Difference between revisions of "Part:BBa K4347007:Design"
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<u>Residues to mutate</u> | <u>Residues to mutate</u> | ||
| − | Since there is little literature on point mutations on Bst, point mutations made in Taq (Klentaq) were sought after. Three notable point mutations were found in the Klentaq thumb domain at positions L505A, L540A and L542A where each Lysine (L) residue was switched to an Arginine residue (R)[[Part:BBa_K4347007 | + | Since there is little literature on point mutations on Bst, point mutations made in Taq (Klentaq) were sought after. Three notable point mutations were found in the Klentaq thumb domain at positions L505A, L540A and L542A where each Lysine (L) residue was switched to an Arginine residue (R)[[Part:BBa_K4347007:Design#References|<sup>[1]</sup>]]. |
How we came up with these residues to mutate. | How we came up with these residues to mutate. | ||
Making point mutations in yasara - talking about the differences in gibbs free energy. | Making point mutations in yasara - talking about the differences in gibbs free energy. | ||
Revision as of 15:11, 12 July 2022
Bst with point mutations for enhanced thermal stability codon optimized for E.coli
- 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]
Design Notes
Sequence alignment
Since Bst is structurally homologous to Klentaq polymerase, we fetched the Bst (6MU6) and Klentaq (6QV4) FASTA sequences from the Protien Data Bank and ran a multiple sequence alignment using Seaview. A good amount of conservation between the amino acid sequences was found between the two polymerases.
Residues to mutate
Since there is little literature on point mutations on Bst, point mutations made in Taq (Klentaq) were sought after. Three notable point mutations were found in the Klentaq thumb domain at positions L505A, L540A and L542A where each Lysine (L) residue was switched to an Arginine residue (R)[1]. How we came up with these residues to mutate. Making point mutations in yasara - talking about the differences in gibbs free energy.
Design Considerations
Considering which residues were in active site or not. Fingers are important for strand displacement and activity in taq(victor to fetch paper) and when mutated only 25% worked compared to thumb and palm domains.
Source
PDB: 6MU5: https://www.rcsb.org/structure/6mu5