Difference between revisions of "Team:Toulouse/Parts"

(Undo revision 362400 by Bpons (talk))
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       <div class="maintitle">
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     <center> <h3>Fun Facts</h3> </center>
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     <center> <h3>Biobricks</h3> </center>
 
     </div>
 
     </div>
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  <center><img src="https://static.igem.org/mediawiki/2015/1/1f/TLSE_immortanBee.png"></center>
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  <center><img src="https://static.igem.org/mediawiki/2015/2/2a/TLSE_brickBee.png" ></center>
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<div class="wrapper row3">
 
<div class="wrapper row3">
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<main class="container clear">
 
  
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<div class="group center">
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<br>
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<p class="text">
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  <main class="container clear">
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10 students in a lab during the whole summer<br>
+
  <div class="title">
 +
      <a href="#main1"><h3>Content</h3></a>
 +
    </div>
 +
<center> 
 +
    <div id="breadcrumb" class="clear" style="float: center;" >
 +
  <ul>
 +
        <li><a href="#1">- Attraction (butyrate pathway)</a></li>
 +
        <li><a href="#2">- Eradication (formate pathway)</a></li>
 +
        <li><a href="#3">- Circadian swich</a></li>
 +
 
 +
      </ul>
 +
    </div>
 +
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1);">
 +
 
 +
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 +
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 +
  <!---- TEST BACKGROUND-COULEUR --->
 +
 
 +
<br><br>
 +
<div class="group center" style="background-color:#E2FAFC;">
 +
<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.
 +
<br>
 +
<br>
 +
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>
−
   
+
 
 +
<div class="group center">
 +
<table class="result">
 +
  <tbody>
 +
    <tr>
 +
      <th>Name</th>
 +
      <th>Type</th>
 +
      <th>Genic construction</th>
 +
      <th>Module</th>
 +
  <th>Length (bp)</th>
 +
  <th>Sequencing</th>
 +
      <th>References</th>
 +
    </tr>
 +
    <tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587000"target=_blank">BBa_K1587000</a></td>
 +
      <td>Composite (with RBS)</td>
 +
      <td>RBS-<i>ho1</i></td>
 +
  <td>Regulate</td>
 +
      <td>744</td>
 +
  <td>Ok</td>
 +
      <td>[13] [14]</td>
 +
    </tr>
 +
  <tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587001"target=_blank">BBa_K1587001</a></td>
 +
      <td>Basic part</td>
 +
      <td><i>tesB</i></td>
 +
  <td>Attract</td>
 +
      <td>861</td>
 +
  <td>Ok</td>
 +
      <td>[3][4][5]</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587002"target=_blank">BBa_K1587002</a></td>
 +
      <td>Composite (with RBS)</td>
 +
      <td>RBS-<i>pcyA</i></td>
 +
  <td>Regulate</td>
 +
      <td>796</td>
 +
  <td>Ok</td>
 +
      <td>[13] [14]</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587003"target=_blank">BBa_K1587003</a></td>
 +
      <td>Basic part</td>
 +
      <td><i>crt</i></td>
 +
  <td>Attract</td>
 +
      <td>786</td>
 +
  <td>Sequenced until 736 pb : ok </td>
 +
      <td>[1][2][3][4][7]</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587004"target=_blank">BBa_K1587004</a></td>
 +
      <td>Device</td>
 +
      <td>pBla-RBS-<i>ccr</i>-RBS-<i>hbd</i>-RBS-<i>crt</i>-RBS-
 +
<i>tesB</i>-RBS-<i>atoB</i>-Terminator
 +
</td>
 +
<td>Attract</td>
 +
      <td>5192</td>
 +
  <td>Ok</td>
 +
      <td>[1][2][3][4][6]</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587005"target=_blank">BBa_K1587005</a></td>
 +
      <td>Device</td>
 +
      <td>pBla-RBS-<i>hbd</i>-RBS-<i>crt</i>-RBS-
 +
<i>tesB</i>-RBS-<i>atoB</i>-Terminator</td>
 +
<td>Attract</td>
 +
      <td>3827</td>
 +
  <td>Ok</td>
 +
      <td>[1][2][3][4][6]</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587006"target=_blank">BBa_K1587006</a></td>
 +
      <td>Device</td>
 +
      <td>P<sub>OmpC</sub>-<i>LacI</i>box-RBS-<i>cI</i></td>
 +
  <td>Regulate</td>
 +
      <td>905</td>
 +
  <td>Ok</td>
 +
      <td>[12][13] [14]</td>
 +
    </tr>
 +
<tr>
 +
      <td><a href="http://parts.igem.org/Part:BBa_K1587007"target=_blank">BBa_K1587007</a></td>
 +
      <td>Device</td>
 +
      <td>RBS-<i>pflB</i>-RBS-<i>pflA</i>-Terminator</td>
 +
  <td>Eradicate</td>
 +
      <td>3093</td>
 +
  <td>Ok</td>
 +
      <td>[7][8][9][10] </td>
 +
    </tr>
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1587008"target=_blank">BBa_K1587008</a></td>
 +
<td>Device</td>
 +
<td><i>cph8</i></td>
 +
<td>Regulate</td>
 +
<td>2288</td>
 +
<td>Ok</td>
 +
<td>[11][13][14]</td>
 +
</tr>
 +
 +
<tr>
 +
<td><a href="http://parts.igem.org/Part:BBa_K1587009"target=_blank">BBa_K1587009</a></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>Eradicate</td>
 +
<td>4006</td>
 +
<td>-</td>
 +
<td>[7][8][9][10][11][12][13][14]</td>
 +
</tr>
 +
 +
  </tbody>
 +
</table>
 +
</div>
 +
 
 
<center>
 
<center>
−
<img src="https://static.igem.org/mediawiki/2015/8/88/VOcQKG.gif" style="width:50%;"/>
+
<div class="title" id="1">
 +
<h3>Attraction (butyrate pathway)</h3>
 +
</div>
 +
</center>
 +
 
 +
 
 
<div class="group center">
 
<div class="group center">
 
<p class="text">
 
<p class="text">
−
What could possibly go wrong?
+
The chassis we used is <i>Escherichia coli</i>, and this bacterium is not able to naturally produce butyrate. That is why we introduced genes from others bacterial strains to synthesize this molecule.
 
</p>
 
</p>
−
</div>
+
</div>
−
</center>
+
  
−
<br>
+
<div class="subtitle">
−
<div class="group center">
+
<h3>Basic parts</h3>
−
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
+
 
</div>
 
</div>
−
<br>
 
  
−
<br>
+
<div class="subsubtitle">
 +
<h3><i>tesB</i> <a href="http://parts.igem.org/Part:BBa_K1587001"target=_blank">(BBa_K1587001)</a></h3>
 +
</div>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2015/3/39/TLSE_Parts_image1b.PNG" style="width:34%;"/>
  
−
<table>
+
<div class="group center">
−
<tbody>
+
−
<tr>
+
−
<td style="width:30%;">
+
−
<center>
+
 
<p class="text">
 
<p class="text">
−
During our summer at iGEM, we often impersonated movie characters or recreated famous scenes from TV shows - sometimes on purpose, sometimes not. Here are a few of our best cosplays !
+
A gene from <i>Escherichia coli</i> (Accession Number: <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10995"target="_blank">EG10995</a>) 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).
 
</p>
 
</p>
−
</center>
+
</div>
−
</td>
+
  
−
<td style="width:30%;">
+
<div class="subsubtitle">
−
<center>  
+
<h3><i>crt</i> <a href="http://parts.igem.org/Part:BBa_K1587003"target=_blank">(BBa_K1587003)</a></h3>
−
<img src="https://static.igem.org/mediawiki/2015/a/ab/Tlse_benhook.jpg" style="width:85%;"/>
+
</div>
−
</center>
+
−
</td>
+
  
−
<td style="width:30%;">
+
<img src="https://static.igem.org/mediawiki/2015/4/4c/TLSE_Parts_image2b.PNG" style="width:37%;"/>
−
<center>
+
−
<img src="https://static.igem.org/mediawiki/2015/thumb/c/c3/TLSE_CSI.jpeg/800px-TLSE_CSI.jpeg" style="width:140%;"/>
+
−
</center>
+
−
</td>
+
−
</tr>
+
  
−
<tr>
+
<div class="group center">
−
<td>
+
<p class="text">
−
</td>
+
A gene from <i>Clostridium acetobutylicum</i> (Accession Number: <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2688"target="_blank">GJIH-2688</a>) introduced in our
−
 
+
bacterium after codon optimization in order to obtain a better
−
<td>
+
expression in our strain. The <i>crt</i> enzyme substrate is 3-hydroxybutyryl CoA,
−
<center>
+
and the product is Crotonyl CoA. This reaction does not need any coenzyme.
−
<p class="legend">
+
−
<a href = "https://www.youtube.com/watch?v=vfol_TLLnIw" target="_blank">Ya'll ready for iGEM? Arrrr !</a>
+
−
</p>
+
−
</center>
+
−
</td>
+
−
 
+
−
<td>
+
−
<center>
+
−
<p class="legend">
+
−
CSI : Toulouse
+
 
</p>
 
</p>
−
</center>
+
</div>
−
</td>
+
−
</tr>
+
  
−
<tr>
+
<div class="subtitle">
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<td style="width:30%;">
+
<h3>Other parts</h3>
−
<center>
+
</div>
−
<img src="https://static.igem.org/mediawiki/2015/8/8e/Trap_ackbar.jpeg" style="width:70%;"/>
+
−
</center>
+
−
</td>
+
  
−
<td style="width:30%;">
+
<div class="subsubtitle">
−
<center><img src="https://static.igem.org/mediawiki/2015/8/83/TLSE_king.jpg" style="width:80%;"/>
+
<h3><i>ccr-Butyrate pathway</i> <a href="http://parts.igem.org/Part:BBa_K1587004"target=_blank">(BBa_K1587004)</a></h3>
−
</center>
+
</div>
−
</td>
+
<img src="https://static.igem.org/mediawiki/2015/5/5b/TLSE_Parts_image3.PNG" style="width:61%;"/>
  
−
<td style="width:30%;">
+
<div class="group center">
−
<center><img src="https://static.igem.org/mediawiki/2015/thumb/e/e2/TLSE_interstellar.jpeg/800px-TLSE_interstellar.jpeg" style="width:100%;"/>
+
<p class="text">
−
</center>
+
−
</td>
+
−
</tr>
+
  
−
<tr>
+
This BioBrick construction is composed of a constitutive promoter p(Bla)
−
<td>
+
(<a href="http://parts.igem.org/Part:BBa_I14018"target=_blank">BBa_I14018</a>) and 5 genes from three different micro-organisms :
−
<center>
+
in yellow are the <i>E. coli</i> genes, in blue those from <i>Clostridium
−
<p class="legend">
+
acetobutylicum</i> and finally, the purple gene is from <i>Streptomyces
−
<a href = "https://www.youtube.com/watch?v=4F4qzPbcFiA" target="_blank">It's a trap !</a>
+
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
−
</p>
+
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. 
−
</center>
+
<br><br>
−
</td>
+
<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
 +
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.
  
−
<td>
 
−
<center>
 
−
<p class="legend">
 
−
<a href ="https://www.youtube.com/watch?v=iQExgALv9wI" target="blank">Theoden and Grima Worm-Tongue</a> (or Charlemagne and Quasimodo, depending on the sources !)
 
 
</p>
 
</p>
−
</center>
+
</div>
−
</td>
+
  
−
<td>
+
<div class="subsubtitle">
−
<center>
+
<h3><i>Butyrate pathway wihout ccr</i> <a href="http://parts.igem.org/Part:BBa_K1587005"target=_blank">(BBa_K1587005)</a></h3>
−
<p class="legend">
+
</div>
−
<a href = "https://www.youtube.com/watch?v=3_nprPycZow" target="_blank">“Do not go gentle into that good night.
+
<img src="https://static.igem.org/mediawiki/2015/6/6f/TLSE_Parts_image4.PNG" style="width:59%;"/>
−
Old age should burn and rave at close of day.
+
−
Rage, rage against the dying of the light.”</a>
+
−
</p>
+
−
</center>
+
−
</td>
+
−
</tr>
+
−
</tbody>
+
−
</table>
+
  
−
<br>
 
 
<div class="group center">
 
<div class="group center">
−
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
+
<p class="text"> 
 +
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>.
 +
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
 +
<i>E. coli</i>, and in blue 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>). 
 +
</p>
 
</div>
 
</div>
−
<br>
 
  
−
<table>
 
−
<tbody>
 
−
<tr>
 
−
<td style="width:30%;">
 
 
<center>
 
<center>
−
<img src="https://static.igem.org/mediawiki/2015/f/f3/TLSE_top_world.jpeg"style="width:70%;"/>
+
<div class="title" id="2">
 +
<h3>Eradication (formate pathway)</h3>
 +
</div>
 
</center>
 
</center>
−
</td>
+
 
−
<td style="width:30%;">
+
<div class="group center">
−
<center>
+
<p class="text">
 +
The second module is dedicated to formate production and composed of two key genes :
 +
<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>. 
 +
  </p>
 +
  </div>
 +
 
 +
 
 +
<div class="subtitle">
 +
<h3>Formate pathway (<a href="http://parts.igem.org/Part:BBa_K1587007"target=_blank">BBa_K1587007</a>)</h3>
 +
</div>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2015/3/3b/TLSE_Parts_image5.PNG" style="width:40%;"/>
 +
 
 +
 
 +
 
 +
 
 +
<div class="group center">
 
<p class="text">
 
<p class="text">
−
At the beginning of the project, we went to a park to take some pictures that we later used for our communication. There was a playground in that park, so we could not resist the temptation to use the toys and take a few pictures !
+
<i>pflB</i> 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.
 +
<br><br>
 +
<i>pflA</i> encodes the pyruvate formate lyase activase, an enzyme
 +
which activates <i>pflB</i>.
 +
 
 +
 
 +
<br><br>
 +
 +
The formate compound is naturally produced by <i>E. coli</i>, that is why we  
 +
decided to overexpress these two genes. For this, <i>pflB</i> and <i>pflA</i> are put
 +
together with two RBS (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>) in front of them to improve the proteic synthesis. A strong terminator (<a href="http://parts.igem.org/Part:BBa_B1006"target=_blank">BBa_B1006</a>) ends up the sequence.
 
</p>
 
</p>
 +
</div>
 +
 
 +
<center>
 +
<div class="title" id="3">
 +
<h3>Circadian swich</h3>
 +
</div>
 +
 
</center>
 
</center>
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<div class="group center">
 
<div class="group center">
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+
<p class="text">
 +
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.
 +
<br><br>
 +
The center of the light sensor is composed of membrane proteins PCB
 +
(chromophore phycocyanobilin) and the hybrid protein Cph8 (EnvZ and Cph1)[11].
 +
<br><br>
 +
PCB protein comes from a cyanobacterium <i>Synechocystis</i> sp PCC 6803
 +
and to be synthesized, it needs the expression of two genes:  
 +
heme oxygenase (<i>Ho1</i>) and biliverdin reductase (<i>PcyA</i>)[11].
 +
</p>
 
</div>
 
</div>
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<br>
 
  
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<center><img src="https://static.igem.org/mediawiki/2015/thumb/0/02/TLSE_Team_laughing.jpeg/800px-TLSE_Team_laughing.jpeg" style="width:75%;"/>
 
 
</center>
 
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+
<div class="subtitle">
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<center><img src="https://static.igem.org/mediawiki/2015/thumb/b/ba/TLSE_instructor_laughing.jpeg/800px-TLSE_instructor_laughing.jpeg" style="width:75%;"/>
+
<h3><i>Ho1</i> with RBS (<a href="http://parts.igem.org/Part:BBa_K1587000"target=_blank">BBa_K1587000</a>)</h3>
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+
</div>
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+
<img src="https://static.igem.org/mediawiki/2015/6/61/TLSE_Parts_image7.PNG" style="width:29%;"/>
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+
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+
A gene required for chromophore synthesis in photosynthetic
−
When we realize that we have been doing our competent cells wrong for 2 weeks
+
light-harvesting complexes, photoreceptors, and circadian clocks.
 +
<i>ho1</i>, along with <i>pcyA</i>, converts heme into the chromophore
 +
phycocyanobillin (PCB).
 +
<br><br>
 +
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>
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+
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+
 
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<td style="width:30%;">
+
 
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<center>
+
<div class="subtitle">
−
<p class="legend">
+
<h3><i>PcyA</i> with RBS (<a href="http://parts.igem.org/Part:BBa_K1587002"target=_blank">BBa_K1587002</a>)</h3>
−
When we tell our instructor that we failed our competent cells
+
</div>
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+
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+
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+
<img src="https://static.igem.org/mediawiki/2015/e/e1/TLSE_Parts_image8.PNG" style="width:27%;"/>
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<div class="group center">
 
<div class="group center">
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<p class="text">
 
<p class="text">
−
With some slowly growing strains of bacteria, we often had difficulties to spot the colonies, even after an overnight incubation. As we were eager to check the results of our cloning, everybody checked every plate with different ways to look at it !
+
A gene required for chromophore synthesis in photosynthetic
 +
light-harvesting complexes, photoreceptors, and circadian clocks.
 +
<br><br>
 +
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 (<a href="http://parts.igem.org/Part:BBa_K566023"target=_blank">BBa_K566023</a>).
 
</p>
 
</p>
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+
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+
 
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<td style="width:30%;">
+
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<center><img src="https://static.igem.org/mediawiki/2015/6/6c/TLSE_petri_instructors.jpeg"style="width:85%;"/>
+
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+
 
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+
<div class="subtitle">
 +
<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>
−
<br>
+
<img src="https://static.igem.org/mediawiki/2015/4/4c/TLSE_Parts_image9b.PNG" style="width:25%;"/>
  
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<center>
+
<div class="group">
 
<p class="text">
 
<p class="text">
−
<font color="#000000">
+
This biobrick contains the OmpC promoter (<a href="http://parts.igem.org/Part:BBa_R0082"target=_blank">BBa_R0082</a>), a lacI box
−
To be lucky in our wet lab work and wiki coding, we built two glove-gods: <b>ATGC</b> for molecular biology and <b>html</b> for informatics ! (Their main inconvenient being that gloves deflate rather quickly)
+
separated from <i>cI</i> gene by a RBS sequence (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>).
−
</font>
+
 
</p>
 
</p>
−
</center>
+
</div>
  
−
<table>
+
<div class="subtitle">
−
<tbody>
+
<h3>Red light biosensor (<a href="http://parts.igem.org/Part:BBa_K1587008"target=_blank">BBa_K1587008</a>)  </h3>
−
<tr>
+
</div>
−
<td style="width:30%;">
+
 
−
<center><img src="https://static.igem.org/mediawiki/2015/thumb/a/a9/TLSE_ATGC_god.jpeg/337px-TLSE_ATGC_god.jpeg"style="width:60%;"/>
+
<img src="https://static.igem.org/mediawiki/parts/1/10/TLSE_Circ3.jpg" style="width:21%;"/>
−
</center>
+
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+
−
<td style="width:30%;">
+
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<center><img src="https://static.igem.org/mediawiki/2015/6/6d/TLSE_HTML_god.jpeg"style="width:60%;"/>
+
−
</center>
+
−
</td>
+
−
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+
−
</tbody>
+
−
</table>
+
  
−
<br>
 
 
<div class="group center">
 
<div class="group center">
−
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
+
<p class="text">
 +
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>
−
<br>
 
  
−
<table>
+
<div class="subtitle">
−
<tbody>
+
<h3>Regulated formate production (<a href="http://parts.igem.org/Part:BBa_K1587009"target=_blank">BBa_K1587009</a>)  </h3>
−
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+
</div>
−
<td style="width:30%;">
+
<img src="https://static.igem.org/mediawiki/2015/6/6e/Ci_formate.png" style="width:50%;"/>
−
<center><img src="https://static.igem.org/mediawiki/2015/5/5f/TLSE_spot.jpg" style="width:110%;"/>
+
−
</center>
+
−
</td>
+
  
−
<td style="width:26%;">
+
<div class="group center">
−
<center>  
+
 
<p class="text">
 
<p class="text">
−
After spending countless hours in the lab during almost four months, our president, Marine, has found a way to make herself at home !
+
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>
−
</center>
+
</div>
−
</td>
+
 
 +
 
 +
  </main>
 +
</div>
  
−
<td style="width:30%;">
+
<div class="wrapper row4">
−
<center><img src="https://static.igem.org/mediawiki/2015/thumb/2/28/TLSE_organized_mess.jpeg/800px-TLSE_organized_mess.jpeg"style="width:100%;"/>
+
−
</center>
+
−
</td>
+
−
</tr>
+
  
−
<tr>
+
<div class="container clear" style="padding-top:30px;">
 +
<center><p class="maintitle">  
 +
References
 +
</p></center>
  
−
<td>
+
<div class="clear">
−
<center>
+
<ul>
−
<p class="legend">
+
−
<a href = "https://www.youtube.com/watch?v=9Wzq2dzJeXo" target="_blank">"That's MY spot !"</a>
+
−
</p>
+
−
</center>
+
−
</td>
+
  
−
<td>
+
<li>
−
</td>
+
[1] Layton DS & Trinh CT (2014) Engineering modular ester fermentative pathways in <i>Escherichia coli</i>. Metabolic Engineering 26: 77–88
 +
</li>
  
−
<td>
+
<li>
−
<center>
+
−
<p class="legend">
+
[2] Saini M, Hong Chen M, Chiang C-J & Chao Y-P (2015) Potential production platform of n-butanol in <i>Escherichia coli</i>. Metabolic Engineering 27: 76–82
−
"It's not messy, I can find everything in there !"
+
</li>
−
</p>
+
−
</center>
+
−
</td>
+
−
</tr>
+
  
−
</tbody>
+
<li>
−
</table>
+
[3] Volker AR, Gogerty DS, Bartholomay C, Hennen-Bierwagen T, Zhu H & Bobik TA (2014) Fermentative production of short-chain fatty acids in <i>Escherichia coli</i>. Microbiology (Reading, Engl.) 160: 1513–1522
 +
</li>
  
−
<br>
+
<li>
−
<div class="group center">
+
[4] Saini M, Wang ZW, Chiang C-J & Chao Y-P (2014) Metabolic Engineering of <i>Escherichia coli</i> for Production of Butyric Acid. J. Agric. Food Chem. 62: 4342–4348
−
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
+
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−
</div>
+
−
<br>
+
  
−
<table>
+
<li>
−
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+
[5] Hunt MC & Alexson SEH (2002) The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41: 99–130
−
<tr>
+
</li>
−
<td style="width:30%;">
+
−
<center>
+
−
<img src="https://static.igem.org/mediawiki/2015/thumb/f/fb/Fungus_contamination.jpeg/320px-Fungus_contamination.jpeg" style="width:70%;"/>
+
−
</center>
+
−
</td>
+
−
<td style="width:30%;">
+
−
<center>
+
−
<p class="text">
+
−
We found several old LB medium in the lab, but this was the most beautiful specimen of fungus contamination we saw !
+
−
</p>
+
−
</center>
+
−
</td>
+
−
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+
−
</tbody>
+
−
</table>
+
  
−
<br>
+
<li>
−
<div class="group center">
+
[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 <i>Clostridium difficile</i> on butyrate production in <i>Escherichia coli</i>. J. Bacteriol. 195: 3704–3713
−
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
+
</li>
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</div>
+
−
<br>
+
  
−
<table>
+
<li>
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<tbody>
+
[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.  
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<tr>
+
</li>
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<td style="width:30%;">
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<center><img src="https://static.igem.org/mediawiki/2015/7/79/Toulouse_selfie_capital.jpeg"style="width:65%;"/>
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−
</center>
+
−
</td>
+
  
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<td style="width:30%;">
+
<li>
−
<center><img src="https://static.igem.org/mediawiki/2015/1/1a/TSLE_because_api.png"style="width:100%;"/>
+
[8]  CEA-CNRS- Aix Marseille Université. February 2015. Paris . Activation d’enzymes bactériennes pour convertir le CO2 en source d’énergie renouvelable. 
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</center>
+
</li>
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</td>
+
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</tr>
+
  
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<tr>
+
<li>
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<td style="width:30%;">
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[9] Crain AV & Broderick JB (2014) Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme. J. Biol. Chem. 289: 5723–5729
−
<center>
+
</li>
−
<p class="legend">
+
−
But first, let me take a selfie !
+
−
</p>
+
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</center>
+
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</td>
+
−
<td style="width:30%;">
+
−
<center>
+
−
<p class="legend">
+
−
<a href="https://www.youtube.com/watch?v=y6Sxv-sUYtM"target="_blank">♪ Because I’m Api ♫</a>
+
−
</p></center>
+
−
</td>
+
−
</tr>
+
−
</tbody>
+
−
</table>
+
  
−
<br>
+
<li>
−
<div class="group center">
+
[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 <i>Escherichia coli</i> for Poly(3-hydroxybutyrate) Production under Microaerobic Condition. BioMed Research International, BioMed Research International 2015, 2015: e789315
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<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
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</li>
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</div>
+
−
<br>
+
  
−
<center>
+
<li>
−
<img src="https://static.igem.org/mediawiki/2015/6/6b/TLSE_mathilde_sasd.png"style="width:45%;"/>
+
[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 <i>Escherichia coli</i> to see light. Nature 438: 441–442
−
<p class="legend">
+
</li>
−
Mathilde's face when she had to cope with our puns and jokes !
+
−
</p>
+
−
</center>
+
  
−
<br>
+
<li>
−
<div class="group center">
+
[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
−
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
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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
 +
</li>
 +
<li>
 +
[14] Gambetta GA & Lagarias JC (2001) Genetic engineering of phytochrome biosynthesis in bacteria. PNAS 98: 10566–10571
 +
</li>
 +
</ul>
 
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Test tableau
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Revision as of 17:49, 18 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 pBla-RBS-ccr-RBS-hbd-RBS-crt-RBS- tesB-RBS-atoB-Terminator Attract 5192 Ok [1][2][3][4][6]
BBa_K1587005 Device pBla-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 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 improve the proteic synthesis. A strong terminator (BBa_B1006) ends up the sequence.

Circadian swich

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 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 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