Part:BBa_K615003

E. coli strain for TolC one-hybrid selection system

This strain is designed to test zinc finger binding by tying the binding event to cell survival, specifically the expression of TolC, a membrane pump that removes toxins from the cell. The omega subunit of RNA polymerase has been knocked out using lambda red to replace the gene with a zeocin cassette, and the strain is designed to be used in conjunction with a zinc finger-omega subunit fusion protein. See below for a summary of features and techniques.

Usage and Biology

This one-hybrid strain uses TolC as both a negative and positive selector. As a membrane pump that removes toxins from the cell, TolC is needed for the cell to survive in media containing SDS. If the zinc finger expressed in the cell successfully binds to the binding site upstream of the TolC gene, TolC will be expressed and the cell will live. If the zinc finger does not bind, TolC will not be expressed and the cell will not be able to pump out the SDS. Additionally, the strain can be grown in media with colicin before zinc fingers are added to the cells. Colicin is a toxin that needs TolC to enter the cell, and so any cells containing constitutively active promoters would die while those without leaky promoters would live.

The strain has been shown to have the proper growth phenotype in SDS. Currently the binding site is for Zif268, but this can be easily changed using lambda red. When Zif268 is diluted into different amounts of negative control zinc fingers, the strain is able to recognize a valid hit in a dilution of no greater than one to one hundred.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 845
  Illegal BglII site found at 965
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal NgoMIV site found at 181
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 121
  Illegal SapI site found at 331
  

Functional Parameters