Team:GreatBay SZ/Contribution
Trouble Shooting
We confronted many setbacks during the experiment, but through the efforts of team members and instructors, those problems were all solved. The following summarized all the difficulties we met, which could be useful for other teams as references in the future.
FimA Knock Out--Different fimA sequencesfimA Knock Out--Different fimA sequences
When we knocked out the fimA gene, we first designed gRNA and upper stream and lowered homologous stream arms based on the fimA sequence on the E.Coli MG1655 genome, but we found that the upper stream and lower stream homologous arms of fimA could not be cloned from E. Coli Top10. Therefore, we speculated that the sequences of fimA of the two E.coli strains were different. Later on, the comparison showed that this was the case, so the fimA knockout in E. Coli Top10 was redesigned and successfully completed.
Pilin Expression--Elimination of pCasPilin Expression--Elimination of pCas
When we transferred the constructed plasmid expressing PilA into the E.Coli Top10 with fimA knocked out, we found a lot of false positives in the cultured bacteria, which did not contain the target plasmid. Later on, we performed PCR tests on these bacteria and found that the pCas plasmid (a thermosensitive plasmid containing the Cas9 gene sequence for fimA knockout) was not eliminated. Since both pCas plasmids and our target plasmids were both Kana resistant, we couldn't use Kana antibiotics to screen out bacteria capable of producing pilin protein. Later, we tried to eliminate pCas plasmid by repeatedly incubating these bacteria in a 42-degree incubator, but it only gave an unideal result. Therefore, we used chloramphenicol resistant plasmid skeleton to reconstruct the plasmid expressing pilin and transferred it to fimA-knockout E.Coli Top10. After the antibiotic screening, E.Coli Top10 (BIOT) with stable PilA expression and no pCas plasmid was successfully obtained.
PilA Mutation--the construction of mutated plasmidPilA Mutation--the construction of mutated plasmid
When we constructed the plasmid expressing mutated PilA for the first time, we encountered many problems. At first, we transferred the mutated plasmid into E.Coli Top10 for amplification and designed a pair of detecting primers for the mutation site. However, when the PCR product was sent to do sequencing, the mutation site was always not detected. We then carried out relevant experiments with wild E.Coli Top10 to confirm that the detection primers could work. It turned out that the problem was not due to the detecting primers. Then, to exclude the effect of the plasmid's inefficient transfer in E.Coli Top10, we first transferred the plasmid into E.coli DH5α. However, the result was still unideal. Therefore, it was estimated that problems occurred during plasmid construction. And then, mutant fragments and skeleton were cloned again, connected, and transferred. Finally, 17 correct mutant plasmids were successfully obtained and transferred into E.Coli Top10 without fimA, and 17 BIOT mutant species were obtained.
Protein Purification ProtocolProtein Purification Protocol
We sorted out a more concise and intuitive protocol based on protein purification in reference [1]. Also, We shot an instructional video for the reference of subsequent iGEM teams and partners. In the instructional video, we fully demonstrated the process of purifying protein and various equipment we used, which did have particular reference value.
see more in our protocol.
At the same time, we also summarized some new precautions based on the original protocol:
References
[1] Ueki, Toshiyuki, et al. An Escherichia Coli Chassis for Production of Electrically Conductive Protein Nanowires. ACS Synthetic Biology, vol.9, no.3, 3 Mar.2020, pp.647–654.