Difference between revisions of "Part:BBa K4347007"
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Bst polymerase Large Fragment is a family I DNA polymerase derived from the thermophilic bacterium <em> Geobacillus stearothermophilus.</em> Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability [[Part:BBa_K4347007#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity [[Part:BBa_K4347007#References|<sup>[2]</sup>]]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP. | Bst polymerase Large Fragment is a family I DNA polymerase derived from the thermophilic bacterium <em> Geobacillus stearothermophilus.</em> Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability [[Part:BBa_K4347007#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity [[Part:BBa_K4347007#References|<sup>[2]</sup>]]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP. | ||
| − | [[File: BBa K4347007--bst.PNG| | + | [[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 taq.PNG|150px|left|thumb|Bst polymerase (green) superimposed with Klentaq fragment (white) from Taq polymerase in Pymol.]] | [[File:BBa K4347007-bst taq.PNG|150px|left|thumb|Bst polymerase (green) superimposed with Klentaq fragment (white) from Taq polymerase in Pymol.]] | ||
[[File:BBa K4347007-bst KF.PNG|150px|right|thumb|Bst polymerase (green) superimposed with Klenow fragment (white) from DNA polymerase 1 from <em> E.coli</em> in Pymol.]] | [[File:BBa K4347007-bst KF.PNG|150px|right|thumb|Bst polymerase (green) superimposed with Klenow fragment (white) from DNA polymerase 1 from <em> E.coli</em> in Pymol.]] | ||
Revision as of 18:48, 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 large fragment is structurally homologous to KlenTaq polymerase (large fragment of Taq polymerase used in PCR) [3] and Klenow fragment (large fragment of DNA polymerase I in E. coli) [4].
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 766
- 1000COMPATIBLE 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