All PCRs were done with mixes made according to the manufacturer's protocol. See (link to protocol here) for details.
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 and Q5 polymerase.
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 approximately 1750 basepairs, for chiB approximately 1560 basepairs. The gel was run for 30 minutes at 100V.
This did not work, so I also tried raising the annealing temperature to 70 degrees Celsius, diluting the templates to 1 ng/μL. That failed as well, so a gradient PCR was performed with OneTaq and 2% DMSO(see Figure 2).
Step
Temperature in °C
Time
Predenaturation
94
30 seconds
Denaturation
94
30 seconds
Annealing
Gradient of 60+-5
60 seconds
Extension
68
120 seconds
Final Extension
68
5 minutes
Table 2: PCR program for chitinase gradient PCR with BioBrick-f and BioBrick-r primers and OneTaq polymerase.
Figure 2. Photo of a 1% TAE gel loaded with chiA gradient PCR products, BBa_I0500 as a positive control and water as a negative control. The expected size for chiA is approximately 1750 basepairs. The gel was run for 30 minutes at 100V.
Figure 3. Photo of a 1% TAE gel loaded with chiB gradient PCR products, BBa_I0500 as a positive control and water as a negative control. The expected size for chiB is approximately 1560 basepairs. The gel was run for 30 minutes at 100V.
As Q5 PCR seemed to fail, both chitinases were digested with EcoRI and PstI and cloned into pSB1C3 with heat shock transformation. Both the PCR products were used as well as the synthetic genes as supplied by IDT. The transformation worked well. Colony PCR to confirm insert size failed, so a miniprep was done for 5 colonies from each transformation. These were analysed with a OneTaq PCR with VF2 and VR primers.
Figure 4. 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 2007 basepairs, for chiB 1816 basepairs. The gel was run for 30 minutes at 100V.
Week 2
Figure 2. 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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