CviJI Endonuclease (+ ssrA deg. tag)
As part of the St Andrews iGEM 2020 kill switch mechanism, an enzyme capable of destroying plasmid inserts was of highest priority to prevent uptake of synthetic genes, whose expression by bacteria in the surrounding environment may have provided a survival advantage. All plasmid insert sequences were subject to commercial enzyme restriction site screening using the SnapGene feature which proposed the CviKI-1/CviJI restriction site to be most common across all sequences. In this case, the small size (4bp) of the restriction site is useful for our project.
The restriction endonuclease CviJI (also known as R.CviJI) is taken natively from the Chlorella virus IL-3A, a double-stranded DNA phycodnavirus that infects unicellular, eukaryotic Chlorella-like green algae. As well as being previously expressed in E.coli by Skowron et al. (1995) and Swaminathan et al. (1996), the restriction endonuclease is also used commercially and is available via NEB as CviKI-1. The enzyme cuts at RG/CY sites (where R = purines, Y = pyrimidines) in the presence of Mg2+. With the addition of ATP, R.CviJI (now R.CviJI*) cleaves at additional restriction sites RG/CN and YG/CY (where N = any nucleotide) but not YG/CR. Both enzymes cleave DNA frequently and therefore possess a variety of functions such as generating numerous sequence-specific oligonucleotides. The sequence to be used in this part is 278 amino acids in length and does not exhibit additional R.CviJI* activity. The 144-235 amino acid region is also suggested to have a recognition/catalytic domain.
R.CviJI will be fused to the ssrA degradation tag AANDENYADAS to prevent plasmid destruction as a result of leaky expression only. Lon protease, native to E.coli, will recognise and degrade the fusion construct. 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.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 316
Illegal AgeI site found at 459
- 1000COMPATIBLE WITH RFC