Difference between revisions of "Part:BBa K4347007"
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===References=== | ===References=== | ||
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| − | 1. https:// | + | 1. Ignatov, K. B., Barsova, E. V., Fradkov, A. F., Blagodatskikh, K. A., Kramarova, T. V., & Kramarov, V. M. (2014). A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques, 57(2), 81–87. https://doi.org/10.2144/000114198 |
| − | 2. | + | 2. Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H (1996). Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. (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. | + | |
3. https://pubs.acs.org/doi/10.1021/acs.biochem.8b00200 | 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 | 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 20:43, 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 reaction temperature. Three point mutations were made in the polymerase thumb domain: K549W, K582L and Q584L. The increased thermal stability from these point mutations was validated using a protien simulation program called YASARA. The overall change in Gibbs free energy of wild-type Bst was calculated to be -150.13 Kcal/mol, and the overall stability of the mutated Bst was calculated to be -151.81 Kcal/mol thus indicative of a more thermally stable protein.
- 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. Ignatov, K. B., Barsova, E. V., Fradkov, A. F., Blagodatskikh, K. A., Kramarova, T. V., & Kramarov, V. M. (2014). A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques, 57(2), 81–87. https://doi.org/10.2144/000114198
2. Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H (1996). Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. (5-6):185-95. PMID: 8740835