Team:Caltech/Project/Conclusions

A Word of Advice for Future iGEM Teams Attempting Gibson Assembly

We made many attempts at the beginning of the summer attempting to clone plasmids containing biobrick prefixes & suffixes at the beginning & end of the insert via Gibson Assembly. However, our colony PCRs were never able to verify properly assembled constructs. Instead, gels with lanes containing colony PCRs of colonies that grew on carbenicillin plates were run and ended up showing bands at roughly 100 base pairs. These bands at ~100bp suggest that the vector does not contain any insert, since a resealed vector containing no insert should yield a fragment of roughly that length when PCRed with our checking primers. On examination of the biobrick prefix and suffix, which were used as the "overlap regions" for Gibson assembly, it was indeed found that there was enough sequence homology between the two to suggest significant binding of the prefix to the suffix without incorporating the insert (resealing of the backbone). In particular, the sequence of similarity (14 base pairs in length [include that sequence]) had a very high estimated melting temperature of 46.2°C, owing to its rich GC-content. Since the melting temperature is so close to the 50°C used as our incubation temperature for Gibson assembly, it can be concluded that a significant number of backbone ends were "chewed back" by exonucleases in the Gibson reaction and then annealed to each other, recircularizing the backbone without the desired insert.

From our analysis of gels run with colony PCR product of the comX-mNeonGreen assemblies, we can conclude that attempting to use biobrick prefixes and suffixes as the overlap regions in Gibson assembly will more likely than not result in failure in assembly. The prefix and suffix sequences share too much sequence homology and anneal at too high a temperature to run efficiently the Gibson assembly. We thus recommend any future iGEM teams that wish to use Gibson assembly in construction of their biobrick parts to assemble their plasmid without the biobrick prefix and suffix. From there, after successful cloning of the construct has been verified, their desired insert can be PCRed back out using primers adding the biobrick prefix and suffix as overhangs. This linear fragment can then finally be "biobricked" via ligation into a standard vector backbone using the standard 3A assembly.