A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions

Sundar Durai, Allen Bosley, Alice Bridgeman Abulencia, Srinivasan Chandrasegaran, Marc Ostermeier

Research output: Contribution to journalArticle

Abstract

We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT). Binding of the zinc finger domain to the target binding site results in a 10-fold increase in chloramphenicol resistance with the CAT reporter and an 8- to 22-fold increase in total cell fluorescence with the GFP reporter. The CAT reporter allows for sequence specific zinc fingers to be isolated in a single selection step whereas the GFP reporter enables quantitative evaluation of libraries using flow cytometry and theoretically allows for both negative and positive selection. Both systems have been used to select for zinc fingers that have affinity for the motif 5′-GGGGCAGAA- 3′ from a library of approximately 2 × 105 variants. The systems have been engineered to report on zinc finger-DNA binding with dissociation constants less than about 1 μM in order to be most applicable for evaluating binding specificity in an in vivo setting.

Original languageEnglish (US)
Pages (from-to)301-311
Number of pages11
JournalCombinatorial Chemistry and High Throughput Screening
Volume9
Issue number4
DOIs
StatePublished - May 1 2006

Keywords

  • Flow cytometry
  • Incremental truncation
  • One-hybrid system
  • Zinc fingers

ASJC Scopus subject areas

  • Drug Discovery
  • Computer Science Applications
  • Organic Chemistry

Fingerprint Dive into the research topics of 'A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions'. Together they form a unique fingerprint.

  • Cite this