Team:Caltech/Project/Conclusions

comX Export

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.