Difference between revisions of "Part:BBa K4477013:Design"

Revision as of 13:17, 12 October 2022

McPC603 anti-oxLDL scFv - complete expression cassette

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]

Design Notes

Amino acid sequences were reverse transcribed and codon optimized for expression in E. coli B (the parent strain of the SHuffle we're using). In addition, further codon optimization was used to remove illegal restriction sites from the construct.

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 McPC603 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.

A two-way terminator was used so that any genes in the vector in which this insert will be inserted that happen to be on the strand opposite to the strand encoding the insert will not be read backward into the insert and interfere with transcription of our antibody of interest.

For detailed annotations of the sequence, 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

3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312285/
4. https://www.pnas.org/doi/epdf/10.1073/pnas.71.11.4298

@@ Line 7: / Line 7: @@
 ===Design Notes===
-Amino acid sequences were reverse transcribed and codon optimized for expression in <em>E. coli B</em? (the parent strain of the SHuffle we're using). In addition, further codon optimization was used to remove illegal restriction sites from the construct.
+Amino acid sequences were reverse transcribed and codon optimized for expression in <em>E. coli B</em> (the parent strain of the SHuffle we're using). In addition, further codon optimization was used to remove illegal restriction sites from the construct.
 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 McPC603 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.