Composite

Part:BBa_K4477013

Designed by: Miranda Khoury   Group: iGEM22_Virginia   (2022-09-06)
Revision as of 02:39, 7 September 2022 by Registry (Talk | contribs)

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McPC603 anti-oxLDL scFv - complete expression cassette

This sequence codes for a short chain variable fragment (scFv) derived from the anti-phosphocholine McPC603 antibody. Specifically, the sequence codes for the following in sequential order: McPC603 variable heavy chain, a (G4S)3 linker sequence, and McPC603 variable light chain. This sequence is one of two antibody sequences designed by Viginia 2022 to bind specifically to oxidized LDL (OxLDL). It is a composite part, complete with a T7 promoter [part number], E. coli-optimized ribosomal binding site (RBS) [part number], coding sequence (CDS) [part number], and terminator [part number]. A strong T7 promoter was included on the 5' end of the sequence, enabling IPTG induction. The RBS was designed using the Salis RBS calcuator for expression in E. coli. The coding sequence is derived from the antibody McPC603 (1), which binds to oxidized phosphocholine on OxLDL. The variable heavy and light chain of McPC603 are connected by a (G4S)3 linker sequence (2). This linker sequence was chosen because it was characterized to enhanced protein expression of recombinant antibodies. Note that the protein resulting from this sequence will only fold correctly in SHuffle E. coli, as there are disulfide bonds present in its structure. As with other Virginia 2022 composite parts, non-illegal restriction sites were included between each part for modular design.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 446
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 752
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None