Difference between revisions of "Part:BBa K1351036:Design"
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===Design Notes===
 
===Design Notes===
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This part contains the native sequences of agrC and agrA from ''Staphylococcus aureus'' N315.
+
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===
 
===Source===
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agrA-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcATGAAAATTTTCATTTGCGAAGACG
 
agrA-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcATGAAAATTTTCATTTGCGAAGACG
 +
 
agrA-rev: gatcctgcagcggccgctactagtattaaccggtTATTTTTTTAACGTTTCTCACCG
 
agrA-rev: gatcctgcagcggccgctactagtattaaccggtTATTTTTTTAACGTTTCTCACCG
−
agrC-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcTTGATTCCAACTTTTTCATCTATC
 
−
agrC-rev: gatcctgcagcggccgctactagtattaaccggtGTTGTTAATAATTTCAACTTTTTGAATAAAG
 
  
−
The two intermediated parts agrA and agrC were then fused by using the restriction sites EcoRI and SpeI for agrA, as well as XbaI and PstI for agrC, thus creating the composite part agrCA.
+
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===
 
===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)

Latest revision as of 16:17, 17 October 2014

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)