Difference between revisions of "Part:BBa K3634021:Design"

Latest revision as of 10:48, 3 September 2020

LacI-Controlled CviJI Endonuclease (+ ssrA deg. tag)

Design Notes

The wt sequence for the lac operon regulatory region was taken from Kalnins A. (1993) [GenBank accession no. J01636]. Within the CAP binding site, bases -66 (G) and -55 (C) of the wt binding region were substituted with A and T respectively to prevent binding of the CAP protein at low glucose concentrations. The wt -10 promoter sequence was also mutated from TATGTT to TATAAT in order to allow σ factor (RpoD) to bind without relying on further activation by the CAP protein (Reznikoff et al., 1978). This produced PL8-UV5.

As the cviJI gene is new to the registry, it must meet BBa assembly standards. A BsaI illegal restriction site was present at position 503. This was subsequently removed by in silico point mutagenesis: t501a (TCT (Ser) -> TCA (Ser)). As with other parts St. Andrews iGEM 2020 have characterised, a codon optimisation step was favourable however upon using the IDT codon optimisation tool, many additional CviJI restriction sites were introduced to the cviJI gene sequence. This was of course an issue as the gene coding for the enzyme would be fragmented, limiting the amount of expression of R.CviJI. As a result, codon optimisation was abandoned.

Additionally, a CviJI restriction site was already present in the initial gene sequence at position 477 (a perfect example of evolution at work given such a small frequency). To prevent the enzyme cutting its own gene sequence, the St Andrews iGEM team 2020 looked at in silico point mutagenesis for this site however unfortunately, no alternative was available as the codon involved in the restriction site was that of tryptophan (TGG/CCT). As this is the only CviJI restriction site present in the gene sequence, it is likely that this Trp residue is vital to the function of the enzyme. This was considered however to follow in the same HGT vein as with introducing R.CviJI in the first place as it meant a functioning endonuclease gene would less likely be transformed into other bacterial species.

As previously mentioned, placing a lon protease ssrA degradation tag onto R.CviJI would mop up any leaky expression of the fusion construct in order to prevent unwanted plasmid fragmentation too early. The strong AANDENYALAA degradation tag was initially suggested however it was thought this sequence was too strong to allow any endonuclease functionality when expressed. As a result, the tag was replaced with a 'moderately fast' AANDENYADAS degradation tag. The TAG stop codon of the R.CviJI gene was removed and replaced with the AANDENYADAS sequence. TAATAA was then added to the 3' end of the the ssrA to terminate translation.

The RBS BBa_J61100 was used as a very weak RBS (strength 4.29% relative to B0034 - Team Warsaw iGEM 2010) as strong expression of the endonuclease is not required for plasmid DNA fragmentation.

Source

The lac operon is found natively in E.coli. The part sequence was taken from BBa_K1695000 who generated the part sequence through data obtained from Reznikoff et al. (1978) and Hirschel et al. (1980). The sequence for R.CviJI, native to Chlorella virus IL-3A, was obtained from Skowron et al. (1995) and confirmed by Swaminathan et al. (1996) (ENA reference: U09001.1). The sequence for the ssrA degradation tag was obtained from registry part BBa_M0052 by the Endy lab (2007).

References

Jacob F., Monod J. 1961. Genetic Regulatory Mechanisms in the Synthesis of Proteins. J. Mol. Biol. 3: p818-356.

Reznikoff W. S., Abelson J. N. 1978. The lac promoter. The operon. 7: p221-243. DOI: 10.1101/0.221-243

Hirschel B.J., Shen V., Schlessinger D. 1980. Lactose Operon Transcription from Wild-Type and L8-UV5 lac Promoters in Escherichia coli Treated with Chloramphenicol. J. Bacteriol. 143(3): p1534-1537.

Skowron PM, Swaminathan N, McMaster K, George D, Van Etten JL, Mead DA. 1995. Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella. Gene. 157: p37-41. DOI: 10.1016/0378-1119(94)00564-9.

Swaminathan N, Mead DA, McMaster K, George D, Van Etten JL, Skowron PM. 1996. Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A. Nucleic Acids Res. 24: p2463-2469. DOI: 10.1093/nar/24.13.2463.

Endy Lab iGEM 2007 - https://parts.igem.org/Part:BBa_M0052