Difference between revisions of "Team:Toulouse/project/attract"

 
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      <center> <p class="maintitle">  
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      <div class="title">
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Attract
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    <center> <h3>Attract</h3> </center>
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</p></center>
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    </div>
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  <center><img src=" https://static.igem.org/mediawiki/2015/5/57/TLSE_Attract_BG.png" style="width:40%;"></center>
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  <center><img src=" https://static.igem.org/mediawiki/2015/5/57/TLSE_Attract_BG.png"></center>
 
    
 
    
 
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      <h3>Content</h3>
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<p class="title">    
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<center>
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    <div id="breadcrumb" class="clear" style="float: center;" >
 +
  <ul>
 +
        <li><a href="#part1">How to Attract</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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    </div>
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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);">
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    <!-- FIRST PARAGRAPH -->
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<center><div class="subtitle" >    
 +
<h3>About varroasis</h3>
 +
</div></center>
  
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</p>
 
−
</div>
 
 
    <div class="group center"> <!-- FIRST PARAGRAPH -->
 
    <div class="group center"> <!-- FIRST PARAGRAPH -->
 
         <p align="justify" style="font-size:15px;">
 
         <p align="justify" style="font-size:15px;">
−
Varroasis occurs with the Varroa entrance in the hive, carried by infected bees:  
+
Varroasis occurs with the varroa entrance in the hive, carried by  
−
the mite can begin its parasitism and attack the brood. When the queen gives birth
+
infected bees:  
−
to new larvaes in honeycombs, the fertilized adult female Varroa mite will come into  
+
the mite can begin its parasitism and attack the brood. When the queen  
−
it before capping, and lay her eggs. The larvaes will develop, increasing the overall  
+
gives birth
−
infection that affects bee population. To tackle this issue, it is necessary to attract  
+
to new larvaes in honeycombs, the fertilized adult female varroa mite  
−
Varroa carried by honeybees before they come into the hive.
+
will come into  
 +
it before capping, and lay her eggs. The larvaes will develop,  
 +
increasing the overall  
 +
infection that affects bee population [1]. To tackle this issue,  
 +
it is necessary to attract  
 +
varroa carried by honeybees before they come into the hive.
 
</p>
 
</p>
 
       </div>
 
       </div>
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   <img src="https://static.igem.org/mediawiki/2015/0/04/TLSE_Attract_fig1.png" />
 
   <img src="https://static.igem.org/mediawiki/2015/0/04/TLSE_Attract_fig1.png" />
 
</div>
 
</div>
 +
<div id="part1"></div><!-- ANCHOR 1 -->
 
<div class="group center">
 
<div class="group center">
 
<br>
 
<br>
−
  <p>Figure 1 : <i>Varroa destructor</i> life cycle, adapted from B. Alexander</p>
+
  <p>Figure 1 : <i>Varroa destructor</i> life cycle,  
 +
adapted from B. Alexander</p>
 
  </div>
 
  </div>
−
 
+
 
 
<div>
 
<div>
 
   
 
   
−
<p class="title">    
+
<div class="subtitle" >    
−
How to attract Varroa ?
+
<h3>How to attract varroa</h3>
−
</p>
+
 
</div>
 
</div>
  
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Just before capping, bee larvaes produce a wide range of molecules,
 
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.   
+
those molecules warn the mite about the upcoming capping and motivate  
−
Of all these molecule, scientific studies have shown that one can signicantly attract Varroa :
+
it to enter the cell [2].   
−
<i>butyrate.</i>
+
Of all these molecule, scientific studies have shown that one can  
 +
significantly attract varroa:
 +
<i>butyrate</i> [3].
 
</p>
 
</p>
 
</div>
 
</div>
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       Butyrate is a volatile acid which is non-toxic for honeybees  
 
       Butyrate is a volatile acid which is non-toxic for honeybees  
 
  nor the human being, because it is already present at physiologic  
 
  nor the human being, because it is already present at physiologic  
−
  concentrations in the digestive tract. Moreover this molecule is naturally  
+
  concentrations in the digestive tract. Moreover this molecule  
−
  produced by some bacterial strains like <i>Clostridium</i>, which is an asset  
+
  is naturally  
−
  for this synthetic biology project. Therefore we decided to modify <i>Apicoli</i>
+
  produced by some bacterial strains like <i>Clostridium</i>,  
 +
  which is an asset  
 +
  for this synthetic biology project [4].</p><div id="part2"></div> <!-- ANCHOR 2 --><p align="justify" style="font-size:15px;"> Therefore we decided to  
 +
  modify Apicoli  
 
  so it will synthesize
 
  so it will synthesize
 
  butyrate in order to attract varroa.  
 
  butyrate in order to attract varroa.  
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<img src="https://static.igem.org/mediawiki/2015/e/e6/TLSE_Attract_fig2.png">
 
<img src="https://static.igem.org/mediawiki/2015/e/e6/TLSE_Attract_fig2.png">
−
<p>Figure 2 : Results of butyrate attraction test with quadrants method
+
<p>Figure 2: Results of butyrate attraction  
 +
test with quadrants method
 
</p>
 
</p>
 
 
 
           </div>
 
           </div>
 
  </div>
 
  </div>
−
 
−
 
−
 
−
 
−
 
−
 
−
<div>
 
−
 
 
−
<p class="title">  
 
−
Butyrate attraction test:
 
−
</p>
 
  
 +
<div class="subtitle" >  
 +
<h3>Butyrate attraction test</h3>
 
</div>
 
</div>
  
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<img src="https://static.igem.org/mediawiki/2015/b/b8/TLSE_Attract_fig3.png">
 
<img src="https://static.igem.org/mediawiki/2015/b/b8/TLSE_Attract_fig3.png">
−
<p>Figure 3 : Butyrate attraction test using T tube, with Varroa mite in the middle
+
<p>Figure 3: Butyrate attraction test using  
 +
T tube, with varroa mite in the middle
  
 
</p>
 
</p>
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  <div class="one_half">
 
  <div class="one_half">
 
  <p align="justify" style="font-size:15px;">
 
  <p align="justify" style="font-size:15px;">
−
To check adequacy and relevance of this study <i>(Figure 2)</i>,  
+
To check adequacy and relevance of this study (Figure 2),  
−
an experiment using a T-tube has been developed <i>(Figure 3)</i>.  
+
an experiment using a T-tube has been developed (Figure 3).  
 
In the first branch, there is a cotton soaked with 50 µL of water,  
 
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  
 
in the second a cotton with  50 µL of butyrate at 4%, and finally the  
−
last one contains the varroa. The butyrate being very volatile, our system  
+
last one contains the varroa.</p> <div id="part3"> <!-- ANCHOR 3 --> </div> <p align="justify" style="font-size:15px;">The butyrate being very volatile, our  
 +
system  
 
used a pump to renew air, producing a concentration gradient.
 
used a pump to renew air, producing a concentration gradient.
 
</p>
 
</p>
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</div>
 
</div>
  
−
<p class="title">    
+
<div class="subtitle" >    
−
How to produce butyrate with E.coli?
+
<h3>How to produce butyrate with <i>E.coli</i>?</h3>
−
</p>
+
</div>
  
 
<div class="group center">
 
<div class="group center">
 
        
 
        
 
  <p align="justify" style="font-size:15px;">
 
  <p align="justify" style="font-size:15px;">
−
     In this project, an Escherichia coli strain is used for its known
+
     In this project, an <i>Escherichia coli</i> strain is used for its known
 
simplicity of genetic manipulation and its adequacy with butyrate
 
simplicity of genetic manipulation and its adequacy with butyrate
 
synthesis. Indeed, among the five enzymes of the butyrate pathway,
 
synthesis. Indeed, among the five enzymes of the butyrate pathway,
 
two enzymes are naturally produced by the bacteria. The following
 
two enzymes are naturally produced by the bacteria. The following
−
engineered butyrate pathway has been designed :
+
engineered butyrate pathway has been designed:
 
</p>   <br>
 
</p>   <br>
 
       </div>
 
       </div>
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<div style="font-size:15px;">  
 
<div style="font-size:15px;">  
 
  <ul>
 
  <ul>
−
   <li><b>atoB</b> present in E. coli, coding for acetyl-CoA acetyltransferase, an acetyltransferase catalyzing the combination of two acetyl-CoA.
+
   <li><b><i>atoB</i></b> present in <i>E.coli</i>, coding for acetyl-CoA  
 +
  acetyltransferase, an acetyltransferase catalyzing the combination  
 +
  of two acetyl-CoA.
 
<br>
 
<br>
 
<div class="group center">
 
<div class="group center">
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   <div class="group center">
 
   <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 5: Reaction catalyzed by acetyl-CoA acetyltransferase </p>
+
  <p class="legend">Figure 5: Reaction catalyzed by acetyl-CoA  
 +
acetyltransferase </p>
 
  </div>
 
  </div>
 
    
 
    
 
   </li>
 
   </li>
 
    
 
    
−
   <li><b>hbd</b> present in Clostridium acetobutylicum coding for 3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing the formation of an alcohol function.
+
   <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>
 
   <br>
 
     <div class="group center">
 
     <div class="group center">
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   <div class="group center">
 
   <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 6: Reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase
+
  <p class="legend">Figure 6: Reaction catalyzed by  
 +
3-hydroxybutyryl-CoA dehydrogenase
 
</p>
 
</p>
 
  </div>
 
  </div>
 
   </li>
 
   </li>
 
    
 
    
−
   <li><b>crt</b> present in C. acetobutylicum coding for 3-hydroxybutyryl-CoA dehydratase, a lyase cleaving carbon-oxygen bond.
+
   <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>
 
  <br>
 
   <div class="group center">
 
   <div class="group center">
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     <div class="group center">
 
     <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 7: Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase
+
  <p class="legend">Figure 7:
 +
Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase
  
 
</p>
 
</p>
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   </li>
 
   </li>
 
    
 
    
−
   <li><b>ccr</b> 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.
+
   <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>
 
<br>
 
   <div class="group center">
 
   <div class="group center">
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   <div class="group center">
 
   <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 8: Reaction catalyzed by crotonyl-CoA reductase
+
  <p class="legend">Figure 8: Reaction  
 +
catalyzed by crotonyl-CoA reductase
 
</p>
 
</p>
 
  </div>
 
  </div>
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   </li>
 
   </li>
 
    
 
    
−
     <li><b>tesB</b> present in E. coli coding for acyl-CoA transferase 2, a thiolase which enables coenzyme A transfer.
+
     <li><b><i>tesB</i></b> present in <i>E.coli</i>
 +
coding for acyl-CoA transferase 2,  
 +
a thiolase which enables coenzyme A transfer.
 
   <br>
 
   <br>
 
   <div class="group center">
 
   <div class="group center">
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<div class="group center">
 
<div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 9: Reaction catalyzed by acyl-CoA transferase 2
+
  <p class="legend">Figure 9: Reaction  
 +
catalyzed by acyl-CoA transferase 2
 
</p>
 
</p>
 
  </div>
 
  </div>
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<div class="wrapper row4">
 
<div class="wrapper row4">
  
 +
<div class="container clear" style="padding-top:30px;">
 
<center><p class="maintitle">    
 
<center><p class="maintitle">    
 
References
 
References
 
</p></center>
 
</p></center>
 
<br>
 
<br>
−
<div class="clear" style="margin-right:50px;margin-left:50px;">
+
<div class="clear">
 
<ul>
 
<ul>
 
<li>
 
<li>
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<li>
 
<li>
 
[3] Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24].  
 
[3] Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24].  
 +
</li>
 +
 +
<li>
 +
[4] 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>
 +
[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>
 +
[6] Wallace KK, Bao Z-Y, 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. European Journal of Biochemistry 233:954–962.
 
</li>
 
</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br>
 
<br>
 +
</div>
 
</div>
 
</div>
 
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Latest revision as of 14:59, 27 August 2015

iGEM Toulouse 2015

Attract


Content

About varroasis

Varroasis occurs with the varroa entrance in the hive, carried by infected bees: the mite can begin its parasitism and attack the brood. When the queen gives birth to new larvaes in honeycombs, the fertilized adult female varroa mite will come into it before capping, and lay her eggs. The larvaes will develop, increasing the overall infection that affects bee population [1]. To tackle this issue, it is necessary to attract varroa carried by honeybees before they come into the hive.



Figure 1 : Varroa destructor life cycle, adapted from B. Alexander

How to attract varroa

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 [2]. Of all these molecule, scientific studies have shown that one can significantly attract varroa: butyrate [3].


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 [4].

Therefore we decided to modify Apicoli so it will synthesize butyrate in order to attract varroa.

Figure 2: Results of butyrate attraction test with quadrants method

Butyrate attraction test

Figure 3: 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 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.

The butyrate being very volatile, our system used a pump to renew air, producing a concentration gradient.

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 4: 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 5: 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 6: 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 7: 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 8: 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 9: Reaction catalyzed by acyl-CoA transferase 2

References


  • [1] Boecking O, Genersch E. 2008. Varroosis – the Ongoing Crisis in Bee Keeping. J. Verbr. Lebensm. 3:221–228.
  • [2] 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.
  • [3] Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24].
  • [4] 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.
    • [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 Z-Y, 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. European Journal of Biochemistry 233:954–962.