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Part:BBa_K777002:Design

Designed by: Bianca Genenncher   Group: iGEM12_Goettingen   (2012-09-21)

Tar receptor under the control of constitutive promoter J23104


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1323
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 152


Design Notes

  • Genomic sequence was amplified using the following primers.
    • Primers PCR: Base pairs in caps represent the respective restriction sites followed by either prefix or suffix. Primers were provided by SIGMA.
      • Fwd: Tar for + prefix: 5´ actGAATTCgcggccgctTCTAGAtgattaaccgtatccgcgtagtc 3´
      • Rev: Tar rev + suffix: 5´ tcCTGCAGcggccgctACTAGTcaaaatgtttcccagtttggatc 3´


  • One XbaI site was found at position 420. The restriction site was changed from TCTAGt to CCTAGt via QuikChange PCR.
    • Primers QuikChange (QC): Primers were provided by SIGMA.
      • Fwd: TarQC for: 5´ ACTGATTGATTAcCTAGATTATGGCAATACTGGAG 3´
      • Rev: TarQC rev: 5´ TGCCATAATCTAGgTAATCAATCAGTTCAGTTAAC 3´
        (the uncapitalized letter induces the mutation for removal of the XbaI site)

Source

  • The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B, complete genome (CP000948.1).
  • J23104 information was taken from the parts.igem and physical DNA from the 2012 distribution kit.

References

  • Derr P., Boder E. and Goulian M. Changing the specificity of a bacterial chemoreceptor. J. Mol. Biol. 2006. 355:923–932.
  • Anderson promoters from the 2006 Berkeley group