Part:BBa_K1673204
T7 Endonuclase (Mutation Optimized)
Repair enzyme that induces double strand breaks at DNA base mismatches
Enzymatic Activity
T7 Endonuclease I is an enzyme which cleaves DNA creating a double-stranded break within 3 basepairs 5’ of the mismatched DNA. In addition to heteroduplexes, the enzyme also cuts cruciform structures, Holliday junctions, and to a lesser extent, nicked dsDNA (Huang et al 2012). T7 has been used as a means of detecting single-nucleotide polymorphisms, including those generated by mutation, because of its ability to cleave heteroduplex DNA.
Mutation Optimization
The coding sequence for K1673204 was generated using the mutation-optimization software developed by Vanderbilt iGEM's 2015 team. The optimization was weighted to remove overall mutation hotspots while maintaining codon usage for proper expression.
Cloning
We first check for the successful integration of our synthesized sequence for K1673204 first by restriction digest. A band was produced at 2.5 kb, as expected for the sequence in pSB1C3. We then sequenced the gene from the forward and reverse ends using VF2 and VR primers. This sequencing conclusively determined that our optimized sequence was present and in the correct orientation.
Expression
We placed T7 nuclease both under a T7 Promoter (K1673224) and IPTG-inducible promoter (K1673214). We ran a coomassie on E. coli lysate to demonstrate expression of S1, although these results were inconclusive. This may be because levels of overexpression high enough to visualize by coomaisse would be lethal to the cell.
References
Huang MC, Cheong WC, Lim LS, Li MH. A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis. Electrophoresis. 2012;33(5):788-96. Sequence and Features
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