Part:BBa_K4347007:Design

Bst with point mutations for enhanced thermal stability codon optimized for E.coli

Assembly Compatibility:

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

  Illegal AgeI site found at 766
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Since Bst is structurally homologous to Klentaq polymerase, we fetched the Bst (6MU6) and Klentaq (6QV4) FASTA sequences from the Protien Data Bank and ran a multiple sequence alignment using Seaview. We found a good amount of conservation between the amino acid sequences between the two polymerases.

Seaview alignment photos

Once the sequences alignment was confirmed, we began searching literature for point mutations made in Klentaq which improved thermal stability such that

How we came up with these residues to mutate. Making point mutations in yasara - talking about the differences in gibbs free energy.

Design Considerations

Considering which residues were in active site or not. Fingers are important for strand displacement and activity in taq(victor to fetch paper) and when mutated only 25% worked compared to thumb and palm domains.

Source

PDB: 6MU5: https://www.rcsb.org/structure/6mu5