Coding

Part:BBa_K4477004:Design

Designed by: Miranda Khoury   Group: iGEM22_Virginia   (2022-09-06)
Revision as of 08:14, 12 October 2022 by Mirandakhoury (Talk | contribs) (→‎Design Notes)

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McPC603 anti-oxLDL scFv antibody fragment


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 355
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 661
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Amino acid sequences reverse transcribed and codon optimized for expression in E. coli B (parent strain of SHuffle). Illegal restriction sites removed via codon optimization.

His tag and TEV cleavage sites were included downstream of the start codon but upstream of the antibody coding sequence. The His tag ensured the entire polypeptide could be purified out of a total protein solution using immobilized metal ion affinity chromatography (IMAC), and the TEV cleavage site ensured that TEV could be used to cleave off the His tag once it was no longer needed.

For detailed annotations as to which nucleotides form the variable light, variable heavy, and linker sequence domains, see Team Virginia 2022's Benchling folder here: https://benchling.com/mrkhoury/f_/YR4zZSUb-virginia-igem-2022-dna-constructs/

Source

1. https://www.sciencedirect.com/science/article/pii/S0769262585800581
2. https://pubmed.ncbi.nlm.nih.gov/14644097/

References