Part:BBa_K615000

E. coli strain for His3-URA3 one-hybrid selection system

This strain is designed to test zinc finger binding using a one-hybrid metabolic selection system. The endogenous genes HisB and PyrF have been knocked out and replaced with the yeast versions His3 and URA3 under the control of a Zif268 binding site. The gene coding for the omega subunit of RNA polymerase (rpoZ) was knocked out by replacing it with a Zeocin cassette, and the strain is designed to be used in conjunction with a zinc finger-omega subunit expression construct.

Usage and Biology

This strain is designed to test zinc finger binding using a one-hybrid selection system. It currently contains the Zif268 binding site, but that can easily be changed using lambda red or MAGE. When Zif268 fused to the omega subunit of RNA polymerase is not present, the genes His3 and URA3 are not transcribed, and thus the cells are incapable of surviving in incomplete media due to their inability to synthesize histidine. If the zinc-finger binding site promoter is leaky in some clones, URA3 can be used as a negative selector in the presence of 5-FOA: URA3 breaks down 5-FOA into a toxin, so only cells without leaky promoters will survive. Zinc finger binding can then be established by growing the strain in incomplete media and looking for a growth phenotype rescue. The sensitivity of the selection can be further manipulated by adding increasing concentrations of 3-AT, a competitive inhibitor of His3. Our results have shown that the strain is able to recognize valid binders even when diluted one in a million and in 10mM 3-AT.

Characterization of BBa_K61500

Sensitivity to hits among background

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 845
  Illegal BglII site found at 965
  Illegal BglII site found at 1478
  Illegal BglII site found at 1538
  Illegal BamHI site found at 1768
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  Illegal NgoMIV site found at 181
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2458
  Illegal SapI site found at 121
  Illegal SapI site found at 331
  Illegal SapI.rc site found at 2305
  

Functional Parameters