Coding

Part:BBa_K3634011:Design

Designed by: Laurence Seeley   Group: iGEM20_St_Andrews   (2020-08-06)
Revision as of 10:58, 6 August 2020 by Ls268 (Talk | contribs) (→‎References)

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ccdB (BsaI Removed)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The ccdB sequence was taken from BBa_K145151 and found to have an illegal BsaI restriction site at 222bp. The appropriate alteration was made to make the part RFC[1000] compatible: c219g (GTC (Val) to GTG (Val)). As native to E.coli, no codon optimisation step was required.

Source

The ccdB toxin is found natively in E.coli and the sequence specified in BBa_K145151 by KULeuven iGEM (2008). This sequence was confirmed against pAG415 GPD-EGFP and pCMV SPORT6ccdB plasmids and in silico site-directed mutagenesis removed a BsaI site so that the part was RFC[1000] standard.

References

Bernard P., Couturier M. 1992. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J. Mol. Biol. 226: p735–745.

KULeuven iGEM 2008 - https://parts.igem.org/Part:BBa_K145151

Vandervelde A., Drobnak I., Hadži S., Sterckx Y.GJ., Welte T., Greve. H.D., Charlier D., Efremov R., Loris R., Lah J. 2017. Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon. NAR. 45(6): p2937-2950. DOI: 10.1093/nar/gkx108