Difference between revisions of "Team:Toulouse/Parts"

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{{Toulouse}}
 
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<html>
 
<html>
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<head>
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<meta charset="utf-8">
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<title>iGEM Toulouse 2015</title>
 +
<link rel="icon" href="https://static.igem.org/mediawiki/2015/2/2e/TLSE_ApiColi.png" type="image/png" style="width:10%;"/>
  
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<h2> Part Documentation</h2>
+
</head>
 +
<body id="top">
 +
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
+
</html>{{Toulouse/nav}}<html>
−
<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
+
  
 +
  
−
<div class="highlightBox">
+
−
<h4>Note</h4>
+
    <!-- ################################################################################################
−
<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
+
 +
    <!-- ################################################################################################ -->
 +
 
 +
 
 +
<!-- ################################################################################################ -->
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<!-- ################################################################################################ -->
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<!-- ######################################  URL DE L'IMAGE EN BG    ########################################################## -->
 +
<div class="wrapper row0 bgded" style="background-image:url('https://static.igem.org/mediawiki/2015/8/8f/TLSE_bg_9.jpg')">
 +
 
 +
 
 +
 
 +
 
 +
  <div class="shout">  
 +
   
 +
    <div class="shout-content clear">
 +
      <div class="maintitle">
 +
    <center> <h3>Biobricks</h3> </center>
 +
    </div>
 +
  <center><img src="https://static.igem.org/mediawiki/2015/2/2a/TLSE_brickBee.png" ></center>
 +
 
 +
</div>
 +
 
 +
  </div>
 +
 
 +
 +
 
</div>
 
</div>
 +
<!-- ################################################################################################ -->
 +
<!-- ################################################################################################ -->
 +
<!-- ################################################################################################ -->
  
 +
<div class="wrapper row3">
  
 +
<br>
 +
  <main class="container clear">
 +
  <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 switch</a></li>
  
−
<h4>Adding parts to the registry</h4>
+
      </ul>
−
<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
+
    </div>
−
<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
+
<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1);">
  
 +
</center>
 +
  <!-- ################################ ICI CONTENEUR OU TU PEUX T'AMUSER A METTRE LES DIVS ############################### -->
 +
 
 +
  <!---- TEST BACKGROUND-COULEUR --->
 +
 
 +
<br><br>
 +
<div class="group center" id="biobricks">
 +
<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>
 +
</div>
  
−
<h4>What information do I need to start putting my parts on the Registry?</h4>
+
<div class="group center">
−
<p>The information needed to initially create a part on the Registry is:</p>
+
<table class="result">
−
<ul>
+
  <tbody>
−
<li>Part Name</li>
+
    <tr>
−
<li>Part type</li>
+
      <th>Name</th>
−
<li>Creator</li>
+
      <th>Type</th>
−
<li>Sequence</li>
+
      <th>Genic construction</th>
−
<li>Short Description (60 characters on what the DNA does)</li>
+
      <th>Module</th>
−
<li>Long Description (Longer description of what the DNA does)</li>
+
  <th>Length (bp)</th>
−
<li>Design considerations</li>
+
  <th>Sequencing</th>
−
</ul>
+
      <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>P(Bla)-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>P(Bla)-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>Strong Promotor-RBS-<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>
 +
<div class="title" id="1">
 +
<h3>Attraction (butyrate pathway)</h3>
 +
</div>
 +
</center>
  
−
<p>
 
−
We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
 
  
 +
<div class="group center">
 +
<p class="text">
 +
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>
 +
</div>
  
 +
<div class="subtitle">
 +
<h3>Basic parts</h3>
 +
</div>
  
 +
<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%;"/>
  
 +
<div class="group center">
 +
<p class="text">
 +
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>
 +
</div>
  
−
<h4>Inspiration</h4>
+
<div class="subsubtitle">
−
<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
+
<h3><i>crt</i> <a href="http://parts.igem.org/Part:BBa_K1587003"target=_blank">(BBa_K1587003)</a></h3>
 +
</div>
  
−
<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
+
<img src="https://static.igem.org/mediawiki/2015/4/4c/TLSE_Parts_image2b.PNG" style="width:37%;"/>
 +
 
 +
<div class="group center">
 +
<p class="text">
 +
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
 +
expression in our strain. The <i>crt</i> enzyme substrate is 3-hydroxybutyryl CoA,
 +
and the product is Crotonyl CoA. This reaction does not need any coenzyme.
 +
</p>
 +
</div>
 +
 
 +
<div class="subtitle">
 +
<h3>Other parts</h3>
 +
</div>
 +
 
 +
<div class="subsubtitle">
 +
<h3><i>ccr-Butyrate pathway</i> <a href="http://parts.igem.org/Part:BBa_K1587004"target=_blank">(BBa_K1587004)</a></h3>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2015/5/5b/TLSE_Parts_image3.PNG" style="width:61%;"/>
 +
 
 +
<div class="group center">
 +
<p class="text">
 +
 
 +
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 :
 +
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
 +
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
 +
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>
 +
<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.
 +
 
 +
</p>
 +
</div>
 +
 
 +
<div class="subsubtitle">
 +
<h3><i>Butyrate pathway wihout ccr</i> <a href="http://parts.igem.org/Part:BBa_K1587005"target=_blank">(BBa_K1587005)</a></h3>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2015/6/6f/TLSE_Parts_image4.PNG" style="width:59%;"/>
 +
 
 +
<div class="group center">
 +
<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>
 +
 
 +
<center>
 +
<div class="title" id="2">
 +
<h3>Eradication (formate pathway)</h3>
 +
</div>
 +
</center>
 +
 
 +
<div class="group 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">
 +
<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 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.
 +
</p>
 +
</div>
 +
 
 +
<center>
 +
<div class="title" id="3">
 +
<h3>Circadian switch</h3>
 +
</div>
 +
 
 +
</center>
 +
 
 +
<div class="group center">
 +
<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 sp.</i> 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>
 +
 
 +
</center>
 +
 
 +
<div class="subtitle">
 +
<h3><i>Ho1</i> with RBS (<a href="http://parts.igem.org/Part:BBa_K1587000"target=_blank">BBa_K1587000</a>)</h3>
 +
</div>
 +
 +
 +
<img src="https://static.igem.org/mediawiki/2015/6/61/TLSE_Parts_image7.PNG" style="width:29%;"/>
 +
 +
<div class="group center">
 +
<p class="text">
 +
A gene required for chromophore synthesis in photosynthetic
 +
light-harvesting complexes, photoreceptors, and circadian clocks.
 +
<i>ho1</i>, along with <i>pcyA</i>, converts heme into the chromophore
 +
phycocyanobillin (PCB). The gene comes from the cyanobacterium <i>Synechocystis sp. </i>PCC 6803.
 +
<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>
 +
</div>
 +
 
 +
 
 +
<div class="subtitle">
 +
<h3><i>PcyA</i> with RBS (<a href="http://parts.igem.org/Part:BBa_K1587002"target=_blank">BBa_K1587002</a>)</h3>
 +
</div>
 +
 +
 +
<img src="https://static.igem.org/mediawiki/2015/e/e1/TLSE_Parts_image8.PNG" style="width:27%;"/>
 +
 +
<div class="group center">
 +
<p class="text">
 +
A gene required for chromophore synthesis in photosynthetic
 +
light-harvesting complexes, photoreceptors, and circadian clocks. The gene comes from the cyanobacterium <i>Synechocystis sp.</i> PCC 6803.
 +
<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>
 +
</div> 
 +
 
 +
 
 +
 
 +
 
 +
<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>
 +
<img src="https://static.igem.org/mediawiki/2015/4/4c/TLSE_Parts_image9b.PNG" style="width:25%;"/>
 +
 
 +
<div class="group">
 +
<p class="text">
 +
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 (<a href="http://parts.igem.org/Part:BBa_B0030"target=_blank">BBa_B0030</a>).
 +
</p>
 +
</div>
 +
 
 +
<div class="subtitle">
 +
<h3>Red light biosensor (<a href="http://parts.igem.org/Part:BBa_K1587008"target=_blank">BBa_K1587008</a>)  </h3>
 +
</div>
 +
 
 +
<img src="https://static.igem.org/mediawiki/parts/1/10/TLSE_Circ3.jpg" style="width:21%;"/>
 +
 
 +
<div class="group center">
 +
<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 class="subtitle">
 +
<h3>Regulated formate production (<a href="http://parts.igem.org/Part:BBa_K1587009"target=_blank">BBa_K1587009</a>)  </h3>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2015/6/6e/Ci_formate.png" style="width:50%;"/>
 +
 
 +
<div class="group center">
 +
<p class="text">
 +
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>
 +
</div>
 +
 
 +
 
 +
  </main>
 +
</div>
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<div class="wrapper row4">
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<div class="container clear" style="padding-top:30px;">
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<center><p class="maintitle">  
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References
 +
</p></center>
 +
 
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<div class="clear">
 
<ul>
 
<ul>
−
<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
 
−
<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
 
−
</ul>
 
  
 +
<li>
 +
[1] Layton DS & Trinh CT (2014) Engineering modular ester fermentative pathways in <i>Escherichia coli</i>. Metabolic Engineering 26: 77–88
 +
</li>
  
 +
<li>
 +
 +
[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
 +
</li>
  
−
<h4>Part Table </h4>
+
<li>
−
</html>
+
[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
−
<groupparts>iGEM015 Example</groupparts>
+
</li>
−
<html>
+
  
 +
<li>
 +
[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
 +
</li>
  
 +
<li>
 +
[5] Hunt MC & Alexson SEH (2002) The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41: 99–130
 +
</li>
  
 +
<li>
 +
[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
 +
</li>
 +
 +
<li>
 +
[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.
 +
</li>
 +
 +
<li>
 +
[8]  CEA-CNRS- Aix Marseille Université. February 2015. Paris . Activation d’enzymes bactériennes pour convertir le CO2 en source d’énergie renouvelable. 
 +
</li>
 +
 +
<li>
 +
[9] Crain AV & Broderick JB (2014) Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme. J. Biol. Chem. 289: 5723–5729
 +
</li>
 +
 +
<li>
 +
[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
 +
</li>
 +
 +
<li>
 +
[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
 +
</li>
 +
 +
<li>
 +
[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
 +
</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>
 
</div>
 
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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