LacI-Controlled CviJI Endonuclease (+ ssrA deg. tag)
The mutant lac promoter PL8-UV5 will regulate the intracellular concentration of R.CviJI through independent lac repressor binding to the operator sites. The ssrA degradation tag AANDENYADAS will prevent unwanted leaky expression of the endonuclease from degrading the plasmid sequences when still required.
The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence.
Reznikoff et al. (1978) mutated the regulatory region in question at three different sites. 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. As a result of these mutations, gene expression mediated by the PL8-UV5 promoter will be independently regulated by intracellular concentrations of the lacI repressor as all CAP-associated regulation has been removed.
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 441
Illegal AgeI site found at 584
- 1000COMPATIBLE WITH RFC