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Dual Bait-Compatible Bacterial Two-Hybrid System 3113.3.2. Additional Confirmations of Positive Interactions,and Specificity Test for Positive Candidates1. A strongly recommended option is to use the AG58(RP28) strain to further characterizepotential interactors isolated in the library screen based on the ability ofinteracting proteins to activate LacZ activity. For this purpose, transform thedesired bait/prey combinations, including controls, in the AG58(RP28) strainessentially as described in Subheading 3.2.1., and then perform quantitativeβ-galactosidase assays (18,19).2. One additional test that may be performed before sequencing the prey-encodingplasmids is to check that the candidate preys interact specifically with the baitused for their selection in the two-hybrid assay. This is accomplished by testingwhether or not the prey can activate the weak promoter reporter in the absence ofthe original bait fusion protein.a. Use 1 µL of purified prey plasmid DNA along with the bait plasmid to cotransformreporter strain cells. In parallel, cotransform the candidate prey plasmidwith the pBaitC-zip and/or pBaitC-Ras control baits (see Notes 21 and 22).Plate transform cells on NM_AC as described in Subheading 3.3.1., step 5and characterize six independent colonies for each transformation as describedin Subheading 3.1.1.b. Analyze growth on the plates. Candidates that demonstrate a specific interactionphenotype on selective medium should certainly have their insertssequenced.5. Notes1. A number of modified versions of the plasmids and bacterial strains exist. Theseinclude a GATEWAY-ready (20,21) pBR-UV5-α(LP) prey vector, and also counterselectablesystems (22) for assessing interaction disruption in bacterial-basedinteraction trap systems.2. See the Stratagene website for Bacteriomatch II-compatible libraries in pTRGseriesvectors [http://www.stratagene.com/products/showCategory.aspx?catId=78].In contrast, KJ1567 libraries must be constructed in Ap R vectors such as the pBR-UV5-αLP vector discussed in this chapter.3. A metal replicator gives more precision, but requires more practice for reproducibleuse (inexperienced users often stab holes in agar plates). However, it is also muchmore expensive to purchase than a plastic replicator; it can easily be homemade (seeFig. 4A). If the user is constructing their own, it is important that all of the spokeshave a flat surface, and that spoke ends are level. The replicator should have 48spokes in a 6 × 8 configuration to fit to a standard 90-mm Petri plate (thus, a plasticreplicator from Bel-Art, made to fit 12 × 8 plate, must be cut in half). A metal replicatorcan be sterilized by autoclaving or by alcohol/flaming; a plastic replicator canbe sterilized by autoclaving or by rinsing with alcohol and air-drying.4. So far, no autoactivating eukaryotic baits have been reported for the cI fusion-basedbacterial two-hybrid system. However, because of the still limited number of