We underwent the Engineering Design Cycle as we designed and tested our primer for csgA knockouts.
The pages for our Primers have been finished ( http://parts.igem.org/Part:BBa_K4436017 and http://parts.igem.org/Part:BBa_K4436666 ) but the cycle was heavily followed in ensuring our success.
Our primer design consisted of the following:
1. Finding 40 nucleotides of homology to the flanking region of the csgA gene (the one we’re trying to knockout)
2. Finding 20 nucleotides of homology to the flanking regions of the FLP recombinase sites in pKD3 plasmid
3. Combing them respectively to make the primer
The primers were built by IDT as part of our collaboration with the company.
We test our primers by conducting the PCR of pKD3 and running it on a gel to see if the amplification worked. More information can be found on our parts pages.
The first time PCR failed, we found homology regions lower in GC content and closer to the sites because we believed that’s why we failed. We then designed a second set of primers.
The second time it failed, we took a closer look and realized we placed the homology regions in the wrong order for the Reverse Primer. We then designed our third set of primers.
When that still wasn’t working, we lowered the annealing temperature and finally got great results, which can be found on our parts registry.
Thanks for the collaborator teams and the sponsor of our university
