Difference between revisions of "Part:BBa K3794004:Design"
 
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The DNA sequenced derived from this protein sequence was codon optimised for expression in E.coli K12.
 
The DNA sequenced derived from this protein sequence was codon optimised for expression in E.coli K12.
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A stop codon was added at the 3' end of the ChABC CDS to allow for translational termination during protein synthesis.
  
  

Latest revision as of 20:04, 19 October 2021


ChABC CDS


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 NgoMIV site found at 2754
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part's DNA sequence was derived from PDB 1HN0, and had its signal peptide sequence (amino acids 1-24) removed. A 6xHis tag + TEV site with upstream start codon were incorporated to allow for transcriptional initiation and subsequent protein purification via a Ni-NTA column.

Due to ChABC's thermal instability, a series of 8 point mutations derived from "Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability" (Hettiaratchi et al., 2020) were incorporated into the protein sequence.


These mutations are as follows:

  • K194E
  • A228K
  • S274P
  • N288D
  • S343N
  • K654D
  • R670T
  • Q781E


The DNA sequenced derived from this protein sequence was codon optimised for expression in E.coli K12.

A stop codon was added at the 3' end of the ChABC CDS to allow for translational termination during protein synthesis.


Source

Synthesised from IDT. Protein sequence obtained from PDB: 1HN0 and signal peptide removed. Mutations obtained from "Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability" (Hettiaratchi et al., 2020)

References

Hettiaratchi, Marian & O'Meara, Matthew & O'Meara, Teresa & Pickering, Andrew & Letko-Khait, Nitzan & Shoichet, Molly. (2020). Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability. Science Advances. 6. eabc6378. 10.1126/sciadv.abc6378.