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312 Serebriiskii et al.published uses of bacterial two-hybrid systems compared with yeast two-hybridsystems, it still remains a possibility. In any case, performance of the autoactivationassay provides an opportunity to work out the conditions for the subsequent screen.In this assay, a combination of interacting proteins is used as a positive control.5. A phage immunity assay can be run to check for both the stability and DNA-bindingcapability inside a bacterial cell, as discussed in ref. 15.6. Standard molecular biology techniques or alternative cloning strategies (i.e., invivo recombination [23] or GATEWAY cloning [20]) can be used. The bait-encodingcDNA should be cloned in-frame with the λcI DBD, and a translation stop codonshould be created in-frame at the end of the bait sequence. If screens in both bacteriaand yeast are planned using a single bait prepared in pGLS23, bait constructionis subject to additional limitations required for use in the yeast system (see commentaryin Chapter 15).7. An efficient transformation yields approx 10 7 transformants per µg of DNA (whentwo plasmids are being simultaneously transformed). Therefore, this experimentalso provides a good chance to assess transformation efficiency, which will be ofconsiderable importance at the time of library transformation. If only a very smallnumber of colonies are obtained, or colonies are not apparent within 24 h, thisimplies that transformation is very inefficient, and results obtained in characterizationexperiments may not be typical. In this case all solutions, media, and conditionsmust be double-checked or prepared fresh and transformation be repeated. Ifvery few transformants containing the bait plasmid appear (compared with thecontrols), or bacteria expressing the bait protein grow noticeably more poorly thancontrols, or if colony population appears much more heterogeneous than control(e.g., presents a mix of large and small colonies), this would suggest that the baitprotein is somewhat toxic to the E. coli.8. If one is planning to use the Bacteriomatch II strain for screening, a pTRG-basedplasmid (pTRG-RGL) should be used instead of pLibB-Raf. Accordingly, ampicillinin the medium should be replaced with Tc throughout the protocol.9. For simplicity, the same prey plasmid pLibB-Raf (or pTRG-RGL) is usedthroughout this experiment, under the assumption that most bait proteins will notinteract with BRaf (or RGL). At the researcher’s discretion (and if the researcheris, e.g., studying Raf-interacting proteins), these plasmids can be substituted forempty vectors in reaction mix (a).10. At this step, the ability of a cI-fused bait to activate transcription should betested on both HIS3 and aadA reporters, whereas potential activation of the LacZreporter (in AG58[RP28] strain) is only optional. The auxotrophic reporter is themost important for the library screening because it allows direct selection forinteraction phenotype. In addition, there is normally a good correlation betweenactivation of the two reporters, so it is very unlikely that a bait that does not activateHIS3 will significantly activate LacZ. Therefore, if no activation is detected onHis-deficient plates, one should proceed further; if the bait causes growth onHis-deficient plates, it should be modified, regardless of its phenotype with theLacZ reporter.