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-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 agrCII and agrA can be  
+
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.
 
yielded by using the NgoMIV or AgeI restriction sites with appropiate restriction sites in the pre- or suffix.
  
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agrA-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcATGAAAATTTTCATTTGCGAAGACG
 
agrA-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcATGAAAATTTTCATTTGCGAAGACG
 +
 
agrA-rev: gatcctgcagcggccgctactagtattaaccggtTATTTTTTTAACGTTTCTCACCG
 
agrA-rev: gatcctgcagcggccgctactagtattaaccggtTATTTTTTTAACGTTTCTCACCG
 +
 
agrC-II-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcTTGATTCCAACTTTTTCATCTATC
 
agrC-II-fwd: gatcgaattccgcggccgcttctagataaggaggagccggcTTGATTCCAACTTTTTCATCTATC
 +
 
agrC-II-rev: gatcctgcagcggccgctactagtattaaccggtGTTGTTAATAATTTCAACTTTTTGAATAAAG
 
agrC-II-rev: gatcctgcagcggccgctactagtattaaccggtGTTGTTAATAATTTCAACTTTTTGAATAAAG
  
−
The two intermediated parts agrA and agrC-II were then fused by using the restriction sites EcoRI and SpeI for agrA, as well as XbaI and PstI for agrC-II, and ligation into EcoRI and PstI cut pSB1C3, thus creating the composite part agrC-IIA.
+
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.
 
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)