Coding

Part:BBa_K3843005:Design

Designed by: Anthony Dang   Group: iGEM21_Waterloo   (2021-10-01)
Revision as of 06:35, 1 October 2021 by Anthneedee (Talk | contribs) (Design Notes)


Affinity-improved monomeric streptavidin (mSA2+)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

^^sequence to be updated - computational rational protein design currently in progress

To obtain the affinity-improved sequence, the original protein was first computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Next, the protein's binding affinity to biotin was assessed using molecular dynamics simulations in GROMACS, which returned an energy score for the biotin+mSA2 complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of mSA2. The mutation and evaluation process was repeated once more to obtain a double mutant of mSA2. The final protein was back-translated and codon optimized for E. coli K12. As well, illegal restriction sites for RFC[10] were removed, and prefixes and suffixes for RFC[10] were added.

Source

Improvement of binding affinity of monomeric streptavidin BBa_K1896000. Sequence obtained through computational rational protein design (describe a bit of the rational protein design process here). The original sequence used as a basis for computational rational protein design was not the DNA sequence, but rather the amino acid sequence of monomeric streptavidin (PDB ID: 4JNJ, https://www.ncbi.nlm.nih.gov/Structure/pdb/4JNJ). After applying the desired mutations to 4JNJ, the mutated amino acid sequence was back-translated and codon-optimized.

References