Part:BBa_K1351036:Design

Quorum sensing two component system (AIP-II sensing histidine kinase agrC, response regulator agrA)

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 1139
  Illegal AgeI site found at 1114
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 475
  

Design Notes

This part contains the native sequences of agrC-II and agrA from Staphylococcus aureus N315. Since these two parts are forming a two component system, this is actually a composite part. The basic parts agrC-II and agrA can be yielded by using the NgoMIV or AgeI restriction sites with appropiate restriction sites in the pre- or suffix.

Source

This part is derived from Staphylococcus aureus N315 gDNA by PCR using primers with modified RFC25 prefix and suffix:

agrA-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcATGAAAATTTTCATTTGCGAAGACG

agrA-rev: gatcctgcagcggccgctactagtattaaccggtTATTTTTTTAACGTTTCTCACCG

agrC-II-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcTTGATTCCAACTTTTTCATCTATC

agrC-II-rev: gatcctgcagcggccgctactagtattaaccggtGTTGTTAATAATTTCAACTTTTTGAATAAAG

The two intermediated parts agrC-II and agrA were then fused by using the restriction sites EcoRI and SpeI for agrC-II, as well as XbaI and PstI for agrA, and ligation into pSB1C3 (linearized with EcoRI and PstI), thus creating the composite part agrC-IIA.

References

Novick, R. P. (2003). "Autoinduction and signal transduction in the regulation of staphylococcal virulence." Molecular Microbiology 48(6): 1429-1449.

The Staphylococcus aureus N315 gDNA was kindly provided by [http://www.bath.ac.uk/bio-sci/contacts/academics/susanne_gebhard/ Dr. Susanne Gebhard] (University of Bath)