Difference between revisions of "Team:Toulouse/test"

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     <div class="shout-content clear" style="margin-top:-155px;">
 
     <div class="shout-content clear" style="margin-top:-155px;">
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      <p class="fl_left"><i>"If the bee disappeared off the surface of the globe then man would only have four years of life left. <br>
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      <center> <p class="maintitle">  
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  No more bees, no more pollination, no more plants, no more animals, no more man."
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Eradicate
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  </i>
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</p></center>
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  <center><img src=" https://static.igem.org/mediawiki/2015/0/0b/TLSE_Eradicate_BG.png" style="width:20%;"></center>
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</div>
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      <p class="fl_right"><a class="btn" href="#"> UNAF, 1994.</a></p>
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     <h2 ><center><img src="https://static.igem.org/mediawiki/2015/2/2e/TLSE_ApiColi.png" style="width:350px;" /></center></h2>
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<p class="title">  
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</p>
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</div>
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    <div class="group center"> <!-- FIRST PARAGRAPH -->
 
         <p align="justify" style="font-size:15px;">
 
         <p align="justify" style="font-size:15px;">
  
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<i>Apis mellifera</i>, the most efficient and widespread honey producing species, is particularly affected by the mite <i>Varroa destructor</i>. This pathogen is actually the second cause of death for european bees. Indeed, according to beekeepers, up to 50 % of the swarms are lost every year, partly because of these parasites, causing tremendous economic damages to beekeepers.<br><br>
+
After having attracted and trapped varroa in a confined device at the hive entrance,  
−
 
+
it is necessary to prevent it from escaping, and to eradicate it. It should be pointed
 +
out that beekeepers already use some treatments to fight against varroa, among them oxalate,
 +
fluvalinate, thymol or formate. All these known treatments use high doses which are toxic
 +
for bees and humans. On the other hand, varroa is developing resistance against these treatments,  
 +
making them ineffective [1].
 +
In the list of the molecules mentioned above, formate has already prove its acaricide property [2],  
 +
and has the advantage  to be naturally synthesized by <i>E.coli</i>.
  
 +
For this part, the project main goal is to synthesize formate at low concentration with the
 +
bacteria during a short time period, in order to reduce doses and minimize the toxicity on bees.
 +
 +
 +
 +
 +
 
</p>
 
</p>
 
       </div>
 
       </div>
 
   
 
   
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  <div class="group center">
 
−
     
 
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      <div class="one_third first">
 
−
       
 
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<p align="center" style="font-size:50px;">40%<br></p>
 
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<p align="center" style="font-size:15px;">Perte d'abeille commerciales aux états-unis depuis 2006<br><br></p>
 
−
     
 
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      </div>
 
 
   
 
   
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      <div class="one_third">
 
−
       
 
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        <p align="center" style="font-size:50px;">25%<br></p>
 
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<p align="center" style="font-size:15px;">Perte d'abeille commerciales en europe depuis 2005<br><br></p>
 
−
 
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      </div>
 
 
   
 
   
−
      <div class="one_third">
+
 
−
       
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<div>
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        <p align="center" style="font-size:50px;">45%<br></p>
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<p align="center" style="font-size:15px;">Perte d'abeille commerciales au royaume-uni depuis 2005*<br><br></p>
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<p class="title">  
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Formate acaricide activity test:
 +
</p>
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</div>
 +
 
 +
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 +
 
 +
<div class="group center">
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      <div class="one_half first">
 +
  <p align="justify" style="font-size:15px;">
 +
  <br>
 +
    Before formate production by our strain, it was necessary
 +
to check the "miticide" activity of the molecule with a specific test.
 +
In this experiment, three varroas are placed in a Petri dish containing
 +
a cotton soaked with 400 µL of formate at different concentrations (50µM, 500µM, 1mM et 10mM).
 +
The experiment runs 6h during and varroa’s death is validated when it does not move anymore even
 +
after a stimulus. The mite observation is realized with a binocular magnifier.
 +
<br>
 +
<br>
 +
</p>
 +
 +
 
 
       </div>
 
       </div>
 +
 +
  <div class="one_half">
 +
  <img src="https://static.igem.org/mediawiki/2015/f/f3/TLSE_Eradicate_fig1.png" style="height=75%;">
 +
<p>Figure 1: Formate miticide activity test
 +
 +
</p>
 +
  </div>
 
   
 
   
 +
  </div>
 
   
 
   
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    </div>
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<!-- <div class="group center">
 
   
 
   
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      <div class="one_half first">
 +
<img src="https://static.igem.org/mediawiki/2015/f/f3/TLSE_Eradicate_fig2.png">
 +
<p>Figure 2: Varroa death tracking depending on formate concentrations
 +
<br>(0M, 50µM, 500µM, 1mM, 10mM et 2M)
 +
<br>X-squared = 4;  df = 3 ; p-value = 0.2615</p>
 +
 +
          </div>
 +
 
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  <div class="one_half" style="padding-top:50%;">
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  <br>
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  <br>
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  <br>
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  <br>
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  <br>
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  <p align="justify" style="font-size:15px;">
 +
The following results are observed, and a chi-squared test is performed to determine the experiment significance.
  
 +
 +
</p>
 +
  <br>
 +
  <br>
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  <br>
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  <br>
 +
  <br>
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  </div>
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  </div>
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  -->
 
 
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<div class="group center">
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<p align="justify" style="font-size:15px;">
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Our team is committed to preserving honeybees by fighting these mites. The aim of our project is to work out a bacterium capable of baiting and eliminating the parasite using the tools offered by synthetic biology. Our bacteria, named ApiColi, will be safely placed in a trap and will express alternatively an attractant and a poison for the <i>Varroa destructor</i>, in agreement with the honeybee life cycle. Our trap will be sealed to avoid any contact of the microorganism with the bees and the environment. We hope that our concept will help reduce the decrease in bee populations and its disastrous consequences.<br><br><br><br>
+
 
 +
 
 +
 
 +
<div>
 +
 
 +
<p class="title">  
 +
How to synthesize formate with <i>E.coli</i>?
 
</p>
 
</p>
 +
 
</div>
 
</div>
  
−
<p align="center" style="font-size:10px;">* Chiffre greenpeace<br><br></p>
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</div>   
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    <div class="group center"> <!-- FIRST PARAGRAPH -->
 +
     
 +
 
 +
 
 +
 +
  <p align="justify" style="font-size:15px;">
 +
Formate is a simple organic acid produced with an <i>E.coli</i> strain.
 +
The initial substrate, glucose, is decomposed into pyruvate during
 +
glycolysis, and formate is naturally synthesized thanks to two key
 +
genes:
 +
</p>
 +
   
 +
 
 +
 
 +
</div>
 +
 
 +
 
 +
<div style="font-size:15px;">
 +
<ul>
 +
  <li><b><i>pflB</i></b> coding for pyruvate formate lyase which catalyzes the cleavageog pyruvate into C1 and C2. This enzyme is sensitive to oxygen and is only active in microaerobic or anaerobic conditions, which is the case within our device [3].
 +
  </li>
 +
 
 +
  <li><b><i>pflA</i></b> coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation [4].
 +
  </li>
 +
 +
 
 +
</ul>
 +
 
 +
</div>
 +
 
 +
<div class="group center">
 +
<br>
 +
  <img src="https://static.igem.org/mediawiki/2015/c/cf/TLSE_Eradicate_fig3.png" />
 +
 
 +
  </div>
 +
  <div class="group center">
 +
  <p>Figure 3: Enzymatic reaction of the formate synthesis
 +
 
 +
 
 +
</p>
 +
</div>
 +
 +
<div class="group center">
 +
<p align="justify" style="font-size:15px;">
 +
The formate being naturally produced in <i>E.coli</i>, the key genes for synthesis are over-expressed. The genetic construction is realized by assembling of these genes, which are placed under the control of P(Bla) constitutive promotor (BBa_I14018). Between genes there are ribosome binding sites (RBS)(BBa_B0030)  to improve protein expression, and a strong terminator (BBa_B1006) to end this construction which is finally cloned into a pSB1C3 vector.
 +
</p>
 +
</div>
 
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  <li><a href="http://www.insa-toulouse.fr/fr/index.html" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/9/95/TLSE_INSA.png" alt=""></a></li>
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References
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  <li><a href="http://www.insa-toulouse.fr/fr/institution/fondation-insa-toulouse/soutenir-l-insa-toulouse.html" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/0/05/TLSE_INSAFondation.png" alt=""></a></li>
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</p></center>
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  <li><a href="http://www.univ-tlse3.fr/" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/9/98/TLSE_UPS.png" alt="" ></a></li>
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<br>
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  <li><a href="http://www.genotoul.fr/" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/5/56/TLSE_Genotoul.png" alt="" ></a></li>
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<div class="clear">
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  <li><a href="http://www.lisbp.fr/fr/index.html" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/1/17/TLSE_LISBP.png" alt="" ></a></li>
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<ul>
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    </ul>
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<li>
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[1] Elzen PJ, Baxter JR, Spivak M, Wilson WT (2000) Control of <i>Varroa jacobsoni</i> Oud. resistant to fluvalinate and amitraz using coumaphos. Apidologie 31: 437–441.
−
    <ul class="clear">
+
 
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  <li><a href="http://www.univ-tlse3.fr/la-vie-etudiante-224240.kjsp?RH=1238398834558" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/d/d5/TLSE_DVE.png" alt="" ></a></li>
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</li>
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  <li><a href="http://www.solvay.fr/fr/index.html" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/5/59/TLSE_UFTMP.png" alt=""></a></li>
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      <li><a href="http://www.consulfrance-losangeles.org/" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/8/86/TLSE_embassade.png" alt="" ></a></li>
+
<li>
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      <li><a href="http://www.toulouse-white-biotechnology.com/" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/1/15/TLSE_TWB.png" alt=""></a></li>
+
[2] Satta A, Floris I, Eguaras M, Cabras P, Garau VL, Melis M. 2005. Formic Acid-Based Treatments for Control of <i>Varroa destructor</i> in a Mediterranean Area. Journal of Economic Entomology 98:267–273. </li>
−
      <li><a href="http://www.solvay.fr/fr/index.html" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/1/10/TLSE_Solvay.png" alt=""></a></li>
+
<li>
−
     
+
[3] Becker A, Fritz-Wolf K, Kabsch W, Knappe J, Schultz S, Volker Wagner AF. 1999. Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat. Struct. Biol. 6:969–975.</li>
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    <!-- ################################################################################################ -->
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<li>
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[4] Crain AV, Broderick JB. 2014. Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme. J Biol Chem 289:5723–5729.</li>
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+
</div>  
−
      <li><a href="http://www.beeopic.com/fr/" target="_blanc"><img src="https://static.igem.org/mediawiki/2015/c/c4/TLSE_Beeopic.png" alt=""></a></li>
+
−
     
+
−
      <li><a href="http://www.untoitpourlesabeilles.fr/"  target="_blanc"><img src="https://static.igem.org/mediawiki/2015/1/1e/TLSE_UnToitPourLesAbeilles.png" alt=""></a></li>
+
−
      <li><a href="http://www.labeilledecompagnie.fr/"  target="_blanc"><img src="https://static.igem.org/mediawiki/2015/f/fc/TLSE_AbeilleDeCompagnie.png" alt=""></a></li>
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Revision as of 08:15, 14 August 2015

iGEM Toulouse 2015

Eradicate


After having attracted and trapped varroa in a confined device at the hive entrance, it is necessary to prevent it from escaping, and to eradicate it. It should be pointed out that beekeepers already use some treatments to fight against varroa, among them oxalate, fluvalinate, thymol or formate. All these known treatments use high doses which are toxic for bees and humans. On the other hand, varroa is developing resistance against these treatments, making them ineffective [1]. In the list of the molecules mentioned above, formate has already prove its acaricide property [2], and has the advantage to be naturally synthesized by E.coli. For this part, the project main goal is to synthesize formate at low concentration with the bacteria during a short time period, in order to reduce doses and minimize the toxicity on bees.

Formate acaricide activity test:


Before formate production by our strain, it was necessary to check the "miticide" activity of the molecule with a specific test. In this experiment, three varroas are placed in a Petri dish containing a cotton soaked with 400 µL of formate at different concentrations (50µM, 500µM, 1mM et 10mM). The experiment runs 6h during and varroa’s death is validated when it does not move anymore even after a stimulus. The mite observation is realized with a binocular magnifier.

Figure 1: Formate miticide activity test

How to synthesize formate with E.coli?

Formate is a simple organic acid produced with an E.coli strain. The initial substrate, glucose, is decomposed into pyruvate during glycolysis, and formate is naturally synthesized thanks to two key genes:

  • pflB coding for pyruvate formate lyase which catalyzes the cleavageog pyruvate into C1 and C2. This enzyme is sensitive to oxygen and is only active in microaerobic or anaerobic conditions, which is the case within our device [3].
  • pflA coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation [4].

Figure 3: Enzymatic reaction of the formate synthesis

The formate being naturally produced in E.coli, the key genes for synthesis are over-expressed. The genetic construction is realized by assembling of these genes, which are placed under the control of P(Bla) constitutive promotor (BBa_I14018). Between genes there are ribosome binding sites (RBS)(BBa_B0030) to improve protein expression, and a strong terminator (BBa_B1006) to end this construction which is finally cloned into a pSB1C3 vector.

References


  • [1] Elzen PJ, Baxter JR, Spivak M, Wilson WT (2000) Control of Varroa jacobsoni Oud. resistant to fluvalinate and amitraz using coumaphos. Apidologie 31: 437–441.
  • [2] Satta A, Floris I, Eguaras M, Cabras P, Garau VL, Melis M. 2005. Formic Acid-Based Treatments for Control of Varroa destructor in a Mediterranean Area. Journal of Economic Entomology 98:267–273.
  • [3] Becker A, Fritz-Wolf K, Kabsch W, Knappe J, Schultz S, Volker Wagner AF. 1999. Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat. Struct. Biol. 6:969–975.
  • [4] Crain AV, Broderick JB. 2014. Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme. J Biol Chem 289:5723–5729.