Difference between revisions of "Part:BBa K4477012:Design"

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===Design Notes===
 
===Design Notes===
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Amino acid sequences were reverse transcribed and codon optimized for expression in E. coli B (the parent strain of SHuffle). In addition, further codon optimization was used to remove illegal restriction sites from the construct.
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Amino acid sequences were reverse transcribed and codon optimized for expression in <em>E. coli</em> 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.
  
−
A two-way terminator was used so that the KanR gene in our intended vector -- which is encoded on the strand opposite our insert -- would not be read backward into the insert and interfere with transcription of our antibody of interest.
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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 IK17 scFv 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.
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 +
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===
 
===Source===

Latest revision as of 13:59, 12 October 2022


IK17 (anti-oxLDL) scFv - complete expression cassette


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 576
  • 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 IK17 scFv 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.jacc.org/doi/full/10.1016/j.jacc.2011.07.017
2. https://pubmed.ncbi.nlm.nih.gov/14644097/

References

3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230644/