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Engineering Cys2His2 Zinc Finger Domains 3213.1. Selection Strain ConstructionSelection strains are constructed in two steps: In an initial step, a targetDNA site of interest is synthesized and then cloned into a multicopy plasmidreporter vector designed in the lab. In a second step, a portion of this reporterplasmid is recombined to a single copy F′ episome in bacterial strain CSH100and the resulting recombinant F′ is then transferred by conjugation to KJ1C,an F – strain in which one can select for HIS3 and aadA expression (13). Themethod of selection strain construction is similar to one previously describedby Whipple (14) but utilizes a counter-selection step that simplifies identificationof desired double-recombinants (see Fig. 5).3.1.1. Reporter Plasmid Construction1. Cut approx 1 µg of the reporter plasmid pKJ1712 with SapI (NEB, New EnglandBiolabs). pKJ1712 contains two closely positioned SapI sites (see Fig. 2), andtherefore, digestion with SapI releases a small 45-bp fragment.a. 1 µg Stuffer plasmid.b. 5 µL 10X NEB buffer 4.c. 5 µL SapI (2 U/µL).d. Fill to 50 µL with H 2O.e. Incubate at 37°C for 2 h.2. Isolate the 8678-bp pKJ1712 vector backbone on either an agarose or polyacrylamidegel using standard methods (Sambrook and Russell, 2001). Resuspend thefinal purified digested vector in 20 µL of ddH 2O.3. Treat the purified vector with Pfu to create extended overhangs. Cloned Pfu DNApolymerase (Stratagene) has a 3′- to 5′-exonuclease activity, and by providing onlyone nucleotide (dCTP) to the reaction, the enzyme will degrade DNA until it reachesa position that can be filled in with dCTP. At this point, the forward synthesis andreverse exonuclease activities will reach equilibrium, thereby leaving extended overhangsas shown in Fig. 2. Reaction conditions for Pfu treatment are as follows.2 µL 10 mM dCTP.2 µL 10X Cloned Pfu buffer (Stratagene).10 µL pKJ1712 SapI-digested vector.1.2 µL Cloned Pfu (2.5 U/µL).4.8 µL H 2O.72°C for 15 min, 4°C for more than 2 min.4. As illustrated in Fig. 3, design a pair of oligonucleotides, which when annealedtogether will form a double-stranded DNA fragment bearing the target DNA-bindingsite and extended overhangs compatible with the pKJ1712 vector prepared in step 3,Subheading 3.1.1.5. Anneal the target DNA-binding site oligonucleotides together as follows:a. 1 µL Oligo 1 (10 pmol/µL).b. 1 µL Oligo 2 (10 pmol/µL).