Difference between revisions of "Team:Toulouse/Description/Attract"

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     <center> <h3>Device</h3></center>
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     <center> <h3>Attract</h3> </center>
 
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  <center><img src="https://static.igem.org/mediawiki/2015/7/75/TLSE_device_bee.png"></center>
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      <h3>Content</h3>
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<center>
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    <div id="breadcrumb" class="clear" style="float: center;" >
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  <ul>
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        <li><a href="#part1">How to attract <i>Varroa destructor?</i></a></li>
 +
        <li><a href="#part2">Butyrate attraction test</a></li>
 +
        <li><a href="#part3">How to produce butyrate with <i>E.Coli</i>?</a></li>
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      </ul>
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    <!-- FIRST PARAGRAPH -->
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  <p class="text">
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Our goal is to create a solution against varroas. In order to use ApiColi to treat varroosis, we designed a trap, named TrApiColi. TrApiColi has been designed in order to take into account ethical
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<div>
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reflection, safety and ease of use for beekeepers.
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  </p>
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<div class="subtitle" >  
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  </div>
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<h3>How to attract <i>Varroa destructor</i>?</h3>
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  <div class="title">
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  <h3>Trap Construction</h3>
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  </div>
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    <div class="group center">
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  <p class="text">
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Since the production of the two pathways are regulated by day light, our bacteria need to be outside of the beehive. Thus, the trap was made to be placed at the entrance of the hive, in order to prevent the entry of the mites.
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  </p>
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  </div>
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<div class="group center">
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<p class="text">
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<strong>TrApiColi is composed of four main parts:</strong>
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</p>
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</div>
 
</div>
  
 +
 +
<div class="group center">
 +
<p align="justify" style="font-size:15px;">
  
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<center>
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Just before capping, bee larvaes produce a wide range of molecules,
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<div style="one-half;padding:10px;">
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those molecules warn the mite about the upcoming capping and motivate
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<img src="https://static.igem.org/mediawiki/2015/6/63/TLSE_Device_Trap_1.png" style="width:70%;" />
+
it to enter the cell [1]. 
 +
Of all these molecules, scientific studies have shown that one can
 +
significantly attract varroa:
 +
<i>butyrate</i> [2].
 +
</p>
 
</div>
 
</div>
  
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<p class="legend">
 
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The four different parts of our trap
 
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</p>
 
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</center>
 
  
  
 
<div class="group center">
 
<div class="group center">
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  <p class="text">
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      <div class="one_half first">
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<strong>1.</strong> A grid in line with the bottom board
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  <p align="justify" style="font-size:15px;">
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<br><br>The bees usually enter the hive by landing on the bottom board before walking inside. Because of the alignment of the trap with the board, it does not disturb the bee’s comings and goings. The holes are big enough to let the varroas fall through them, but not the bees.
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  <br>
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</p>
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      Butyrate is a volatile acid which is non-toxic for honeybees
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   </div>
+
  nor the human being, because it is already present at physiologic
−
 
+
  concentrations in the digestive tract. Moreover this molecule
 +
  is naturally
 +
  produced by some bacterial strains like <i>Clostridium</i>,
 +
  which is an asset
 +
  for this synthetic biology project [3].</p><div id="part2"></div> <!-- ANCHOR 2 --><p align="justify" style="font-size:15px;"> Therefore, based on the patent US 8647615, we decided to
 +
  modify <i>E. coli</i>
 +
  so it will synthesize
 +
  butyrate in order to attract varroa [4].  
 +
</p>    
 +
      </div>
  
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      <div class="one_half">
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<img src="https://static.igem.org/mediawiki/2015/e/e6/TLSE_Attract_fig2.png">
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<div class="group">
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<p>Figure 1: Results of butyrate attraction
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<p class="text">
+
test with quadrants method, US 8647615 B1 [4].
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<strong>2.</strong> A funnel, to channel all the falling varroas
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</p>
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  </div>
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<center>
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<div style="one-half;padding:10px;">
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<img src="https://static.igem.org/mediawiki/2015/e/e2/TLSE_Device_Trap_2.jpg" style="width:40%;" />
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</div>
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+
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<p class="legend">
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The grid and the funnel
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</p>
 
</p>
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</center>
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 +
          </div>
 +
  </div>
  
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<div class="group">
+
<div class="subtitle" >  
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<p class="text">
+
<h3>Butyrate attraction test</h3>
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<strong>3.</strong> A transparent collector, containing the bacteria confined in a special bag
+
</div>
−
<br><br>It is designed like a fish bottle trap: the tube from the funnel goes inside the collector to ease the entry of the varroas in the collector while preventing them from exiting. The special bag is described in "TPX bag" part
+
−
</p>
+
−
  </div>
+
  
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<div class="group"> 
+
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<p class="text">
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    <div class="group center"> <!-- FIRST PARAGRAPH -->
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<strong>4.</strong> A roof, to protect the trap from the rain
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</p>
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  <div class="one_half first">
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  </div>
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−
 
+
<img src="https://static.igem.org/mediawiki/2015/b/b8/TLSE_Attract_fig3.png">
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  <div class="group center">
+
<p>Figure 2: Butyrate attraction test using
−
<p class="text">
+
T tube, with varroa mite in the middle
−
The dimensions of the trap allow it to be plugged to almost every beehives. Indeed, most of the hive types have the exact same entrance. Thanks to that, the trap can be perfectly plugged to the hive by the beekeepers without drilling or cutting it.
+
−
</p>
+
−
  </div>
+
−
 
+
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<div class="group center">
+
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<p class="text">
+
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The trap was designed using Catia and then 3D printed in order to build a prototype. It is used as a demonstration device for the beekeepers and the general public. This trap could not be tested because the porous plastic used for 3D printing is permeable to liquids and gases. Moreover, the modeling showed that this version of the trap is yet to be optimized to ensure a proper diffusion of our molecules, see more in <a target="_blank" href="https://2015.igem.org/Team:Toulouse/Modeling#part5"> "Modelling" </a> part.
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</p>
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  </div>
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<center>  
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<table>
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<tbody>
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<td>
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<div style="one-half;padding:10px;">
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</p>
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<img src="https://static.igem.org/mediawiki/2015/c/c2/TLSE_Device_Trap_3.jpg" style="width:80%;" />
+
  </div>
 +
 
 +
  <div class="one_half">
 +
  <p align="justify" style="font-size:15px;">
 +
To check adequacy and relevance of this study (Figure 2),
 +
an experiment using a glass T-tube has been developed (Figure 3).
 +
In the first branch, there is a cotton soaked with 50 µL of water,
 +
in the second a cotton with  50 µL of butyrate at 4%, and finally the
 +
last one contains the varroa.</p> <div id="part3"> <!-- ANCHOR 3 --> </div> <p align="justify" style="font-size:15px;"> Butyrate being very volatile, our
 +
system
 +
used a pump to renew air, producing a concentration gradient as seen <a href="https://2015.igem.org/Team:Toulouse/Results#varrotest">here</a>.
 +
</p>
 +
      </div>
 +
 
 
</div>
 
</div>
  
−
</td>
+
<div class="subtitle" >  
 +
<h3>How to produce butyrate with <i>E.coli</i>?</h3>
 +
</div>
  
 +
<div class="group center">
 +
     
 +
  <p align="justify" style="font-size:15px;">
 +
    In this project, an <i>Escherichia coli</i> strain is used for its known
 +
simplicity of genetic manipulation and its adequacy with butyrate
 +
synthesis. Indeed, among the five enzymes of the butyrate pathway,
 +
two enzymes are naturally produced by the bacteria. The following
 +
engineered butyrate pathway has been designed:
 +
</p>   <br>
 +
      </div>
  
 +
 
 +
  <div class="group center"> <!-- CENTERED FIGURE -->
 +
  <img src="https://static.igem.org/mediawiki/2015/0/02/TLSE_Attract_fig4.png" />
 +
</div>
 +
  <div class="group center">
 +
<figcaption>Figure 3: Engineered butyrate pathway</figcaption>
 +
 
 +
</div>
 +
 
 +
<div class="group center">
 +
     
 +
  <p align="justify" style="font-size:15px;">
 +
  <br>
 +
    The initial substrate is glucose which is decomposed into
 +
acetyl-CoA during glycolysis. Finally, butyrate pathway
 +
begin with acetyl-CoA: five genes are required with two
 +
homologous and three heterologous genes.
 +
</p>  
  
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<td>
 
−
 
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<div style="one-half;padding:10px;">
 
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<img src="https://static.igem.org/mediawiki/2015/0/08/TLSE_Device_Trap_4.jpg" style="width:80%;" />
 
 
</div>
 
</div>
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<br>
  
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</td>
 
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</tr>
 
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</tbody>
 
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</table>
 
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</center>
 
  
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<center>
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<div style="font-size:15px;">
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<p class="legend">
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<ul>
−
The 3D printer used to construct our trap and the result of 3D print: TrApiColi
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  <li><b><i>atoB</i></b> present in <i>E.coli</i>, coding for acetyl-CoA
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</p>
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  acetyltransferase, an acetyltransferase catalyzing the combination
−
</center>  
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  of two acetyl-CoA.
−
 
+
<br>
−
 
+
<div class="group center">
 +
<br>
 +
  <img src="https://static.igem.org/mediawiki/2015/c/c5/TLSE_Attract_fig5.png" />
 +
</div>
 +
  <div class="group center">
 +
<br>
 +
<p class="legend">Figure 4: Reaction catalyzed by acetyl-CoA
 +
acetyltransferase </p>
 +
</div>
 
    
 
    
 +
  </li>
 
    
 
    
 +
  <li><b><i>hbd</i></b> present in <i>Clostridium acetobutylicum</i> coding for
 +
  3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing
 +
  the formation of an alcohol function.
 +
  <br>
 +
    <div class="group center">
 +
<br>
 +
  <img src="https://static.igem.org/mediawiki/2015/d/d6/TLSE_Attract_fig6.png" />
 +
</div>
 +
  <div class="group center">
 +
<br>
 +
<p class="legend">Figure 5: Reaction catalyzed by
 +
3-hydroxybutyryl-CoA dehydrogenase
 +
</p>
 +
</div>
 +
  </li>
 
    
 
    
 +
  <li><b><i>crt</i></b> present in <i>C.acetobutylicum</i>
 +
  coding for 3-hydroxybutyryl-CoA dehydratase,
 +
  a lyase cleaving carbon-oxygen bond.
 +
<br>
 +
  <div class="group center">
 +
<br>
 +
  <img src="https://static.igem.org/mediawiki/2015/6/67/TLSE_Attract_fig7.png" />
 +
</div>
 +
    <div class="group center">
 +
<br>
 +
<p class="legend">Figure 6:
 +
Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase
  
 +
</p>
 +
</div>
 +
  </li>
 
    
 
    
 +
  <li><b><i>ccr</i></b> present in
 +
  <i>Streptomyces collinus</i> coding
 +
  for crotonyl-CoA reductase,
 +
  an oxidoreductase acting on
 +
  CH=CH double bond. This enzyme
 +
  is also in <i>C.acetobutylicum</i> with
 +
  <b>bcd</b> gene coding for butyryl-CoA dehydrogenase,
 +
  with the disadvantage
 +
  to run with Electron Transfer
 +
  Flavoprotein (ETF) which complicates the reaction [6].
 +
<br>
 +
  <div class="group center">
 +
<br>
 +
  <img src="https://static.igem.org/mediawiki/2015/5/57/TLSE_Attract_fig8.png" />
 +
</div>
 +
  <br>
 +
  <div class="group center">
 +
<br>
 +
<p class="legend">Figure 7: Reaction
 +
catalyzed by crotonyl-CoA reductase
 +
</p>
 +
</div>
 
    
 
    
 +
  </li>
 
    
 
    
−
<div class="title">
+
    <li><b><i>tesB</i></b> present in <i>E.coli</i>  
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  <h3>ApiColi confinement and culture</h3>
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coding for acyl-CoA transferase 2,
−
  </div>
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a thiolase which enables coenzyme A transfer.
−
 
+
  <br>
 
   <div class="group center">
 
   <div class="group center">
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  <p class="text">
 
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The use of genetically modified organisms in a field, and because our project is associated with
 
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edible products, underlies both applying of regulations and public interest.
 
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In this context, we searched a solution being able to isolate our engineered bacteria from
 
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the environment, but allowing its growth, metabolism and gas diffusion. We found the
 
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project of the iGEM Groeningen 2012 team which used the polymer TPX® in
 
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order to contain their bacteria separated of the meat [1].
 
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TPX® is in fact Polymethylpentene a porous polymer sold by the company MitsuiChemicals (4-methylpentene-1 based polyolefin, Mitsui Chemicals, Inc.),
 
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In order to perform our experiments, we contacted MitsuiChemicals who offered us some samples of TPX®.
 
−
 
 
<br>
 
<br>
 +
  <img src="https://static.igem.org/mediawiki/2015/3/34/TLSE_Attract_fig9.png" />
 +
</div>
 +
<div class="group center">
 
<br>
 
<br>
−
To check the feasability and safety of our device, several tests have been performed:
+
<p class="legend">Figure 8: Reaction
 +
catalyzed by acyl-CoA transferase 2
 
</p>
 
</p>
 +
</div>
 +
 
 +
  </li>
 +
 
 +
</ul>
 +
 
 
</div>
 
</div>
 +
<div class="group center">
 +
  <p align="justify" style="font-size:15px;">Concerning heterologous genes (hbd, crt and ccr), a codon optimization has been performed in order to enable a good expression of these genes in <i>E. coli</i>.
 +
The genetic construction is then done by assembling the five genes presented earlier, which are placed under the control of P(Bla) constitutive promoter (BBa_I14018). In between the genes are placed ribosome binding sites (RBS) (BBa_B0030) to improve protein expression, and a strong terminator (BBa_B1006) is used to end this construction, which is to be cloned into a pSB1C3 vector (<a href="https://static.igem.org/mediawiki/2015/9/9e/PSB1C3_ccr_butyrate.xdna.png">here</a>).</p>
 +
</div>
  
 +
<center><img src="https://static.igem.org/mediawiki/2015/2/20/TLSE_Attract_fig10.png" style="width:65%;"/></center>
  
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<ul style="font-size:15px;margin-bottom:10px;">
 
−
<li>Safety test: Impermeability of the bag of TPX® to the bacteria
 
−
</li>
 
−
<li>Gas diffusion tests: Permeability of butyric acid and formic acid through the bag of TPX®
 
−
</li>
 
−
<li>Growth tests in TPX®: Culture of the strain <i>E. coli</i> BW25113 in a TPX® bag (<i>ie.</i> in microaerobic conditions, without agitation and miming batch culture condition), as it would be in the field
 
−
</li>
 
−
<li>Bacterial survival over 15 days in microaerobic condition
 
−
</li>
 
−
<li>Carbone source test: choice of Carbon source to produce acids during 10 days
 
−
</li>
 
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<li>Acid toxicity on <i>E. coli</i>
 
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</li>
 
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</ul>
 
  
 
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<a href="https://2015.igem.org/Team:Toulouse/Description/Attract">
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<a href="https://2015.igem.org/Team:Toulouse/Description/Eradicate">
 
<div class="title">
 
<div class="title">
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<h3>Attract</h3>
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<h3>Eradicate</h3>
 
</div>
 
</div>
 
</a>
 
</a>
 
</div>
 
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<div style="width:20%;">
 
<div style="width:20%;">
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<a href="https://2015.igem.org/Team:Toulouse/Description/Eradicate">
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<a href="https://2015.igem.org/Team:Toulouse/Description/Regulation">
 
<div class="title">
 
<div class="title">
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<h3>Eradicate</h3>
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<h3>Regulation</h3>
 
</div>
 
</div>
 
</a>
 
</a>
 
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<div style="width:20%;">
 
<div style="width:20%;">
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<a href="https://2015.igem.org/Team:Toulouse/Description/Regulation">
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<a href="https://2015.igem.org/Team:Toulouse/Design">
 
<div class="title">
 
<div class="title">
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<h3>Regulation</h3>
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<h3>Device: TrApiColi</h3>
 
</div>
 
</div>
 
</a>
 
</a>
 
</div>
 
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<div style="width:20%;">
 
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References
 
References
 
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[1] Le Conte Y, Arnold G, Trouiller J, Masson C, Chappe B & Ourisson G (1989) Attraction of the parasitic mite varroa to the drone larvae of honey bees by simple aliphatic esters. Science 245: 638–639 </li>
  
 
<li>
 
<li>
−
[1] REFERENCE 1 Vers la page Groeningen 2012
+
[2] <b>Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24]. </b>
 
</li>
 
</li>
  
 
<li>
 
<li>
−
+
[3] Louis P & Flint HJ (2009) Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 294: 1–8</li>
−
[2] REFERENCE 2 AVEC UN LIEN <a href="http://www.google.com/patents/US8647615">See more</a>
+
 
 +
<li>
 +
[4] <b>Peter EA Teal, Adrian J. Duehl, Mark J. Carroll. The United States Of America, As Represented By The Secretary Of Agriculture. 2014. Methods for attracting honey bee parasitic mites, US 8647615 B1. </b>
 
</li>
 
</li>
  
 +
[5] Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T & Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 10: 305–311
 
<li>
 
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[3] REFERENCE 2 AVEC UN LIEN qui ouvre dans une nouvelle fenêtre <a href="http://www.google.com/patents/US8647615">See more</a>
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[6] Wallace KK, Bao ZY, Dai H, Digate R, Schuler G, Speedie MK & Reynolds KA (1995) Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. Eur. J. Biochem. 233: 954–962
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Revision as of 22:49, 17 September 2015

iGEM Toulouse 2015

Attract


Content

How to attract Varroa destructor?

Just before capping, bee larvaes produce a wide range of molecules, those molecules warn the mite about the upcoming capping and motivate it to enter the cell [1]. Of all these molecules, scientific studies have shown that one can significantly attract varroa: butyrate [2].


Butyrate is a volatile acid which is non-toxic for honeybees nor the human being, because it is already present at physiologic concentrations in the digestive tract. Moreover this molecule is naturally produced by some bacterial strains like Clostridium, which is an asset for this synthetic biology project [3].

Therefore, based on the patent US 8647615, we decided to modify E. coli so it will synthesize butyrate in order to attract varroa [4].

Figure 1: Results of butyrate attraction test with quadrants method, US 8647615 B1 [4].

Butyrate attraction test

Figure 2: Butyrate attraction test using T tube, with varroa mite in the middle

To check adequacy and relevance of this study (Figure 2), an experiment using a glass T-tube has been developed (Figure 3). In the first branch, there is a cotton soaked with 50 µL of water, in the second a cotton with 50 µL of butyrate at 4%, and finally the last one contains the varroa.

Butyrate being very volatile, our system used a pump to renew air, producing a concentration gradient as seen here.

How to produce butyrate with E.coli?

In this project, an Escherichia coli strain is used for its known simplicity of genetic manipulation and its adequacy with butyrate synthesis. Indeed, among the five enzymes of the butyrate pathway, two enzymes are naturally produced by the bacteria. The following engineered butyrate pathway has been designed:


Figure 3: Engineered butyrate pathway


The initial substrate is glucose which is decomposed into acetyl-CoA during glycolysis. Finally, butyrate pathway begin with acetyl-CoA: five genes are required with two homologous and three heterologous genes.


  • atoB present in E.coli, coding for acetyl-CoA acetyltransferase, an acetyltransferase catalyzing the combination of two acetyl-CoA.


    Figure 4: Reaction catalyzed by acetyl-CoA acetyltransferase

  • hbd present in Clostridium acetobutylicum coding for 3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing the formation of an alcohol function.


    Figure 5: Reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase

  • crt present in C.acetobutylicum coding for 3-hydroxybutyryl-CoA dehydratase, a lyase cleaving carbon-oxygen bond.


    Figure 6: Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase

  • ccr present in Streptomyces collinus coding for crotonyl-CoA reductase, an oxidoreductase acting on CH=CH double bond. This enzyme is also in C.acetobutylicum with bcd gene coding for butyryl-CoA dehydrogenase, with the disadvantage to run with Electron Transfer Flavoprotein (ETF) which complicates the reaction [6].



    Figure 7: Reaction catalyzed by crotonyl-CoA reductase

  • tesB present in E.coli coding for acyl-CoA transferase 2, a thiolase which enables coenzyme A transfer.


    Figure 8: Reaction catalyzed by acyl-CoA transferase 2

Concerning heterologous genes (hbd, crt and ccr), a codon optimization has been performed in order to enable a good expression of these genes in E. coli. The genetic construction is then done by assembling the five genes presented earlier, which are placed under the control of P(Bla) constitutive promoter (BBa_I14018). In between the genes are placed ribosome binding sites (RBS) (BBa_B0030) to improve protein expression, and a strong terminator (BBa_B1006) is used to end this construction, which is to be cloned into a pSB1C3 vector (here).

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References


    [1] Le Conte Y, Arnold G, Trouiller J, Masson C, Chappe B & Ourisson G (1989) Attraction of the parasitic mite varroa to the drone larvae of honey bees by simple aliphatic esters. Science 245: 638–639
  • [2] Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24].
  • [3] Louis P & Flint HJ (2009) Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 294: 1–8
  • [4] Peter EA Teal, Adrian J. Duehl, Mark J. Carroll. The United States Of America, As Represented By The Secretary Of Agriculture. 2014. Methods for attracting honey bee parasitic mites, US 8647615 B1.
    • [5] Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T & Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 10: 305–311
  • [6] Wallace KK, Bao ZY, Dai H, Digate R, Schuler G, Speedie MK & Reynolds KA (1995) Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. Eur. J. Biochem. 233: 954–962