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Wageningen UR iGEM 2016

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. The same program was used as in Table 2, except the annealing temperature was 51 degrees Celsius and the extension time was 210 seconds.

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.

I picked the first colonies for both transformants which had been transformed with digested IDT oligo's. They were verified through sequencing. I also worked with the fifth chiB colony for some time, which turned out to have a 500 bp deletion.

Week 2

I tried to add RBS BBa_B0032 to the chitinases with reverse primer VR in combination with the following forward primers and Q5 polymerase:
chiA: 5' CCGATGAATTCGCGGCCGCTTCTAGtcacacaggaaagtaCTAGATGCGCAAGTTCAATAAACC
chiB: 5'CCGgTGAATTCGCGGCCGCTTCTagAtcacacaggaaagtaCTAGATGTCCACACGTAAAGCCGTTATTG
An annealing temperature of 62 degrees Celsius was used. The PCR failed, only the positive control showed bands.

To figure out why the PCR was not working, I did another gradient PCR with OneTaq polymerase. The same program as in Table 2 was used, except the extension time was set to 4 minutes and the annealing temperate was 47 +- 5 degrees Celsius. Figure 5 also shows the wrong size of chiB, but at this point I did not quite realize it.

Figure 5. 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 1913 basepairs, for *chiB* 1722 basepairs. The gel was run for 30 minutes at 100V.

Though the OneTaq gradient PCR clearly worked, I had to make a PCR work with a polymerase that has proofreading activity. I did one PCR with Pfu polymerase, but that failed as even my positive control showed no band. I repeated the Q5 PCR to add BBa_B0032 one more time, but now with an annealing temperature at 52 degrees Celsius, 10 degrees below the NEB recommended temperature. The results are shown in Figure 6.

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.

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Team:Wageningen UR/Notebook/Chitinases

Main Results