Difference between revisions of "Part:BBa K3814009:Design"

Revision as of 14:37, 21 October 2021

5' Homology Arm for Cluster 2

Design Notes

In USYD 2021's project, we have used recombineering to insert 5kb chunks of DNA into E. coli. The particular recombineering strategy we have employed in our design is the bacteriophage λ Red recombineering system, and we are inserting our gene clusters into the fliK gene.

- In a study by Juhas and Ajioka (2016), the fliK gene in the E. coli was shown to be an optimal location for recombineering, and 15kb was successfully inserted there in one iteration. - The bacteriophage λ Red recombineering system is described in the diagram below, and many strains of E. coli have these systems already in place (Sharan et al., 2009). We have decided to use the JM109 strain and the recombineering functions were going to be brought in by the pKD46 plasmid.

Figure 1. Recombineering system using the bacteriophage λ Red system. According to Sharan et al. (2009), the homology arms need to be only 50bp for successful recombination, Additionally, only three genes, gam, bet and exo, are involved. The gene product of gam “prevents an E. coli nuclease, RecBCD, from degrading linear DNA fragments”, which allows for linear DNA to survive in vivo for recombination. The roles of exo and bet are shown above, with the gene product of bet, Beta, being an “ssDNA binding protein” and exo having “5′ to 3′dsDNA exonuclease activity”.

We devised a strategy called Babushka Blocks. See below an images that showcase how the homology arm (red and purple) would help in inserting a section of DNA into the fliK gene:

Additional care was taken to ensure that the homology arm did not form self-dimers or hairpins. Self-dimerisation and hairpin formation metrics were calculated on PrimerSelect. Lowest values are as follows:

Self-dimerisation: -2.4kc/m (worst possible is -86.6kc/m) Hairpin formation: 0.9kc/m

Source

iGEM USYD 2021

References

Juhas, M., & Ajioka, J. W. (2016). Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome. Microbial Cell Factories, 15(1). https://doi.org/10.1186/s12934-016-0571-y

Sharan, S. K., Thomason, L. C., Kuznetsov, S. G., & Court, D. L. (2009). Recombineering: a homologous recombination-based method of genetic engineering. Nature Protocols, 4(2), 206–223. https://doi.org/10.1038/nprot.2008.227