Difference between revisions of "Part:BBa K3794004:Design"

 
 
(One intermediate revision by the same user not shown)
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
Mutations ... etc
+
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:
 +
<ul>
 +
<li>K194E</li>
 +
<li>A228K</li>
 +
<li>S274P</li>
 +
<li>N288D</li>
 +
<li>S343N</li>
 +
<li>K654D</li>
 +
<li>R670T</li>
 +
<li>Q781E</li>
 +
</ul>
 +
 
 +
 
 +
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.
  
  
Line 13: Line 33:
 
===Source===
 
===Source===
  
−
Synthesised from IDT. DNA sequence obtained from PDB: 1HN0. Mutations ...
+
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===
 
===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.

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.