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Dual Bait-Compatible Bacterial Two-Hybrid System 295pTRG series, whereas the KJ1567 strain requires a library based on ampicillinresistant(Ap R ) plasmids. It should be noted that both versions of the system arequite new, reflected by limited publications to date (e.g., ref. 8). When choosingwhich strain to use, one factor to be considered is availability and affordability.The Bacteriomatch II system is predominantly available commercially, whereasa basic set of plasmids described herein is available free of charge from theauthors on request. If a library screen is intended, the investigator should checkwhether or not the appropriate library exists and is affordable, or has to be constructed(see Note 2). Constructing a new library in a pTRG plasmid may beeasier using a commercially available kit available from Stratagene, La Jolla,CA. Conversely, pAC-UV5-αLP encodes an f1 phage origin, so a library constructedin this vector can easily be converted into a library of infectious transducingphage, and subsequently introduced into selection strain cells by simplephage infection. This may permit the reproducible plating of more than 10 8 librarymembers on a single selection plate (15).Besides availability/affordability considerations, both strains should producecomparable results and should be equally well suited to test pair-wiseinteractions between the targeted proteins. Throughout this protocol, the use ofthe KJ1567 strain is described; at the appropriate steps, notes indicate theminimal changes in the protocol to be made to instead use Bacteriomatch IIreporters.1.2.2. The LacZ ReporterIn a two-hybrid screen, the primary advantages of the LacZ reporter are that itsexpression can be measured quantitatively, and thus one can readily assess themagnitude of activation seen with potential positives. Further, activation of LacZby a bait-interactor combination provides independent verification that activationof HIS3 is not owing to a nonspecific mutation (e.g., in the HIS3 promoterregion). The HIS3/aadA double reporter described earlier is more stringent aselection than the single auxotrophic reporter used in yeast. Nevertheless, theAG58(RP28) LacZ reporter strain (Fig. 1C) is available to provide additionaltools for studying the interaction between defined pairs of proteins or to furthercharacterize potential interactors isolated in a library screen using HIS3/aadAselection.1.3. SummaryUsing auxotrophic, drug-resistant, and colorimetric bacterial reporter strainsas described earlier, one can successfully screen large libraries for candidatesthat interact with a protein of interest. This selection system has been successfullyused to isolate candidates of interest from cDNA, randomized and/or mutagenizedlibraries. However, direct comparison of the yeast and bacterial two-hybrid