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

 
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<center><div class="subtitle" >  
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<h3>About varroasis</h3>
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</div></center>
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    <div class="group center"> <!-- FIRST PARAGRAPH -->
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        <p align="justify" style="font-size:15px;">
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Varroasis occurs with the <i>Varroa destructor</i> entrance in the hive, carried by
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−
infected bees:
+
−
the mite can begin its parasitism and infest the brood. When the queen
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−
gives birth
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−
to new larvaes in honeycombs, the fertilized adult female varroa mite
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−
will come into
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−
it before capping, and lay her eggs. The larvaes will develop,
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−
increasing the overall
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−
infection that affects bee population [1]. To tackle this issue,
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−
it is necessary to attract
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varroa carried by honeybees before they come into the hive.
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</p>
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      </div>
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  <div class="group center">
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<br>
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  <img src="https://static.igem.org/mediawiki/2015/0/04/TLSE_Attract_fig1.png" />
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</div>
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<div id="part1"></div><!-- ANCHOR 1 -->
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<div class="group center">
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<br>
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<p>Figure 1 : <i>Varroa destructor</i> life cycle,
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−
adapted from B. Alexander</p>
+
−
</div>
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−
 
+
 
<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  
 
those molecules warn the mite about the upcoming capping and motivate  
−
it to enter the cell [2].   
+
it to enter the cell [1].   
 
Of all these molecules, scientific studies have shown that one can  
 
Of all these molecules, scientific studies have shown that one can  
 
significantly attract varroa:
 
significantly attract varroa:
−
<i>butyrate</i> [3].
+
<i>butyrate</i> [2].
 
</p>
 
</p>
 
</div>
 
</div>
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  <br>
 
  <br>
 
       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 for the human being, because it is already present at physiologic  
−
  concentrations in the digestive tract. Moreover this molecule  
+
  concentrations in their digestive tract. Moreover this molecule  
 
  is naturally  
 
  is naturally  
−
  produced by some bacterial strains like <i>Clostridium</i>,  
+
  produced by some bacterial strains,  
 
  which is an asset  
 
  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  
+
  for identifying key enzymatic activities 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 (Figure 1), we decided to  
 
  modify <i>E. coli</i>  
 
  modify <i>E. coli</i>  
 
  so it will synthesize
 
  so it will synthesize
−
  butyrate in order to attract varroa.  
+
  butyrate in order to attract varroa [4].  
 
</p>    
 
</p>    
 
       </div>
 
       </div>
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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  
+
<p>Figure 1: Results of butyrate attraction  
−
test with quadrants method
+
test with quadrants method, US 8647615 B1 [4].
 
</p>
 
</p>
 
 
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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  
+
<p>Figure 2: Butyrate attraction test using  
 
T tube, with varroa mite in the middle
 
T tube, with varroa mite in the middle
  
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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 (Figure 2),
+
To confirm this previous finding and make sure we can handle such experiment, a new protocol using a custom-made glass T-tube has been developed (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 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.</p> <div id="part3"> <!-- ANCHOR 3 --> </div> <p align="justify" style="font-size:15px;"> Butyrate being very volatile, our  
 
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  
 
system  
−
used a pump to renew air, producing a concentration gradient.
+
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>
 
</p>
 
       </div>
 
       </div>
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     In this project, an <i>Escherichia coli</i> 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 [5]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA,
 
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:
Line 199: Line 166:
 
</div>
 
</div>
 
  <div class="group center">
 
  <div class="group center">
−
<figcaption>Figure 4: Engineered butyrate pathway</figcaption>
+
<figcaption>Figure 3: Engineered butyrate pathway</figcaption>
 
   
 
   
 
</div>
 
</div>
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  <br>
 
  <br>
 
     The initial substrate is glucose which is decomposed into  
 
     The initial substrate is glucose which is decomposed into  
−
acetyl-CoA during glycolysis. Finally, butyrate pathway  
+
acetyl-CoA during glycolysis. Butyrate pathway  
−
begin with acetyl-CoA: five genes are required with two
+
begins with acetyl-CoA: five genes are required with two
 
homologous and three heterologous genes.
 
homologous and three heterologous genes.
 
</p>    
 
</p>    
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<div style="font-size:15px;">  
 
<div style="font-size:15px;">  
 
  <ul>
 
  <ul>
−
   <li><b><i>atoB</i></b> present in <i>E.coli</i>, coding for acetyl-CoA  
+
   <li><b><i>atoB</i></b> present in <i>E.coli</i> (Accession Number: <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11672"target="_blank">EG11672</a>), coding for acetyl-CoA  
 
   acetyltransferase, an acetyltransferase catalyzing the combination  
 
   acetyltransferase, an acetyltransferase catalyzing the combination  
 
   of two acetyl-CoA.
 
   of two acetyl-CoA.
Line 229: Line 196:
 
   <div class="group center">
 
   <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 5: Reaction catalyzed by acetyl-CoA  
+
  <p class="legend">Figure 4: Reaction catalyzed by acetyl-CoA  
 
  acetyltransferase </p>
 
  acetyltransferase </p>
 
  </div>
 
  </div>
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   </li>
 
   </li>
 
    
 
    
−
   <li><b><i>hbd</i></b> present in <i>Clostridium acetobutylicum</i> coding for  
+
   <li><b><i>hbd</i></b> present in <i>Clostridium acetobutylicum</i> (Accession Number: <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2684"target="_blank">GJIH-2684</a>) coding for  
 
   3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing  
 
   3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing  
 
   the formation of an alcohol function.
 
   the formation of an alcohol function.
Line 245: Line 212:
 
   <div class="group center">
 
   <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 6: Reaction catalyzed by  
+
  <p class="legend">Figure 5: Reaction catalyzed by  
 
  3-hydroxybutyryl-CoA dehydrogenase
 
  3-hydroxybutyryl-CoA dehydrogenase
 
</p>
 
</p>
Line 251: Line 218:
 
   </li>
 
   </li>
 
    
 
    
−
   <li><b><i>crt</i></b> present in <i>C.acetobutylicum</i>
+
   <li><b><i>crt</i></b> present in <i>C.acetobutylicum</i> (Accession Number: <a href ="http://www.biocyc.org/CACE272562/NEW-IMAGE?type=GENE&object=GJIH-2688"target="_blank">GJIH-2688</a>)
 
   coding for 3-hydroxybutyryl-CoA dehydratase,
 
   coding for 3-hydroxybutyryl-CoA dehydratase,
 
   a lyase cleaving carbon-oxygen bond.
 
   a lyase cleaving carbon-oxygen bond.
Line 261: Line 228:
 
     <div class="group center">
 
     <div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 7:
+
  <p class="legend">Figure 6:
 
  Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase
 
  Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase
  
Line 269: Line 236:
 
    
 
    
 
   <li><b><i>ccr</i></b> present in  
 
   <li><b><i>ccr</i></b> present in  
−
   <i>Streptomyces collinus</i> coding
+
   <i>Streptomyces collinus</i> (Accession Number: <a href ="http://www.uniprot.org/uniprot/Q53865"target="_blank">Q53865-1</a>) coding
 
   for crotonyl-CoA reductase,
 
   for crotonyl-CoA reductase,
 
   an oxidoreductase acting on
 
   an oxidoreductase acting on
−
   CH=CH double bond. This enzyme
+
   CH=CH double bond and successfully used in <i>E. coli</i> [6].
−
  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  
+
  <p class="legend">Figure 7: Reaction  
 
  catalyzed by crotonyl-CoA reductase
 
  catalyzed by crotonyl-CoA reductase
 
</p>
 
</p>
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   </li>
 
   </li>
 
    
 
    
−
     <li><b><i>tesB</i></b> present in <i>E.coli</i>  
+
     <li><b><i>tesB</i></b> present in <i>E.coli</i> (Accession Number: <a href ="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10995"target="_blank">EG10995</a>)
 
coding for acyl-CoA transferase 2,  
 
coding for acyl-CoA transferase 2,  
 
a thiolase which enables coenzyme A transfer.
 
a thiolase which enables coenzyme A transfer.
Line 303: Line 265:
 
<div class="group center">
 
<div class="group center">
 
<br>
 
<br>
−
  <p class="legend">Figure 9: Reaction  
+
  <p class="legend">Figure 8: Reaction  
 
  catalyzed by acyl-CoA transferase 2
 
  catalyzed by acyl-CoA transferase 2
 
</p>
 
</p>
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</div>
 
</div>
 
<div class="group center">
 
<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>.  
+
  <p align="justify" style="font-size:15px;">Concerning heterologous genes (<i>hbd</i>, <i>crt</i> and <i>ccr</i>), codon optimizations have been performed in order to enable a correct 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 (here).</p>
+
The genetic construction is then done by assembling the five genes presented earlier, which are placed under the control of the P(Bla) constitutive promoter (BBa_I14018). Ribosome binding sites (RBS ; BBa_B0030) are added before the coding sequences to ensure protein expression. A strong terminator (BBa_B1006) is used to end this construction. The final biobrick was cloned into a pSB1C3 vector (<a href="https://static.igem.org/mediawiki/2015/9/9e/PSB1C3_ccr_butyrate.xdna.png"target="_blank">here</a>).</p>
 
</div>
 
</div>
  
−
<center><img src="https://static.igem.org/mediawiki/2015/2/20/TLSE_Attract_fig10.png" style="width:70%;"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2015/6/6f/TLSE_Parts_image4.PNG" style="width:65%;"/></center>
 +
 
 +
 
 +
<center>
 +
<div class="title">
 +
<h3>READ MORE</h3>
 +
</div> </center>
 +
 
 +
<div class="group center">
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Description">
 +
<div class="title">
 +
<h3>Project Description</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Description/Eradicate">
 +
<div class="title">
 +
<h3>Eradicate</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Description/Regulation">
 +
<div class="title">
 +
<h3>Regulation</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Design">
 +
<div class="title">
 +
<h3>Device: TrApiColi</h3>
 +
</div>
 +
</a>
 +
</div>
 +
<div style="width:20%;">
 +
<a href="https://2015.igem.org/Team:Toulouse/Results">
 +
<div class="title">
 +
<h3>Results</h3>
 +
</div>
 +
</a>
 +
</div>
 +
</div>
 +
 
 
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     <!-- / container body -->
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<div class="clear">
 
<div class="clear">
 
<ul>
 
<ul>
−
<li>
 
−
[1] Boecking O, Genersch E. 2008. Varroosis – the Ongoing Crisis in Bee Keeping. J. Verbr. Lebensm. 3:221–228.</li>
 
  
−
<li>
+
[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>
−
[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.</li>
+
  
 
<li>
 
<li>
−
[3] Methods for attracting honey bee parasitic mites. [accessed 2015 Jul 24].  
+
[2] 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.
 
</li>
 
</li>
  
 
<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.
+
[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>
 +
 
 +
<li>
 +
[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.
 
</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.
+
 
 +
[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>
 
<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.
+
[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  
 
</li>
 
</li>
 
</ul>
 
</ul>

Latest revision as of 01:46, 19 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 for the human being, because it is already present at physiologic concentrations in their digestive tract. Moreover this molecule is naturally produced by some bacterial strains, which is an asset for identifying key enzymatic activities for this synthetic biology project [3].

Therefore, based on the patent US 8647615 (Figure 1), 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 confirm this previous finding and make sure we can handle such experiment, a new protocol using a custom-made glass T-tube has been developed (Figure 2). 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 [5]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA, 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. Butyrate pathway begins with acetyl-CoA: five genes are required with two homologous and three heterologous genes.


  • atoB present in E.coli (Accession Number: EG11672), 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 (Accession Number: GJIH-2684) 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 (Accession Number: GJIH-2688) 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 (Accession Number: Q53865-1) coding for crotonyl-CoA reductase, an oxidoreductase acting on CH=CH double bond and successfully used in E. coli [6].



    Figure 7: Reaction catalyzed by crotonyl-CoA reductase

  • tesB present in E.coli (Accession Number: EG10995) 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), codon optimizations have been performed in order to enable a correct 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 the P(Bla) constitutive promoter (BBa_I14018). Ribosome binding sites (RBS ; BBa_B0030) are added before the coding sequences to ensure protein expression. A strong terminator (BBa_B1006) is used to end this construction. The final biobrick was cloned into a pSB1C3 vector (here).

READ MORE

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] 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.
  • [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