Part:BBa_K3814017:Design
5' Homology Arm for Cluster 4
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 5
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 5
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 5
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 5
- 1000COMPATIBLE WITH RFC[1000]
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:
Figure 2. Babushka block design. To insert Cluster 1 into the fliK landing pad, Cluster 1 must be first hybridised with the primer, which contains the red homology arm (Step 1). Afterwards, there will be two matching homology arms between the landing pad and Cluster 1: the red and purple arms. As a result, Cluster 1 is able to be inserted into the landing pad, and the end result has the genes of interest inside the landing pad (Step 2).
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: -1.6kc/m (worst possible is -83.9kc/m) Hairpin formation: 1.2kc/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