <i>pcyA</i>  
−
coding region (BBa_K566023).
+
coding region (<a href="http://parts.igem.org/Part:BBa_K566023"target=_blank">BBa_K566023</a>).
 
</p>
 
</p>
 
</div>   
 
</div>   
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<div class="subtitle">
 
<div class="subtitle">
−
<h3>P<sub>OmpC</sub>-<i>LacIbox</i>-RBS-<i>cI</i> (BBa_K1587006) </h3>
+
<h3>P<sub>OmpC</sub>-<i>LacIbox</i>-RBS-<i>cI</i> (<a href="http://parts.igem.org/Part:BBa_K1587006"target=_blank">BBa_K1587006</a>) </h3>
 
</div>
 
</div>
 
<img src="https://static.igem.org/mediawiki/2015/4/4c/TLSE_Parts_image9b.PNG" style="width:25%;"/>
 
<img src="https://static.igem.org/mediawiki/2015/4/4c/TLSE_Parts_image9b.PNG" style="width:25%;"/>
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<div class="group">
 
<div class="group">
 
<p class="text">
 
<p class="text">
−
This biobrick contains OmpC promoter (BBa_R0082), a lacI box  
+
This biobrick contains the OmpC promoter (<a href="http://parts.igem.org/Part:BBa_R0082"target=_blank">BBa_R0082</a>), a lacI box  
−
separated from <i>cI</i> gene by a RBS sequence (BBa_B0030).  
+
separated from <i>cI</i> gene by a RBS sequence (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>).  
 
</p>
 
</p>
 
</div>
 
</div>
  
 
<div class="subtitle">
 
<div class="subtitle">
−
<h3>Red light biosensor (BBa_K1587008)  </h3>
+
<h3>Red light biosensor (<a href="http://parts.igem.org/Part:BBa_K1587008"target=_blank">BBa_K1587008</a>)  </h3>
 
</div>
 
</div>
  
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<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
Strong promoter BBa_J23119. Strong RBS BBa_B0030 Chimeric Red light receptor cph8 BBa_I15010. It is a chimeric Red light receptor cph8 genetic construction. This chimeric Red light receptor <i>cph8</i> (cph1-envZ) is inactivated by phycobilin PCB photosynthetic light-harvesting, preventing the phosphorylation of ompR</p>
+
This is an assembly between the strong promoter <a href="http://parts.igem.org/Part:BBa_J23119"target=_blank">BBa_J23119</a>, an RBS (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>), and the chimeric Red light receptor Cph8 <a href="http://parts.igem.org/Part:BBa_I15010"target=_blank">BBa_I15010</a>.</p>
 
</div>
 
</div>
−
<center><img src="https://static.igem.org/mediawiki/2015/0/04/TLSE_Parts_image10.PNG" style="width:60%;"/></center>
+
 
 
<div class="subtitle">
 
<div class="subtitle">
−
<h3>Regulated formate production (BBa_K1587009)  </h3>
+
<h3>Regulated formate production (<a href="http://parts.igem.org/Part:BBa_K1587009"target=_blank">BBa_K1587009</a>)  </h3>
 
</div>
 
</div>
 
<img src="https://static.igem.org/mediawiki/2015/6/6e/Ci_formate.png" style="width:50%;"/>
 
<img src="https://static.igem.org/mediawiki/2015/6/6e/Ci_formate.png" style="width:50%;"/>
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<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
Uses a positively regulated promoter (P<sub>ompC</sub>) and a lacI binding site to regulate the expression of cI. This construction was designed to be regulated by <i>cph8</i> red light receptor (cph1-envZ chimeric protein). The purpose was to create a circadian switch to alternate the expression of two sets of genes (formate/butyrate).
+
An assembly between biobricks <a href="http://parts.igem.org/Part:BBa_K1587006"target=_blank">BBa_K1587006</a>  
 +
and <a href="http://parts.igem.org/Part:BBa_K1587007"target=_blank">BBa_K1587007</a>, which have been both successfully sequenced, for the regulation of formate
 +
production by the circadian switch system.
 
</p>
 
</p>
 
</div>
 
</div>
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<li>
 
<li>
 
[13] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable gene expression in <i>Escherichia coli</i> using a miniaturized photobioreactor. PLoS ONE 8: e52382  
 
[13] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable gene expression in <i>Escherichia coli</i> using a miniaturized photobioreactor. PLoS ONE 8: e52382  
 +
</li>
 +
<li>
 +
[14] Gambetta GA & Lagarias JC (2001) Genetic engineering of phytochrome biosynthesis in bacteria. PNAS 98: 10566–10571
 
</li>
 
</li>
 
</ul>
 
</ul>

Latest revision as of 02:34, 19 September 2015

iGEM Toulouse 2015

Biobricks


Content


In the list below you will find an overview over the BioBrick parts created by the iGEM Toulouse 2015 team and added to the IGEM registry.

For our project, we worked on three main biological modules : Attract, Eradicate, and Regulate to alternatively produce the two molecules of interest (butyrate by day, formate by night).

Name Type Genic construction Module Length (bp) Sequencing References
BBa_K1587000 Composite (with RBS) RBS-ho1 Regulate 744 Ok [13] [14]
BBa_K1587001 Basic part tesB Attract 861 Ok [3][4][5]
BBa_K1587002 Composite (with RBS) RBS-pcyA Regulate 796 Ok [13][14]
BBa_K1587003 Basic part crt Attract 786 Sequenced until 736 pb : ok [1][2][3][4][7]
BBa_K1587004 Device P(Bla)-RBS-ccr-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator Attract 5192 Ok [1][2][3][4][6]
BBa_K1587005 Device P(Bla)-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator Attract 3827 Ok [1][2][3][4][6]
BBa_K1587006 Device POmpC-LacIbox-RBS-cI Regulate 905 Ok [12][13][14]
BBa_K1587007 Device RBS-pflB-RBS-pflA-Terminator Eradicate 3093 Ok [7][8][9][10]
BBa_K1587008 Device Strong Promotor-RBS-cph8 Regulate 2288 Ok [11][13][14]
BBa_K1587009 Composite (with RBS) POmpC-LacIbox-RBS-cI-RBS-pflB-RBS-pflA-Terminator Eradicate 4006 - [7][8][9][10][11][12][13][14]

Attraction (butyrate pathway)

The chassis we used is Escherichia coli, and this bacterium is not able to naturally produce butyrate. That is why we introduced genes from others bacterial strains to synthesize this molecule.

Basic parts

tesB (BBa_K1587001)

A gene from Escherichia coli (Accession Number: EG10995) involved in the butyrate pathway that enables its production directly from acyl-coAs. This group of enzymes catalyzes the hydrolysis of acyl-CoAs into free fatty acid (in our case, butyryl-coA into butyrate) plus reduced coenzyme A (CoA-SH).

crt (BBa_K1587003)

A gene from Clostridium acetobutylicum (Accession Number: GJIH-2688) introduced in our bacterium after codon optimization in order to obtain a better expression in our strain. The crt enzyme substrate is 3-hydroxybutyryl CoA, and the product is Crotonyl CoA. This reaction does not need any coenzyme.

Other parts

ccr-Butyrate pathway (BBa_K1587004)

This BioBrick construction is composed of a constitutive promoter P(Bla) (BBa_I14018) and 5 genes from three different micro-organisms : in yellow are the E. coli genes, in blue those from Clostridium acetobutylicum and finally, the purple gene is from Streptomyces collinus. A Ribosome Binding Site (RBS; represented by a green circle; BBa_B0030), is added between each gene in order to improve the proteic synthesis. Finally, a strong terminator BBa_B1006) represents the end of the sequence.

tesB and crt have been described above. ccr encodes a crotonyl-CoA reductase (an oxidoreductase which acts on the double bond CH=CH). hbd from Clostridium acetobutylicum encodes a 3-hydroxybutyryl-CoA dehydrogenase (an oxidoreductase which catalyses the formation of alcohol function). atoB, from E. coli, encodes an acetyl-CoA acetyltransferase which catalyses the condensation of two acetyl-CoA.

Butyrate pathway wihout ccr (BBa_K1587005)

This BioBrick construction is the same as the previous one, but for the fact that it does not contain the ccr gene from Streptomyces collinus. It is composed of a constitutive promoter P(Bla) (BBa_I14018) and of 4 of the 5 genes present in the previous construction. Genes are issued from two micro-organisms : in yellow from E. coli, and in blue from Clostridium acetobutylicum. The green circles correspond to the strong RBS sequence (BBa_B0030) based on Ron Weiss thesis and the red one is the terminator (BBa_B1006).

Eradication (formate pathway)

The second module is dedicated to formate production and composed of two key genes : pflA and pflB.

Formate pathway (BBa_K1587007)

pflB encodes a pyruvate formate lyase, an enzyme which catalyses the cutting between C1 and C2 carbons of pyruvate. This enzyme is oxygen-sensitive and is only active in microaerobic or anaerobic conditions.

pflA encodes the pyruvate formate lyase activase, an enzyme which activates pflB.

The formate compound is naturally produced by E. coli, that is why we decided to overexpress these two genes. For this, pflB and pflA are put together with two RBS (BBa_B0030) in front of them to allow the proteic synthesis. A strong terminator (BBa_B1006) ends up the sequence.

Circadian switch

We aimed to produce butyric acid in our trap during the day and formic acid during the night. We designed a light response system which is improved to obtain an on/off switch of genic expression.

The center of the light sensor is composed of membrane proteins PCB (chromophore phycocyanobilin) and the hybrid protein Cph8 (EnvZ and Cph1)[11].

PCB protein comes from a cyanobacterium Synechocystis sp. PCC 6803 and to be synthesized, it needs the expression of two genes: heme oxygenase (Ho1) and biliverdin reductase (PcyA)[11].

Ho1 with RBS (BBa_K1587000)

A gene required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks. ho1, along with pcyA, converts heme into the chromophore phycocyanobillin (PCB). The gene comes from the cyanobacterium Synechocystis sp. PCC 6803.

The biobrick is composed of a strong RBS (BBa_B0030) and Ho1 coding region (BBa_K566022).

PcyA with RBS (BBa_K1587002)

A gene required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks. The gene comes from the cyanobacterium Synechocystis sp. PCC 6803.

The biobrick is composed of a strong RBS (BBa_B0030) and pcyA coding region (BBa_K566023).

POmpC-LacIbox-RBS-cI (BBa_K1587006)

This biobrick contains the OmpC promoter (BBa_R0082), a lacI box separated from cI gene by a RBS sequence (BBa_B0030).

Red light biosensor (BBa_K1587008)

This is an assembly between the strong promoter BBa_J23119, an RBS (BBa_B0030), and the chimeric Red light receptor Cph8 BBa_I15010.

Regulated formate production (BBa_K1587009)

An assembly between biobricks BBa_K1587006 and BBa_K1587007, which have been both successfully sequenced, for the regulation of formate production by the circadian switch system.

References

  • [1] Layton DS & Trinh CT (2014) Engineering modular ester fermentative pathways in Escherichia coli. Metabolic Engineering 26: 77–88
  • [2] Saini M, Hong Chen M, Chiang C-J & Chao Y-P (2015) Potential production platform of n-butanol in Escherichia coli. Metabolic Engineering 27: 76–82
  • [3] Volker AR, Gogerty DS, Bartholomay C, Hennen-Bierwagen T, Zhu H & Bobik TA (2014) Fermentative production of short-chain fatty acids in Escherichia coli. Microbiology (Reading, Engl.) 160: 1513–1522
  • [4] Saini M, Wang ZW, Chiang C-J & Chao Y-P (2014) Metabolic Engineering of Escherichia coli for Production of Butyric Acid. J. Agric. Food Chem. 62: 4342–4348
  • [5] Hunt MC & Alexson SEH (2002) The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41: 99–130
  • [6] Aboulnaga E-H, Pinkenburg O, Schiffels J, El-Refai A, Buckel W & Selmer T (2013) Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli. J. Bacteriol. 195: 3704–3713
  • [7] Thesis: SUNYA Sirichai. July 2012. Dynamique de la réponse physiologique d’Escherichia coli à des perturbations maîtrisées de son environnement : vers le développement de nouveaux outils de changement d’échelle. Ingénieries Enzymatique et Microbienne.
  • [8] CEA-CNRS- Aix Marseille Université. February 2015. Paris . Activation d’enzymes bactériennes pour convertir le CO2 en source d’énergie renouvelable.
  • [9] Crain AV & Broderick JB (2014) Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme. J. Biol. Chem. 289: 5723–5729
  • [10] Wei X-X, Zheng W-T, Hou X, Liang J, Li Z-J, Wei X-X, Zheng W-T, Hou X, Liang J & Li Z-J (2015) Metabolic Engineering of Escherichia coli for Poly(3-hydroxybutyrate) Production under Microaerobic Condition. BioMed Research International, BioMed Research International 2015, 2015: e789315
  • [11] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM & Voigt CA (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442
  • [12] Okada K (2009) HO1 and PcyA proteins involved in phycobilin biosynthesis form a 1:2 complex with ferredoxin-1 required for photosynthesis. FEBS Lett. 583: 1251–1256
  • [13] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable gene expression in Escherichia coli using a miniaturized photobioreactor. PLoS ONE 8: e52382
  • [14] Gambetta GA & Lagarias JC (2001) Genetic engineering of phytochrome biosynthesis in bacteria. PNAS 98: 10566–10571