Plasmid

Part:BBa_K617001:Design

Designed by: Andreas Haldimann, Barry Wanner   Group: iGEM11_UIUC-Illinois   (2011-09-27)

pINT-ts


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 878
    Illegal SpeI site found at 3313
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 878
    Illegal NheI site found at 2183
    Illegal SpeI site found at 3313
    Illegal NotI site found at 3632
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 878
    Illegal BamHI site found at 1939
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 878
    Illegal SpeI site found at 3313
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 878
    Illegal SpeI site found at 3313
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4278


Design Notes

pINT-ts is a temperature sensitive CRIM helper plasmid required for integration. It was not designed by our iGEM group, but is part of the CRIM system (Haldimann, Wanner 2001) and is required for the integration of BBa_K617000 into the Lambda attB site of the E. coli chromosome.

Since pINT-ts is temperature sensitive it most be propagated at 30 degrees Celsius. The plasmid has three ORFs: cI repressor, lambda integrase, and the ampicillin resistance gene bla. Lambda integrase is under a temperature sensitive promoter. Thus, when a culture containing this plasmid is moved from 30 to 37 degrees the plasmid expresses lambda integrase transiently and is then cured (eliminated).

A full description of how to use this plasmid is described by Haldimann and Wanner (2001).

Source

Plasmid was orginally designed by Haldimann and Wanner (2001) at Purdue University.

References

1. Haldimann, A., Wanner, B. L. 2001. Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria. Journal of Bacteriology. 183:21 6384-6393. 2. Metcalf, W. W., Weihong, J., Wanner, B. L. 1994. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kϒ origin plasmids at different copy numbers. Gene. 138: 1-7.