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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 2834. Notes1. Some of the control plasmids described in this protocol have inserts cloned in apGLS22 background: pGLS22 is identical to pGLS23 but lacks an EcoRI site inthe polylinker. The pGLS series of plasmids uses the HIS5 gene in the histidinesynthesis pathway: the PRT50 yeast strains used for selection are doubly mutatedin HIS5 and a second gene in this pathway, HIS3, which is used to select thesecond bait (producing the LexA-fusion). It is thus important to simultaneouslyintroduce both baits into the PRT50 yeast (otherwise no yeast will grow on His −dropout plates): practically, this saves about a week by skipping transformationstep used to combine two different baits.2. Standard molecular biology techniques (restriction digests) or alternative cloningstrategies (i.e., in vivo recombination of PCR products ref. 26 or GATEWAYcloning ref. 27) can be used. Whatever approach is used, it is a good idea toinclude a translational stop sequence at the carboxy-terminal end of the baitsequence. It is also important to keep in mind that the assay depends on the abilityof the bait to enter the nucleus, and requires the bait to be a transcriptional nonactivator.Hence, obvious membrane localization motifs, or known transcriptionalADs should be removed in the cloning process. Using two-hybrid systems to findassociating partners for proteins that are normally extracellular, even though suchstrategies have apparently worked in a limited number of cases, should be regardedas extremely high risk.3. A number of modified versions of the plasmid exists, which contains additional restrictionsites, altered reading frames, and alternative antibiotic resistance markers (seehttp://www.fccc.edu/research/labs/golemis/interactiontrapinwork.html for details).4. It is important to use a fresh (thawed from −70°C and streaked to single colony lessthan ~7 d previously) colony and maintain sterile conditions throughout all subsequentprocedures.5. An efficient transformation yields approx 10 3 transformants/µg of DNA (whenthree plasmids are being simultaneously transformed). Therefore, this experimentalso provides a good chance to assess transformation efficiency, which will be ofmuch higher importance by the time of library transformation. If only a very smallnumber of colonies are obtained, or colonies are not apparent within 3–4 d, thisimplies that transformation is for some reason very inefficient, and that resultsobtained in characterization experiments may not be typical. In this case allsolutions/media/conditions must be double-checked or prepared fresh, and transformationbe repeated. In library transformations, sssDNA is often used as carrierDNA to boost transformation frequency. sssDNA must be of very high quality,whether obtained from a commercial vendor or homemade (22) (also see http://www.umanitoba.ca/faculties/medicine/biochem/gietz/Solutions.html). As a separateissue, if very few transformants containing the bait plasmid appear (comparedwith the controls), or yeast expressing the bait protein grow noticeably morepoorly than control yeast, or if colony population appears much more heterogeneousthan control (e.g., presents a mix of large and small colonies), this wouldsuggest that the bait protein is somewhat toxic to the yeast.