Chapter 2
Engineering Single Cys2His2 Zinc Finger Domains Using
a Bacterial Cell-Based Two-Hybrid Selection System
Stacey Thibodeau-Beganny, Morgan L. Maeder,
and J. Keith Joung
Abstract
Individual synthetic Cys2His2 zinc finger domains with novel DNA-binding specificities can be identified
from large randomized libraries using selection methodologies such as phage display. We have 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 such libraries. In this chapter we provide updated,
detailed protocols for performing zinc finger selections using the bacterial two-hybrid system.
Key words: Bacterial two-hybrid, zinc finger selection.
1. Introduction
Artificial Cys2His2 zinc finger domains (C2H2 ZFs) with engi-
neered DNA-binding specificities have shown promise for appli-
cations in both biological research and gene therapy (1–7).
Selection-based methods for altering the specificities of individ-
ual C2H2 ZFs typically involve randomization of amino acid
residues in the DNA recognition helix to generate large libraries
followed by use of a selection method to identify variants with
desired DNA-binding specificities. Early studies utilized phage
display for the selection method (8–10) but more recent studies
have demonstrated that a bacterial cell-based two-hybrid (B2H)
system works as well as phage display and may be, in certain cases,
more effective (11, 12). In addition, the B2H system is somewhat
This is a revised version of a protocol that was originally published as Thibodeau-
Beganny S, Joung JK. Engineering Cys2His2 zinc finger domains using a bacterial
cell-based two-hybrid system, Methods Mol Biol. 2007, 408: 317–34.
J.P. Mackay, D.J. Segal (eds.), Engineered Zinc Finger Proteins, Methods in Molecular Biology 649,
DOI 10.1007/978-1-60761-753-2_2, ? Springer Science+Business Media, LLC 2010
31

32 Thibodeau-Beganny, Maeder, and Joung
easier to use than phage display because it directly selects for
proteins in an in vivo, cellular context whereas phage display
requires multiple rounds of in vitro selection.
In this chapter, we describe detailed methods for using
the B2H system to identify individual C2H2 ZFs with desired
DNA-binding specificities from randomized libraries >10
8
in size.
Although we have outlined similar protocols in earlier publica-
tions (11, 12), the overall approach has evolved in our labora-
tory as we have gained experience with the method. The protocol
described in detail here is the most up-to-date method currently
utilized by our laboratory for selections of individual ZFs.
The B2H system, as used in this protocol, links the occur-
rence of a protein–DNA interaction to the activation of two
reporter genes: the yeast HIS3 and the bacterial aadA genes.
These genes code for an enzyme that is essential for histidine syn-
thesis and a gene that confers resistance to streptomycin, respec-
tively. These genes are used to create a “selection strain” harbor-
ing a co-cistronic HIS3/aadA reporter on a single copy episome.
The reporter also contains a target DNA site of interest posi-
tioned just upstream of the weak promoter directing HIS3/aadA
expression. If a zinc finger domain capable of binding the tar-
get DNA site of interest (and fused to a fragment of the yeast
Gal11P protein) is expressed in the selection strain, this leads
to recruitment of RNA polymerase to the weak promoter and
subsequent activated expression ofHIS3/aadA transcription; this
occurs because the Gal11P fragment interacts with a yeast Gal4
protein fragment that is fused to a subunit of the E. coli RNA
Fig. 2.1. Schematic overview of the bacterial two-hybrid selection system. A selection
strain harboring the HIS3/aadA reporter and a kanamycin resistance gene on a single
copy recombinant F’ is transformed with plasmids encoding a hybrid alphaGal4 protein
(harboring chloramphenicol resistance [CAM
R
]) and a ZF domain-Gal11P hybrid protein
(harboring carbenicillin resistance [AMP
R
]). If the ZF domain binds to the target DNA
site present on the F’ reporter (black box), transcription of the HIS3/aadA reporter gene
cistron is activated via recruitment of RNA polymerase to the reporter promoter mediated
by interaction of the Gal11P and Gal4 domains. See text for additional details.