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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 265Table 1Expected Phenotype for Control InteractionsBait Prey LEU2 LacZ LYS2 GusA ExplanationcI-Ras + Krit + + − − Prey interacts withLexA −LexA-fused bait onlyKrev1 RalGDS + + + + Prey interacts with both baitsRaf1 − − + + Prey interacts with cI-fusedbait onlycI-bait1 + Random − − − − Prey does not interactLexA − bait2specifically with baitAdapted from ref. 23.Each feature marked as positive (+) should be also galactose-dependent, as in Table 2, toprow. Comparing the phenotype of new baits of interest to this set of controls should help to assesswhether an isolated prey interacts with one or both baits.2. Clone the DNA encoding the second protein of interest into the polylinker ofpMW103 (Fig. 3B) to enable synthesis of an in-frame protein fusion to LexA(see Notes 2 and 3). If the main research goal is to use the second bait as alibrary counterselection, rather than to perform a second library screen, a wellcharacterizedLexA-fusion such as pEG202-Krev1 can be used in place of anewly cloned fusion.3. Select a colony of PRT50 (see Note 4) and grow a 5 mL culture in liquid YPDmedium overnight at 30°C in a shaking incubator.4. Dilute into 50–60 mL of YPD liquid medium such that the culture has an opticaldensity (OD) 600nm of approx 0.15. Continue to incubate at 30°C on an orbitalshaker until the culture has reached an OD 600nm of 0.5–0.7. This is sufficient yeastfor 10 transformations.5. Transfer culture to a sterile 50-mL Falcon tube, and centrifuge for 5 min at1000–1500g at room temperature. Gently resuspend the pellet in 5 mL of sterile water.6. Centrifuge the cells for 5 min at 1000–1500g. Pour off the water and resuspendthe yeast pellet in 0.5 mL of TE/0.1 M lithium acetate.7. Aliquot 1 µg of freshly sheared, denatured salmon sperm DNA to 1.5-mL Eppendorftubes.8. Add 50 µL of competent yeast cells from step 6 to each tube. Add the followingcombinations of cI-fusion, LexA-fusion, and reporter plasmids (100–500 ng each):a. pGLS23-Bait1 + pLacGus + pMW103-Bait2 (test for autoactivation).b. pGLS22-Ras + pLacGus + pEG202-Krev1 (negative controls for autoactivation).c. pGLS22-EE 12345L + pLacGus + pEG202-Krit (strong positive controls forautoactivation).9. To each tube, add 300 µL of sterile 40% (w/v) PEG 4000/0.1 M lithium acetate/TEbuffer, pH 7.5. Invert several times to mix (do not vortex). Incubate the tubes at30°C for 30–60 min.10. Add 40 µL of dimethyl sulfoxide to each tube, mix by inversion. Heat shock thetubes by incubating at 42°C (in a heat block) for 10 min.