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

Sequence alignment

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. A good amount of conservation between the amino acid sequences was found between the two polymerases.

Protien sequence alignment of Bst (6MU5) and Klentaq (6QV4) in Seaview.

Residues to mutate

Since there is little literature on point mutations on Bst, point mutations made in Taq (Klentaq) were sought after. Three notable point mutations were found in the Klentaq thumb domain at residues

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