Difference between revisions of "Team:Wageningen UR/Notebook/Chitinases"

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<h1><b>July</b></h1>
 
<h1><b>July</b></h1>
 
<h2><b>Week 1</b></h2>
 
<h2><b>Week 1</b></h2>
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<p>This week, we received our IDT order; this contained chitinase A and B from <i>Serratia marcescens</i> GEI strain, a strain which has been reported to cause higher mortality in <i<Varroa destructor</i> than in worker bees<sup><a href="#ch1" id="refch1">18</a></sup>. The chitinases were optimized for BioBrick use, as is described in their respective BioBrick pages: <a href="http://parts.igem.org/Part:BBa_K1913000"><i>chiA</i></a> and <a href="http://parts.igem.org/Part:BBa_K1913001"><i>chiB</i></a>.Initially, I tried to do a PCR to amplify the synthetic genes, but this resulted in a too small product for <i>chiB</i> (Figure 1).</p>
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<p>This week, we received our IDT order; this contained chitinase A and B from <i>Serratia marcescens</i> GEI strain, a strain which has been reported to cause higher mortality in <i<Varroa destructor</i> than in worker bees<sup><a href="#ch1" id="refch1">18</a></sup>. The chitinases were optimized for BioBrick use, as is described in their respective BioBrick pages: <a href="http://parts.igem.org/Part:BBa_K1913000"><i>chiA</i></a> and <a href="http://parts.igem.org/Part:BBa_K1913001"><i>chiB</i></a>.Initially, I tried to do a PCR to amplify the synthetic genes, but this resulted in a too small product for <i>chiB</i> (Figure 1). The PCR was done with Q5 polymerase and the program below was used.
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<figure>
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<table>
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  <tr>
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      <th>Step</th>
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      <th>Temperature in °C</th>
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      <th>Time</th>
 +
  </tr>
 +
  <tr>
 +
      <td>Predenaturation</td>
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      <td>98</td>
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      <td>30 seconds</td>
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  </tr>
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  <tr>
 +
      <td>Denaturation</td>
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      <td>98</td>
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      <td>7 seconds</td>
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  </tr>
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  <tr>
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      <td>Annealing</td>
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      <td>60</td>
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      <td>20 seconds</td>
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  </tr>
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  <tr>
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      <td>Extension</td>
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      <td>72</td>
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      <td>60 seconds</td>
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  </tr>
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  <tr>
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      <td>Final Extension</td>
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      <td>72</td>
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      <td>5 minutes</td>
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    </tr>
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</table>
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<figcaption>Table 1: PCR program for chitinase amplification with BioBrick-f and BioBrick-r primers.</figcaption></figure></p>
 
<p><figure>
 
<p><figure>
 
<img src="https://static.igem.org/mediawiki/2016/e/ea/T--Wageningen_UR--chipcr1.jpg">
 
<img src="https://static.igem.org/mediawiki/2016/e/ea/T--Wageningen_UR--chipcr1.jpg">

Revision as of 08:28, 11 October 2016

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Wageningen UR iGEM 2016

 

 

 

Main Results

July

Week 1

This week, we received our IDT order; this contained chitinase A and B from Serratia marcescens GEI strain, a strain which has been reported to cause higher mortality in than in worker bees18. The chitinases were optimized for BioBrick use, as is described in their respective BioBrick pages: chiA and chiB.Initially, I tried to do a PCR to amplify the synthetic genes, but this resulted in a too small product for chiB (Figure 1). The PCR was done with Q5 polymerase and the program below was used.

Step Temperature in °C Time
Predenaturation 98 30 seconds
Denaturation 98 7 seconds
Annealing 60 20 seconds
Extension 72 60 seconds
Final Extension 72 5 minutes
Table 1: PCR program for chitinase amplification with BioBrick-f and BioBrick-r primers.

Figure 1. Photo of a 1% TAE gel loaded with chiA and chiB PCR products, BBa_J04450 as a positive control and water as a negative control. The expected size for chiA is 1760 basepairs, for chiB 1560 basepairs.

Week 2

Figure 1. Photo of the first plate with colonies (no. 15). These mites were not sterilized, so this was probably contamination.

Week 3

Week 4

May

Week 5

Week 6

Figure 5. Protein extracts of Bt HD350, E. coli with Cry1 and Cry2 and isolates V46, V47. The red arrow indicates a band that is probably Cry1. The ladder is a BioRad Precision Plus Protein Standard, the gel is a MiniProtean TGX 12%.

Week 7

Week 8

Figure 7. Cellular debris of E. coli BL21, the failed Cry1 and Cry2 clones, Bacillus subtilis, Bt HD350 and Bt tenebrionis, as well as isolates V46 and V47. The red and blue arrrow indicate bands which are probably Cry1Aa (133 kDa) and Cry3Aa (73 kDa). The ladder is a BioRad Precision Plus Protein Standard, the gel is a MiniProtean TGX 12%.

References

      1. Tu, S., Qiu, X., Cao, L., Han, R., Zhang, Y., & Liu, X. (2010). Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite. Journal of invertebrate pathology, 104(2), 75-82. ↩