Difference between revisions of "Part:BBa K4347007:Design"
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*K540 in Klentaq = K582 in Bst
 
*K540 in Klentaq = K582 in Bst
 
*K542 in Klentaq = Q584 in Bst
 
*K542 in Klentaq = Q584 in Bst
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To confirm if these residues in Bst were suitable to mutate, the amino acid stability was estimated using a protien simulation software YASARA. A position scan was ran for each residue to measure the change in free energy (Kcal/mol) if the other 19 amino acid residues were mutated into that position. A mutation is classified as stabilizing if the change in free energy is ≤-1 kcal/mol, it is classified as destabilizing if the change is ≥1 kcal/mol, and neutral if it falls between these values [[Part:BBa_K4347007:Design#References|<sup>[2]</sup>]]. The changes in free energy for mutations at residues K549, K582 and K584 are shown below:  
 
To confirm if these residues in Bst were suitable to mutate, the amino acid stability was estimated using a protien simulation software YASARA. A position scan was ran for each residue to measure the change in free energy (Kcal/mol) if the other 19 amino acid residues were mutated into that position. A mutation is classified as stabilizing if the change in free energy is ≤-1 kcal/mol, it is classified as destabilizing if the change is ≥1 kcal/mol, and neutral if it falls between these values [[Part:BBa_K4347007:Design#References|<sup>[2]</sup>]]. The changes in free energy for mutations at residues K549, K582 and K584 are shown below:  
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[[File:BBa K4347007 K549.PNG|350px|center|thumb|YASARA position scan results for possible point mutations at residue K549.]][[File:BBa K4347007 K582.PNG|350px|center|thumb|YASARA position scan results for possible point mutations at residue K582.]]
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[[File:BBa K4347007 K549.PNG|200px|left|thumb|YASARA position scan results for possible point mutations at residue K549.]]
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[[File:BBa K4347007 Q584.PNG|350px|center|thumb|YASARA position scan results for possible point mutations at residue Q584.]]
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[[File:BBa K4347007 K582.PNG|200px|center|thumb|YASARA position scan results for possible point mutations at residue K582.]]
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[[File:BBa K4347007 Q584.PNG|200px|right|thumb|YASARA position scan results for possible point mutations at residue Q584.]]
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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 18:23, 12 July 2022

Bst with point mutations for enhanced thermal stability codon optimized for E.coli


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 766
  • 1000
    COMPATIBLE 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.

Protien sequence alignment of Bst (6MU5) and Klentaq (6QV4) in Seaview.

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 K505A, K540A and K542A where each Lysine (K) residue was switched to an Arginine residue (R)[1]. After superimposing and aligning the structures and sequences in Pymol, the corresponding positions in Bst were found:

  • K505 in Klentaq = K549 in Bst
  • K540 in Klentaq = K582 in Bst
  • K542 in Klentaq = Q584 in Bst


To confirm if these residues in Bst were suitable to mutate, the amino acid stability was estimated using a protien simulation software YASARA. A position scan was ran for each residue to measure the change in free energy (Kcal/mol) if the other 19 amino acid residues were mutated into that position. A mutation is classified as stabilizing if the change in free energy is ≤-1 kcal/mol, it is classified as destabilizing if the change is ≥1 kcal/mol, and neutral if it falls between these values [2]. The changes in free energy for mutations at residues K549, K582 and K584 are shown below:

YASARA position scan results for possible point mutations at residue K549.
YASARA position scan results for possible point mutations at residue K582.
YASARA position scan results for possible point mutations at residue Q584.

Making point mutations in yasara - talking about the differences in gibbs free energy.

Design Considerations

Since the goal of these point mutations was to increase thermal stability and not necessarily improve polymerase function, residues in the polymerase active site and fingers domain were avoided.

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

References


1. https://patents.google.com/patent/WO2009155464A2/en

2. https://www.frontiersin.org/articles/10.3389/fbioe.2020.558247/full