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320 Thibodeau-Beganny and JoungFig. 1. Schematic overview of the bacterial two-hybrid selection system A selectionstrain harboring the HIS3/aadA reporter and a kanamycin-resistance (Kan R ) gene ona single copy recombinant F′ is transformed with plasmids encoding a hybrid αGal4protein (harboring chloramphenicol resistance [Cam R ]) and a zinc finger domain–Gal11Phybrid protein (harboring carbenicillin resistance [Amp R ]). If the zinc finger domainbinds to the target DNA site present on the F′ reporter (black box), transcription of theHIS3/aadA reporter gene cistron is activated through recruitment of RNA polymerase tothe reporter promoter mediated by interaction of the Gal11P and Gal4 domains. See textfor additional details.the yeast Gal11P protein) is expressed in the selection strain, this leads torecruitment of RNA polymerase to the weak promoter and subsequent activatedexpression of HIS3/aadA transcription; this occurs because the Gal11P fragmentinteracts with a yeast Gal4 protein fragment that is fused to a subunit of theEscherichia coli RNA polymerase α-subunit (a hybrid protein referred to as theαGal4 protein) (see Fig. 1) (13). Cells harboring such a zinc finger domain canbe identified on medium lacking histidine and containing the antibiotic streptomycin.The stringency of the HIS3 or aadA selections can be increased byadding higher concentrations of 3-AT (a competitive inhibitor of the HIS3enzyme) or streptomycin, respectively, to the medium.In this section, first the methods for engineering “selection strains” harboringtarget DNA sequences of interest are described. Then the methods for using thesestrains to identify zinc finger variants of interest from large randomized librariesare described.