View - ResearchGate

296 Serebriiskii et al.systems has shown that both systems identified physiological interactors forcommon a bait; nonidentical interactors were also obtained from the screens inthe different host organisms (8). Thus, the bacterial two-hybrid system can beconsidered to both complement and expand on the yeast two-hybrid system.This chapter provides detailed protocols for using bacterial two-hybrid to analyzeprotein–protein interactions with the HIS3-selection system. It first detailsmethods to construct and characterize a selection strain harboring a “bait”fusion protein. It next describes methods for introducing a library of preyfusionproteins into the selection strain and protocols for performing the selection.Finally, it suggests additional experiments for validating potentialpositives from the selection, including characterization of the candidate’s specificityby testing their interaction with nonrelated bait, and estimating the interactionstrength using LacZ assay. An overview of the various stages, as well asestimated time frames for each step, is given in Fig. 2.2. Materials2.1. Specific Solutions for Media PreparationAmino acid mixture: 17 different amino acids (no His, Met, or Cys). Makethe following six mixtures first; all percentages are (w/v):1. Phe 0.99%, Lys 1.1%, and Arg 2.5% in water.2. Gly 0.2%, Val 0.7%, Ala 0.84%, and Trp 0.41% in water.3. Thr 0.71%, Ser 8.4%, Pro 4.6%, and Asn 0.96% in water.4. Asp 1.04% and Gln 14.6% in 3% hydrochloric acid.5. Glu 18.7% and Tyr 0.36% in water with 4 g NaOH.6. Ile 0.79% and Leu 0.79% in water.Mix together equal volumes of solutions (1–6) and filter-sterilize through a0.2-µm nylon filter. Wrap in foil to protect from light and store at 4°C for up to1 mo. For each 500 mL minimal media, 15 mL of amino acid mixture is required.2.2. Antibiotics and SupplementsSee Table 1 for preparation and concentrations of antibiotic stocks and supplements.Filter all solutions through 0.2-µm nylon filter and store antibioticand isopropyl-beta-D-thiogalactopyranoside (IPTG) at −20°C, 3-AT foil wrappedat +4°C.2.3. Media PreparationStandard size plates (100- or 90 mm) are used throughout this protocol.1. Liquid NM medium: to make 500 mL, mix reagents listed next, and filter-sterilizethrough a 0.2-µm filter: