Difference between revisions of "Team:Toulouse/Design"

 
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   <div class="title">
 
   <div class="title">
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<h3>Device : physical tests</h3>
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      <a href="#main1"><h3>Content</h3></a>
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</div>
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  <div class="subtitle">
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  <h3>Trap Construction</h3>
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   </div>
 
   </div>
 
    
 
    
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   <div class="group center">
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   <center> 
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  <p class="text">
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    <div id="breadcrumb" class="clear" style="float: center;" >
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  (Benoit le boa)
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  <ul>
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  </p>
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        <li><a href="#trapconstruct">- Trap Device </a></li>
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  </div>
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        <li><a href="#tpx">- ApiColi containment and culture</a></li>
 +
      </ul>
 +
    </div>
 +
 +
  </center>
  
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<div class="subtitle">
 
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  <h3>TPX Bag</h3>
 
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  </div>
 
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  <div class="subsubtitle">
 
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  <h3>Safety tests</h3>
 
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  </div>
 
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   <div class="group center">
 
   <div class="group center">
 
   <p class="text">
 
   <p class="text">
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  (Mathile Bléraud)
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Our goal is to create a solution against hives infestation by varroas. In order to get the best out of ApiColi in the treatment of varroosis, we designed a trap named TrApiColi. TrApiColi was thought up to take into account ethical reflection, safety and ease of use for beekeepers.
 
   </p>
 
   </p>
 
   </div>
 
   </div>
−
    
+
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  <div class="subsubtitle">
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   <div id = "trapconstruct"></div>
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   <h3>Gas diffusion tests</h3>
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  <div class="title" >
 +
   <h3>Trap Device</h3>
 
   </div>
 
   </div>
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  <div class="group center">
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  <p class="text">
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  In order to know if our test that we described in “Protocol” test functions, we made a control with acids solutions in a falcon and we sample gas in balance.
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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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  For butyric acid we did not detect gas butyric acid by NMR,
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  the solution we used to solubilize gas should be not basic enough.
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  So we made a test with a TPX bag containing butyric acid into a
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  solution of sodium bicarbonate. As a control we sampled directly a
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  solution of 4% (V/V) of butyric acid.
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  </p>
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  </div>
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  <div class="group center">
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  <p style="text-align:center">
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  (IMAGE)
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</p>
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  </div>
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  <div class="group center">
 
  <div class="group center">
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  <p class="legend">
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<p class="text">
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Since the switch between the production of the two molecules is regulated by light, our bacteria need to be outside of the beehive. Thus, the trap was designed to be placed at the entrance of the hive, in order to act before infection and try to prevent the entry of as many mites as possible.
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  Figure 1: NMR Spectrum of butyric acid liquid control
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  in red and butyric acid liquid which passed through
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  TPX bag in blue. Red curve is zoomed 1340 times more
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  than blue curve. Each condition was tested in two replicates.
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  </p></div>
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  <div class="title">
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  <h3>Device: biological tests</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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  In the end, our objective is to have a bag which contains bacteria to produce alternately butyric acid
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and formic acid during at least ten days in order to be practical for beekeeper.  
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<br>
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So we faced with some biological questions as:
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   </p>
 
   </p>
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  </div>
 
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  <div class="group center">
 
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  <div class="one_quarter">
 
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  </div>
 
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  <div class="three_quarter">
 
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  <ul align="justify" style="font-size:15px;">
 
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<li>Could bacteria live during ten days in micro-aerobic condition?<html></li>
 
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<li>Which carbon source could we have to produce continuously acids?</li>
 
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<li>Would acids be toxic for E.coli?</li>
 
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</ul>
 
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</div>
 
 
   </div>
 
   </div>
 
    
 
    
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  <div class="subtitle">
 
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  <h3>Characteristics of E.coli growth</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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  In order to know better the E.coli strain we would use for our project, we made a culture in aerobic
 
  
−
and micro-aerobic conditions. We sampled OD and supernatant as it is explained <a target="_blank" href="https://2015.igem.org/Team:Toulouse/Experiments#erlencult">here</a> to
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<center>  
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<div style="one-half;padding:10px;">
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see what happen in it.
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<img src="https://static.igem.org/mediawiki/2015/6/63/TLSE_Device_Trap_1.png" style="width:70%;" />
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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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  Micro-aerobic condition is obtained thanks to cultivation in specific falcons with holes recovered by a
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−
 
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membrane into the plug which let pass oxygen without opening the falcon. They were incubated at 37°C
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without agitation to best correspond to our real condition.
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  </p>
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  </div>
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  <div class="group">
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  <p class="text">
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  Aerobic condition is obtained with classic Erlenmeyer incubated at 37°C with agitation.
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  </p>
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  </div>
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+
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  <div class="group center">
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−
  <p class="text">
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−
For the medium, we use a minimal medium M9 because we want to follow acids production by NMR. And
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we choose a standard glucose concentration, 15mM.
+
−
  </p>
+
−
  </div>
+
−
 
+
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  <div class="subsubtitle">
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  <h3>Biomass, substrate and products</h3>
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  </div>
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−
 
+
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−
 
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−
 
+
−
<!-- OD TO CONCENTRATION -->
+
−
 
+
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  <div class="group center">
+
−
  <p class="text">
+
−
In order to plot biomass concentration it is necessary to convert the OD measured. <br> This equation was used:
+
−
  </p>
+
−
  </div>
+
−
 
+
−
  <div class="group center">
+
−
<p style="font-size:15px;">
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−
$$ X=OD_{600nm}\times 0,4325 $$
+
−
</p>
+
 
</div>
 
</div>
  
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  <p style="font-size:15px;">
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<p class="legend">
−
Where X is the cell concentration (g.L<SUP>-1</SUP>)
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The four different parts of our trap
 
</p>
 
</p>
−
<!-- OD TO CONCENTRATION -->
+
</center>
−
 
+
  
 
<div class="group center">
 
<div class="group center">
 
   <p class="text">
 
   <p class="text">
−
For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.  
+
TrApiColi is composed of four main parts:<br><br>
−
<br>
+
<strong>1.</strong> A grid lined up with the bottom board
−
Then, we calculated concentration with this equation:
+
<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 will not disturb the bees' comings and goings. The holes are big enough to let the varroas fall through them, but not the bees.
−
  </p>
+
</p>
−
  </div>
+
</div>
−
 
+
<br><br>
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<!-- NMR TO CONCENTRATION -->
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−
  <div class="group center">
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<div class="group">
−
  <p style="font-size:15px;">
+
<p class="text">
−
$$[A]=\frac{Area_{molecule}}{Area_TSP} \times [TSP] \times \frac{\textrm{TSP proton number}}{\textrm{A proton number}} \times DF $$
+
<strong>2.</strong> A funnel, to channel all the falling varroas
−
  </p>
+
</p>  </div>
 +
 +
<center>
 +
<div style="one-half;padding:10px;">
 +
<img src="https://static.igem.org/mediawiki/2015/e/e2/TLSE_Device_Trap_2.jpg" style="width:40%;" />
 
</div>
 
</div>
−
<!-- NMR TO CONCENTRATION -->
 
−
 
 
−
<ul style="font-size:15px;">
 
−
<li>[A] = concentration of molecule in our solution in mM</li>
 
−
<li>Area<SUB>TSP</SUB> = 1</li>
 
−
<li>[TSP] = 1.075mM <br>concentration of Trimethylsilyl propanoic acid in NMR tube, internal reference for
 
  
−
quantification</li>
+
<p class="legend">
−
<li>TSP proton number = 9</li>
+
The grid and the funnel
−
<li>DF = Dilution Factor = 1.25</li>
+
</p></center>
−
</ul>
+
−
 
+
−
 
+
−
+
−
<center>
+
−
  <p class="text">
+
−
Thanks to these calculations we were able to plot biomass, substrate and products depending on
+
−
time.
+
−
  </p>
+
  
−
 
+
<div class="group">
−
 
+
−
<img src="https://static.igem.org/mediawiki/2015/0/08/TLSE_Devicebio_image1.PNG" style="width:100%;"/>
+
−
<p class="legend">Figure 1: Results of aerobic culture. Culture of BW25113 in M9 medium with [glucose] = 15 mM, in Erlenmeyer at 37°C </p>
+
−
+
−
 
+
−
<img src="https://static.igem.org/mediawiki/2015/2/2b/TLSE_Devicebio_image2.PNG" style="width:100%;"/>
+
−
  <p class="legend">Figure 2: Results of micro-aerobic culture. Culture of BW25113 in M9 medium with [glucose] = 15 mM, in Falcon at 37°C </p>
+
−
</center>
+
−
 
+
−
 
+
−
+
−
+
−
<div class="group center">
+
 
  <p class="text">
 
  <p class="text">
−
Glucose is consumed approximately at the same rate for both conditions but it is not use for the same thing at all. In aerobic condition biomass reaches 3 g/L whereas in micro-aerobic condition there is 6 times less biomass. Inversely, there are far less products in aerobic conditions, and bacteria consume them when there is not glucose anymore, than in micro-aerobic condition.
+
<strong>3.</strong> A transparent collector, containing the bacteria confined in a special bag
 +
<br><br>It is designed like a fish bottle trap: the tube from the funnel goes on for a few centimeters inside the collector to ease the entry of the varroas in the collector while preventing them from exiting. The special bag is described in the <a href="https://2015.igem.org/Team:Toulouse/Design#tpx">"ApiColi containment and culture"</a> part below.
 +
</p>  </div>
 +
<br><br>
  
−
  </p>
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  <div class="group">
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</div>
+
−
+
−
+
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  <div class="group center">
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  <p class="text">
 
  <p class="text">
−
For our objective to produce acids in a microporous bag, it is a really interesting results to have naturally bacteria which have slow growth and fermentation products.
+
<strong>4.</strong> A roof, to protect the trap from the rain
 +
<br><br> Associated with pannels blocking the sides of the entrance, it forces the bees to walk on the grid. This channeling does not disturbs the bees.
 +
</p>
 +
</div>
 +
 
 +
  <div class="group center"> 
 +
<p class="text">
 +
<br>
 +
The dimensions of the trap allow it to adapt to almost every beehive. Indeed, most of the hive types have the exact same entrance. Thanks to that, the trap can be easily and perfectly installed on the hive by the beekeepers without drilling or cutting.
 
  </p>
 
  </p>
−
</div>
+
  </div>
−
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  <br><br>
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  <div class="group center">
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<div class="group center">
 
  <p class="text">
 
  <p class="text">
−
We can convert formate
+
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 without coating 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"> "Modeling" </a> part.
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concentration into formic
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</p>  </div>
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acid to know how much more
+
 
−
we will have to produce to
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  <center> <table><tbody><tr><td>
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kill varroa. Indeed, the bacteria
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<div style="one-half;padding:10px;">
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produce a base but it is the acid that  
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<img src="https://static.igem.org/mediawiki/2015/c/c2/TLSE_Device_Trap_3.jpg" style="width:80%;" />
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interests us.  
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</div>
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</td>
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<br>The formula below
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<td>
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is used:
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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/2015/0/08/TLSE_Device_Trap_4.jpg" style="width:80%;" />
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  </div>
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<!-- pH equation -->  
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  <div class="group center">
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  <p style="font-size:15px;">
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$$ pH=pKa+log \left(\frac{C_{b}}{C_{a}} \right) $$
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  </p>
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</div>
 
</div>
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<!-- pH equation -->  
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</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</center>
  
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  <ul style="font-size:15px;">
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<center>
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<li>pH: medium used is buffered with a low concentration in acid. pH = 7.
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<p class="legend">
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</li>
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The 3D printer used to construct our trap and the result of 3D printing: TrApiColi
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<li>pKa: 3.7 for formic acid and 4.81 for butyric acid</li>
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</p></center>  
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<li>C<SUB>b</SUB>: base concentration</li>
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<li>C<SUB>a</SUB>: acid concentration</li>
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<div id = "tpx"></div>
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</ul>  
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<div class="title">
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  <h3>ApiColi containment and culture</h3>
 +
  </div>
 
    
 
    
 
   <div class="group center">
 
   <div class="group center">
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<p class="text">
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  <p class="text">
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As it is said in the “Eradicate” part, our goal is to produce 50µM of formic acid to kill varroa, thanks to the equation (3) we know it corresponds
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The use of genetically modified organisms in a field associated with edible products requires to carefully consider the issues of safety, national legislation, and public opinion. In this context, we looked for a solution that would isolate our engineered bacteria from the environment, while still allowing its growth and gas diffusion.<br>
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to 77,7mM of formate.
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We found the solution in the <a href="https://2012.igem.org/Team:Groningen/Sticker"target="_blank">iGEM Groeningen 2012 project</a> that used a special polymer called TPX® in order to keep their bacteria from contaminating meat, their edible product.<br>
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</p>
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TPX® is composed of Polymethylpentene, a porous polymer, and is commercialized by the company MitsuiChemicals (4-methylpentene-1 based polyolefin, Mitsui Chemicals, Inc.). In order to perform our experiments, we contacted MitsuiChemicals who offered us some samples of TPX®.  
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</div>
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</p></div>
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    <div class="group center">
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<p class="text">
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At the maximum the bacteria
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produces 32mmol/L of formate.  
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It is necessary to add genes
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involved in formate production
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to regulate production and  
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multiply it by 2.4. For a perfect
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regulation it would be necessary to
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delete pfl-B in E.coli genome not to  
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have formate production during the day.
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</p>
+
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</div>  
+
  
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<div class="subsubtitle">
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<center>  
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<h3>Bacteria survival</h3>
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<div style="one-half;padding:10px;">
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</div>
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<img src="https://static.igem.org/mediawiki/2015/thumb/2/29/TLSE_TPX_bag.jpeg/433px-TLSE_TPX_bag.jpeg" style="width:40%;" />
−
+
−
   
+
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    <div class="group center">
+
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<p class="text">
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−
As it is explained <a target="_blank" href="https://2015.igem.org/Team:Toulouse/Experiments#platecult">here</a> we plated bacteria on Petri dish to know if they were alive or not because OD measure cannot discriminate alive bacteria from dead. This test show us that wild type bacteria can easily survive during at least 15 days. So if we bring them a carbon source during this period they should survive even better.
+
−
</p>
+
−
</div>
+
−
 
+
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<img src="https://static.igem.org/mediawiki/2015/5/56/TLSE_Devicebio_image3.PNG" style="width:100%;"/>
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  <p class="legend">Figure 3: Bacteria survival results from culture test with BW25113 on M9 with 15mM of glucose during 15 days to mime real
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survival condition. </p>
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  </main>
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</div>
 
</div>
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<p class="legend">
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<div class="wrapper row4">
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A TPX containing 6 ml of LB medium
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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
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</p></center>
 
</p></center>
  
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<div class="clear">
 
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<ul>
 
  
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<li>
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<div class="group center">
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[1] REFERENCE 1
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<p class="text">
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</li>
+
As in the Groeningen 2012 project, our bacteria need to be separated from the growth medium until the beekeeper decides to use the trap. In order to do that, freeze-dryed bacteria are contained in a small polyvynil chloride bag placed inside a bigger TPX® bag. Since polyvynil chlorid is fragile, the user have to break it in order to start the growth.
 +
<br><br>
 +
To check the feasability and safety of our device, several tests have been performed:
 +
</p></div>
  
−
<li>
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<ul style="font-size:15px;margin-bottom:10px;">
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<li><a href="https://2015.igem.org/Team:Toulouse/Results#TPX"target="_blank">Growth tests in TPX®</a>: 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
−
[2] REFERENCE 2 AVEC UN LIEN <a href="http://www.google.com/patents/US8647615">See more</a>
+
 
</li>
 
</li>
 +
<li><a href="https://2015.igem.org/Team:Toulouse/Results#gaz"target="_blank">Gas diffusion tests</a>: Permeability of butyric acid and formic acid through the TPX® bag
 +
</li>
 +
<li><a href="https://2015.igem.org/Team:Toulouse/Results#safety"target="_blank">Safety test</a>: Impermeability of the bag of TPX® to the bacteria
 +
</li>
 +
<li><a href="https://2015.igem.org/Team:Toulouse/Results#biolo"target="_blank">Bacterial survival</a> over 15 days in microaerobic conditions
 +
</li>
 +
<li><a href="https://2015.igem.org/Team:Toulouse/Results#carbon"target="_blank">Carbone source test</a>: choice of a carbon source to produce acids during at least 10 days
 +
</li>
 +
<li><a href="https://2015.igem.org/Team:Toulouse/Results#tox"target="_blank">Acid toxicity</a> on <i>E. coli</i>
 +
</li>
 +
</ul>
 +
 +
<center>
 +
<div class="title">
 +
<h3>READ MORE</h3>
 +
</div> </center>
  
−
<li>
+
<div class="group center">
−
[3] REFERENCE 2 AVEC UN LIEN qui ouvre dans une nouvelle fenêtre <a href="http://www.google.com/patents/US8647615">See more</a>
+
<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/Attract">
 +
<div class="title">
 +
<h3>Attract</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">
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<h3>Regulation</h3>
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Latest revision as of 22:16, 18 September 2015

iGEM Toulouse 2015

Device


Content

Our goal is to create a solution against hives infestation by varroas. In order to get the best out of ApiColi in the treatment of varroosis, we designed a trap named TrApiColi. TrApiColi was thought up to take into account ethical reflection, safety and ease of use for beekeepers.

Trap Device

Since the switch between the production of the two molecules is regulated by light, our bacteria need to be outside of the beehive. Thus, the trap was designed to be placed at the entrance of the hive, in order to act before infection and try to prevent the entry of as many mites as possible.

The four different parts of our trap

TrApiColi is composed of four main parts:

1. A grid lined up with the bottom board

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 will not disturb the bees' comings and goings. The holes are big enough to let the varroas fall through them, but not the bees.



2. A funnel, to channel all the falling varroas

The grid and the funnel

3. A transparent collector, containing the bacteria confined in a special bag

It is designed like a fish bottle trap: the tube from the funnel goes on for a few centimeters inside the collector to ease the entry of the varroas in the collector while preventing them from exiting. The special bag is described in the "ApiColi containment and culture" part below.



4. A roof, to protect the trap from the rain

Associated with pannels blocking the sides of the entrance, it forces the bees to walk on the grid. This channeling does not disturbs the bees.


The dimensions of the trap allow it to adapt to almost every beehive. Indeed, most of the hive types have the exact same entrance. Thanks to that, the trap can be easily and perfectly installed on the hive by the beekeepers without drilling or cutting.



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 without coating 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 "Modeling" part.

The 3D printer used to construct our trap and the result of 3D printing: TrApiColi

ApiColi containment and culture

The use of genetically modified organisms in a field associated with edible products requires to carefully consider the issues of safety, national legislation, and public opinion. In this context, we looked for a solution that would isolate our engineered bacteria from the environment, while still allowing its growth and gas diffusion.
We found the solution in the iGEM Groeningen 2012 project that used a special polymer called TPX® in order to keep their bacteria from contaminating meat, their edible product.
TPX® is composed of Polymethylpentene, a porous polymer, and is commercialized by the company MitsuiChemicals (4-methylpentene-1 based polyolefin, Mitsui Chemicals, Inc.). In order to perform our experiments, we contacted MitsuiChemicals who offered us some samples of TPX®.

A TPX containing 6 ml of LB medium

As in the Groeningen 2012 project, our bacteria need to be separated from the growth medium until the beekeeper decides to use the trap. In order to do that, freeze-dryed bacteria are contained in a small polyvynil chloride bag placed inside a bigger TPX® bag. Since polyvynil chlorid is fragile, the user have to break it in order to start the growth.

To check the feasability and safety of our device, several tests have been performed:

  • Growth tests in TPX®: Culture of the strain E. coli BW25113 in a TPX® bag (ie. in microaerobic conditions, without agitation and miming batch culture condition), as it would be in the field
  • Gas diffusion tests: Permeability of butyric acid and formic acid through the TPX® bag
  • Safety test: Impermeability of the bag of TPX® to the bacteria
  • Bacterial survival over 15 days in microaerobic conditions
  • Carbone source test: choice of a carbon source to produce acids during at least 10 days
  • Acid toxicity on E. coli

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