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302 Serebriiskii et al.2.7. Miscellaneous1. Sterile glass balls, 3–4 mm, Thomas Scientific (Swedesboro, NJ) 5663L19 orThermo Fisher Scientific, (Waltham, MA) no. 11-312A (autoclave in jar to sterilize).2. Sterile toothpicks (to sterilize, autoclave foil-wrapped toothpicks, or in a jar).3. Insert grid from a rack of pipet tips (Rainin Instrument [Oakland, CA]) RT series,200 µL capacity).4. A metal replicator for the transfer of multiple colonies (e.g., Dankar Scientific[Reading, MA] no. MC48) (see Fig. 5) (see Note 3), or alternatively, a plasticreplicator (Bel-Blotter, Bel-Art Products (Pequannock, NJ) no. 378776-0002 orFisher no. 1371213) (see Note 3).5. Antibody to cI (commercially available from Invitrogen Corp [Carlsbad, CA]),and other reagents for Western blotting.6. 2X Laemmli sample buffer: 0.125 M Tris-HCl, 4% (w/v) sodium dodecyl sulfate,20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, and 0.002% (w/v) bromophenolblue (pH 6.8). Add 2-mercaptoethanol immediately before use.3. Methods3.1. Construction and Characterization of a BaitThe protein to be screened for interactors is fused to the bacteriophage λ repressor(λcI protein) DNA-binding protein. Two tests of this bait fusion protein are thenperformed. First, an activation assay verifies that the bait fusion does not activate thereporter promoter on its own (see Note 4). Second, a Western blot assay establishes theexpression levels and stability of bait fusion protein inside a bacterial cell (see Note 5).For these assays, expression of the bait-λcI fusion is induced by IPTG, and proteinlevels can be increased or decreased by adjusting the IPTG concentration inthe medium. If the expression level of the bait fusion is too low (it cannot bedetected by Western analysis, under circumstances in which the positive control cIfusedprotein is clearly seen in bacterial lysate) or if the bait is degraded (seen asa ladder or smear of less than the expected molecular weight), then it is not appropriatefor use in bacterial two-hybrid screens. In these circumstances, the bait constructneeds to be redesigned (e.g., by truncating the bait to smaller domains).Transcriptional activation of the reporters by the bait (in the absence of anyinteracting partner) is checked by assaying the expression of a HIS3 and/orLacZ reporter gene directed by a weak promoter bearing an upstream λcI-bindingsite. Baits should not activate expression of the HIS3 or LacZ reporter genes inthe absence of the interacting RNAP-prey fusion.3.1.1. Constructing and Transforming a lcI Bait1. Insert DNA encoding the protein of interest (your favorite gene, [YFG]) into thepolylinker of the λcI-encoding expression vector, pBaitC to create an in-framecI-fusion protein (see Note 6). For simplicity, this plasmid will be referred to aspBaitC-YFG in this protocol.