Part:BBa_K1673203

S1 Nuclease (Mutation Optimized)

DNA modifying enzyme that converts single-strand breaks into double strand breaks.

Enzymatic Activity

Nuclease S1 is a DNA repair enzyme from Aspergillus oryzae. Its primary enzymatic activity is to cleave nicked dsDNA as well as DNA that has gaps, mismatches, or loops (Bai et al 2003). The nicked dsDNA will be hydrolyzed to form a double strand break.

Mutation Optimization

The coding sequence for K1673203 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 K1673203 first by restriction digest. A band was produced at 3.0 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 S1 nuclease both under a T7 Promoter (K1673223) and IPTG-inducible promoter (K1673213). 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

Bai YL, Yang ZL, Qiao MQ, Zhang XM, Zhou J, Gao CC. The action of S1 nuclease and a cloning strategy for microcircular DNAs. Sheng Wu Gong Cheng Xue Bao. 2003;19(2):240-3.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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