Difference between revisions of "Part:BBa K4347007"
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[[File: BBa K4347007--bst.PNG|300.px|center|thumb|Bst polymerase derived from thermophilic bacterium <em> Geobacillus stearothermophilus </em> used in LAMP modelled on PyMol.]]
 
[[File: BBa K4347007--bst.PNG|300.px|center|thumb|Bst polymerase derived from thermophilic bacterium <em> Geobacillus stearothermophilus </em> used in LAMP modelled on PyMol.]]
  
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Bst polymerase large fragment is structurally homologous to KlenTaq polymerase used in PCR and Klenow fragment of DNA polymerase I in E. coli.  
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Bst polymerase large fragment is structurally homologous to KlenTaq polymerase (large fragment of Taq polymerase used in PCR) and Klenow fragment (large fragment of DNA polymerase I in E. coli).  
  
 
===Enhanced Thermal stability===
 
===Enhanced Thermal stability===
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2. https://pubmed.ncbi.nlm.nih.gov/8740835/  
 
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
 
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
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3. https://pubs.acs.org/doi/10.1021/acs.biochem.8b00200
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4. https://www.researchgate.net/publication/14191850_Crystal_Structure_of_a_Thermostable_Bacillus_DNA_Polymerase_I_Large_Fragment_at_21_A_Resolution

Revision as of 18:05, 11 July 2022

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 (large fragment of Taq polymerase used in PCR) and Klenow fragment (large 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

3. https://pubs.acs.org/doi/10.1021/acs.biochem.8b00200

4. https://www.researchgate.net/publication/14191850_Crystal_Structure_of_a_Thermostable_Bacillus_DNA_Polymerase_I_Large_Fragment_at_21_A_Resolution