Difference between revisions of "Part:BBa K4347011"
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===A more thermally stable and processive polymerase=== | ===A more thermally stable and processive polymerase=== | ||
| − | This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006). The modified Bst polymerase contains three point mutations in the polymerase thumb domain: K549W, K582L and Q584L, which have been proven to improve thermal stability in Bst homologue Taq polymerase[[Part:BBa_K4347011#References|<sup>[1]</sup>]]. | + | This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006). The modified Bst polymerase contains three point mutations in the polymerase thumb domain: K549W, K582L and Q584L, which have been proven to improve thermal stability in Bst homologue Taq polymerase[[Part:BBa_K4347011#References|<sup>[1]</sup>]]. 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. |
| − | + | ||
| − | 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. | + | |
[[File:BBa K43470011 bst fusion stability.PNG|500px|center|thumb|Thermal stability of Bst fusion protien computed from YASARA. | [[File:BBa K43470011 bst fusion stability.PNG|500px|center|thumb|Thermal stability of Bst fusion protien computed from YASARA. | ||
]] | ]] | ||
| + | Along with an increased thermal stability, the mutated polymerase was fused to a DNA binding protien Sac7e to increase polymerase processivity during the LAMP reaction. Sac7e is isolated from thermoacidophilic archaeon (em>Sulfolobus acidocaldarius</em> and | ||
Lab results and pictures | Lab results and pictures | ||
Revision as of 17:21, 20 July 2022
Bst fusion with Sac7e and point mutations for enhanced thermal stability codon optimized for E.coli
This fusion protien was designed by linking the N-terminus of a modified Bst polymerase with thermostable DNA binding protien Sac7e using a flexible (GGGGS)4 linker to increase polymerase thermostability and processivity in LAMP reaction.
Usage and Biology
Usage of bst and Sac7e in biology. Can summarize what was written for "bst with point mutations" and "sac7e" pages. This bst version DOES include the point mutations
A more thermally stable and processive polymerase
This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006). The modified Bst polymerase contains three point mutations in the polymerase thumb domain: K549W, K582L and Q584L, which have been proven to improve thermal stability in Bst homologue Taq polymerase[1]. 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.
Along with an increased thermal stability, the mutated polymerase was fused to a DNA binding protien Sac7e to increase polymerase processivity during the LAMP reaction. Sac7e is isolated from thermoacidophilic archaeon (em>Sulfolobus acidocaldarius</em> and
Lab results and pictures
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 5
Illegal XhoI site found at 209 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1015
- 1000COMPATIBLE WITH RFC[1000]
References
1. Xi, L. (2009, December 23). WO2009155464A2 - mutated and chemically modified thermally stable DNA polymerases. Google Patents. Retrieved July 12, 2022, from https://patents.google.com/patent/WO2009155464A2/en