During our research, we have encountered many obstacles and undergone a process of trials and errors. Therefore, we developed a comprehensive guide to help other iGEM teams.
Transformation
When transforming our plasmid, we initially conducted two groups of experiments using two bacteria simultaneously, which was SHuffle and Escherichia coli strain BL21. SHuffle is widely regarded as the superior and more commonly used strain by most researchers. In our result, BL21 Shows a better transformation efficiency. Click the result to see our result.
The main reason is due to the fact that SHuffle expresses the desired protein to a very large extent as it will have a higher tendency to form disulfide bonds which waste most energy to express the protein and reserve almost no energy to express antibiotic resistance genes, thus SHuffle dies.
On the other hand, BL21 expresses the protein to a smaller extent, thus requiring less energy to synthesise our desired antibodies. BL21 can reserve more energy to express antibiotic resistance genes and survive for a longer time.
Protein Induction
During protein induction, when preparing an IPTG solution, try for a wider range of concentrations. This can have different effects on the transformed protein. It should be noted that an IPTG solution with excessively high concentration can exhibit toxicity towards the transformed bacteria, while low concentration of IPTG will lead to low protein expression which lead to low protein yield.
Protein expression
During protein expression, it is of key significance to culture the bacteria in order to replicate more proteins. In some situations, culturing the bacteria for one day may not produce enough proteins, so it is recommended to grow for two days or more.
Biobricking
In Biobricking, use shorter chains instead of longer ones. Purchase the genes separately, avoid using long chains and ligate them together after PCR. If the chains performing PCR are too long, they may risk being denatured.
Primers should contain
50% GC content.
GC clamp at the end of primers.
Codon Optimisation
Purchased genes can be highly complex. Codon Optimization via computer modelling/ by manual manipulation can lower its complexity. This can simplify the production process of the gene.