Intermediate

Part:BBa_K3128016:Design

Designed by: Melodie Dupre   Group: iGEM19_Grenoble-Alpes   (2019-09-18)
Revision as of 20:35, 7 October 2019 by Pinerol (Talk | contribs) (→‎Source)


COMP fused with T25 subpart of Bordetella Pertussis adenylate cyclase under lactose promoter


Euromedex BACTH kit contains the pKNT25 plasmid with a MCS cassette followed by the T25 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T25 subpart. To respect RFC rules, the plasmid has been amplificated by PCR using primers that bind from either site of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the protein in the system. The plasmid was digested by the corresponding enzymes and the protein gene was inserted.

BBa K3128016 design1.png
BBa K3128016 design2.png


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1597
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Source

T18 is from bordetella pertussis
Ompx is from Escherichia coli
pUT18 plasmid from Euromedex BACTH kit was used (containing the T18 subpart and the leucine zipper gene).
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.

References