Revision as of 08:28, 11 October 2016

MENU ▤

Wageningen UR iGEM 2016

Overview

Description

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 from Table 1 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.

↩

@@ Line 10: / Line 10: @@
 <h1><b>July</b></h1>
 <h2><b>Week 1</b></h2>
-<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.
+<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 from Table 1 was used.
 <figure>
 <table>

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