Protocol

Gene Function Analysis

Volume 408 of the series Methods in Molecular Biology™ pp 317-334

Engineering Cys2His2 Zinc Finger Domains Using a Bacterial Cell-Based Two-Hybrid Selection System

  • Stacey Thibodeau-BegannyAffiliated withMassachusetts General Hospital
  • , J. Keith JoungAffiliated withMassachusetts General Hospital

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Abstract

Synthetic Cys2His2 zinc finger domains with novel DNA-binding specificities can be identified from large randomized libraries using selection methodologies such as phage display. It has been previously demonstrated that a bacterial cell-based two-hybrid system is at least as effective as phage display for selecting zinc fingers with desired specificities from these libraries. In this chapter the authors provide updated and detailed protocols for performing zinc finger selections using the bacterial two-hybrid system.

Key Words

Artificial transcription factor bacterial two-hybrid Cys2His2 zinc finger zinc finger nuclease gene therapy gene targeting protein engineering synthetic biology