Part:BBa_K3634019:Design

CcaR-Mediated CcdA Expression System

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

In the initial sequence obtained from Tabor's pJT122 plasmid, an illegal EcoR1 site is found. Uppsala-Sweden iGEM 2011 removed the site by carrying out point mutagenesis of A4C and A6C to obtain the part BBa_K592002. The part sequence was then subject to codon optimisation using the IDT codon optimisation tool to allow optimum expression of ccaR in the E.coli chassis. No new illegal restriction sites were introduced by this step. PcpcG2-172 sequence remained unchanged from pJT119 and the same for ccdA. All other promoters/RBS were chosen from the respective Anderson catalogues from the iGEM registry based off the design of pSR58.6 shown by Ong and Tabor (2018).

Source

ccaR & PcpcG2-172 can be found in the genome of Synechocystis PCC 6803. The sequences can be obtained from BBa_K592002 (Uppsala-Sweden iGEM, 2011 - initially from Tabor's pJT122 plasmid) and BBa_K2012015 (Huazhong Agricultural University iGEM, 2016 - initially from Tabor's pJT119 plasmid) respectively. The part sequence of BBa_K592002 was then fully optimised for our chosen chassis organism, E.coli, using the IDT codon optimisation tool. The ccdA antitoxin is found natively in E.coli with sequence obtained from the pCoo plasmid (NCBI Accession Number: NC_007635.1). This was confirmed with sequence from NCBI Accession Number: NC_019000.

References

IDT Codon Optimisation Tool - https://eu.idtdna.com/CodonOpt

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.

Hirose Y., Shimada T., Narikawa R., Katayama M., Ikeuchi M. 2008. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. PNAS. 105(28): p9528-9533.

Tabor J.J., Levskaya A., Voigt C.A. 2011. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 405(2): p315–324.

Uppsala-Sweden iGEM 2011 - https://parts.igem.org/Part:BBa_K592001

Schmidl S.R., Sheth R.U., Wu A., Tabor J.J. 2014. Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems. ACS Synth Biol. 3: p820-831.

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.

Ong NT., Tabor JJ. 2018. A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range. ChemBioChem. 19: p1255-1258.