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Dual Bait-Compatible Bacterial Two-Hybrid System 307Fig. 5. Detection of bait expression by Western blot. Transformation (lane 1),cI–HRas fusion (pBaitC-Ras); Transformations (lanes 2–4), various leucine zipperfusions being evaluated for expression level (marked with * ); transformation (lane 5),control pBaitC-zip; M, marker lane with the approximate size in kilodaltons. Two independenttransformants are assayed for each transformation. Equivalent quantities oftotal lysate for each bacterial culture are loaded in each lane on the gel, and the subsequentmembrane immunoblotted with α-cI primary antibody. Note that different baitsare expressed at different levels.Following plating of the transformation on selective plates, potential candidatesappear as colonies over the following 24–48 h. These colonies are subjectedto an initial confirmatory test (see Subheading 3.2.2.) that looks not onlyfor increased expression of the primary selectable marker HIS3, but also of theaadA secondary reporter (see Note 15). Prey candidates that pass this initial testare carried through to additional testing in Subheading 3.3.3.2.1. Introducing the Library into Selection Strains,and Performing the Selection1. Prepare highly competent KJ1567 cells by a standard high-efficiency protocol.Alternatively, use manufacturer’s instructions to render the Bacteriomatch II strainhighly competent. Aim for a transformation frequency of more than 10 8 . Transform50–100 µL aliquot of bacteria with a mixture of library and bait plasmid (50 ngeach). At the same time, perform a parallel transformation with pBaitC-YFG andcontrol library plasmid: carrying yeast transformed with these two plasmidsthroughout the subsequent steps provides a negative control for the library screen.2. After the transformation, bacteria typically recover 90–120 min in 1 mL of richmedium broth. After recovery, transfer the culture to sterile tubes and spin in amicrocentrifuge at 2000g for 10 min (room temperature). Remove the medium and