Part:BBa_K4347007

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

This part is an improvement from Bst Polymerase 1 (large fragment) for E.coli from the 2021 iGEM Fudan team: https://parts.igem.org/Part:BBa_K3790000.

Usage and Biology

Bst polymerase Large Fragment is a family I DNA polymerase derived from the thermophilic bacterium Geobacillus stearothermophilus. Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability ^[1]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity ^[2]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP.

Bst polymerase derived from thermophilic bacterium Geobacillus stearothermophilus used in LAMP modelled on PyMol.

Bst polymerase large fragment is structurally homologous to KlenTaq polymerase used in PCR and Klenow fragment of DNA polymerase I in E. coli.

Enhanced Thermal stability

The origional codon optimized Bst polymerase (Part BBa_K3790000) was improved upon to further enhance the polymerases thermal stability such that it can carry out LAMP at a higher temperature. Three point mutations were made in the polymerases thumb domain

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]

References

1. https://www.tandfonline.com/doi/full/10.1080/10826068.2022.2095573?src=

2. https://pubmed.ncbi.nlm.nih.gov/8740835/ Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H. Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Genet Anal. 1996 Mar;12(5-6):185-95. PMID: 8740835