Difference between revisions of "Part:BBa K4347007:Design"
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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. We found a good amount of conservation between the amino acid sequences between the two polymerases. | 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. We found a good amount of conservation between the amino acid sequences between the two polymerases. | ||
| − | Seaview alignment photos | + | Seaview alignment photos File:BBa K4347007--seaview.PNG |
Once the sequences alignment was confirmed, we began searching literature for point mutations made in Klentaq which improved thermal stability such that | Once the sequences alignment was confirmed, we began searching literature for point mutations made in Klentaq which improved thermal stability such that | ||
Revision as of 14:44, 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
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. We found a good amount of conservation between the amino acid sequences between the two polymerases.
Seaview alignment photos File:BBa K4347007--seaview.PNG
Once the sequences alignment was confirmed, we began searching literature for point mutations made in Klentaq which improved thermal stability such that
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