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. 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.
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