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Engineering Cys2His2 Zinc Finger Domains 319b. While agar is cooling, mix together the following components in a sterile flask:100 mL 10X M9 salts, 20 mL 20% glucose, 10 mL 20 mM adenine, 30 mL 200Xamino acid mixture (see below), 1 mL 1 M MgSO 4, 1 mL 10 mg/mL thiamine,1 mL 10 mM ZnSO 4, 1 mL 100 mM CaCl 2, and antibiotics, IPTG, and 3-AT asdesired.c. When agar has reached a temperature of approx 65°C, add the above mixtureto the agar, stir well, and pour plates.d. NM/CCKI plates are NM agar plates supplemented with carbenicillin(100 µg/mL), chloramphenicol (30 µg/mL), kanamycin (30 µg/mL), andIPTG (50 µM).10. 200X Amino acid mixture: each of the six solutions below should be made separatelywith ingredients mixed together in the order listed. The six solutions are thenmixed together, filter-sterilized, and stored at 4°C (see Note 1). This yields a 200Xstock containing all amino acids except histidine, cysteine, and methionine.a. Solution I (100 mL): dissolve 0.99 g phenylalanine, 1.10 g lysine, and 2.50 garginine in H 2O.b. Solution II (100 mL): dissolve 0.20 g glycine, 0.70 g valine, 0.84 g alanine,and 0.41 g tryptophan in H 2O.c. Solution III (100 mL): dissolve 0.71 g threonine, 8.40 g serine, 4.60 g proline,and 0.96 g asparagine in H 2O.d. Solution IV (100 mL): add 9.1 mL 36.5% HCl to 80 mL H 2O, dissolve 1.04 gaspartate and 14.60 g glutamine. Adjust final volume to 100 mL with H 2O.e. Solution V (100 mL): dissolve 18.70 g K-glutamate in 80 mL H 2O, dissolve0.36 g tyrosine and 4 g NaOH pellets, and then adjust final volume to 100 mLwith H 2O to 100 mL.f. Solution VI (100 mL): dissolve 0.79 g isoleucine and 0.79 g leucine in H 2O.11. Final concentrations of antibiotics and other additives in plates.a. Carbenicillin (100 µg/mL); stock is 50 mg/mL in ddH 2O.Note: Carbenicillin is used at a final concentration of 50 µg/mL in liquid media.b. Chloramphenicol (30 µg/mL); stock is 30 mg/mL in EtOH.c. Kanamycin (30 µg/mL); stock is 30 mg/mL in ddH 2O.d. Tetracycline (12.5 µg/mL); stock is 12.5 mg/mL in 80% EtOH.e. Sucrose (5%); stock is 50% in ddH 2O.f. IPTG (50 µM); stock is 50 mM in ddH 2O.g. 3-AT (varies: 10–60 mM); stock is 1 M in ddH 2O (see Note 2).h. Streptomycin (varies: 20–80 µg/mL); stock is 100 mg/mL in ddH 2O.3. MethodsThe B2H system, as used in this protocol, links the occurrence of a protein-DNA interaction to the activation of two reporter genes: the yeast HIS3 andthe bacterial aadA genes. To do this, a “selection strain” harboring a cocistronicHIS3/aadA reporter on a single copy episome is constructed. This reporteralso contains a target DNA site of interest positioned just upstream of theweak promoter directing HIS3/aadA expression. If a zinc finger domaincapable of binding the target DNA site of interest (and fused to a fragment of