Part:BBa_K615004

Zif268 binding site-His3-URA3 construct for one-hybrid selection

This is one of the components necessary for a metabolic His3-URA3 one-hybrid selection system to test for zinc finger binding. When used in conjunction with a ∆HisB∆pyrF∆rpoZ E. coli strain and a zinc finger-omega subunit of RNA polymerase fusion protein, this part ties zinc finger binding to cell survival. As developed by Meng et al (2005), His3 provides a positive selective force while URA3 provides a negative selective force. Without His3 the cell is unable to synthesize histidine, so growth in incomplete media is only possible if the proper zinc finger-omega subunit protein is present to recruit RNA polymerase and initiate His3 expression. Adding 3-AT, a competitive inhibitor of His3, can be used to measure the strength of the zinc finger binding interaction. To prevent leaky promoters from skewing the results, cells can be grown in media containing 5-FOA, which URA3 breaks down into a toxin. If the promoter has a mutation making it constitutively on, URA3 will be expressed and the cell will not survive.

References: Xiangdong Meng, Michael H Brodsky, Scot A Wolfe. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. (2005). Nature Biotechnology, 23(8): 988-994.

Usage

The ZFB-His3-URA3 biobrick could be used both for plasmid- and genome-based selection. After combining the construct with an antibiotic cassette, it can be easily inserted onto the genome using lambda red, and the resulting selection system has been shown to be highly sensitive (see part BBa_K615000 and the [http://2011.igem.org/Team:Harvard/Results/One-Hybrid_Selection Harvard iGEM 2011 One-Hybrid Selection page]). The plasmid version (which was also used by Meng and others) also works successfully: when the proper zinc fingers are present, the growth phenotype is rescued completely in incomplete media (see graph below).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 15
  Illegal BglII site found at 528
  Illegal BglII site found at 588
  Illegal BamHI site found at 818
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1508
  Illegal SapI.rc site found at 1355