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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 273the stack horizontally until all the colonies are in suspension (1–2 min). Using asterile pipet, collect yeast slurry from each plate (by tilting the plates) and pool theresuspended transformants into a sterile 50-mL conical tube.2. Repeat for further sets of five plates of transformants, resulting in a total of up to150 mL of suspension split between three 50-mL tubes (see Note 18).3. Fill each tube containing yeast to the top with sterile TE or water, and vortex/invertto suspend the cells. Spin cells down for 5 min at 1000–1500g at room temperature,and discard the supernatants. Repeat this step. After the second wash, thecumulative pellet volume should be approx 25 mL of cells derived from up toapprox 10 6 transformants.4. Resuspend each pellet in one volume of glycerol solution. Combine the contentsof the three tubes and mix well. Freeze in 0.2–1-mL aliquots at –70°C. (Thesealiquots are stable for more than 1 yr. Refreezing a thawed aliquot results in theloss of viability, and is not advised.)3.3.3. Mating the Bait Strain and the Pretransformed LibraryOnce the bait strain has been made and characterized, and the library strainhas been transformed and frozen in aliquots, the next step is an interactionmating between the bait strain and an aliquot of the pretransformed librarystrain, followed by selection of positive interactors. In parallel, the bait strainis mated with a frozen aliquot of the negative control strain. When matingoccurs, individual haploid cells of the bait strain fuse with individual haploidcells of the library strain to form a diploid yeast strain containing bait–preycombinations.Practically, to mate the two strains, the bait strain and a pretransformedthawed aliquot of library or a control strain are mixed together, and incubatedovernight on rich medium. To select for interactors, the diploids, along unmatedhaploid strains held in reserve as controls, are then recovered from the matingplates, and replated on media on which only diploids can grow (as described inSubheading 3.3.4.). In practice, a few aliquots of the diploid/haploid mixtureare generally frozen in reserve, to allow tittering of mating efficiency, andrepeated platings at various dilutions. Perform the mating with negative controlstrain (generated in Subheading 3.3.1.) at the same time as setting up thelibrary interaction mating. For both matings, use the same techniques, and treatthem identically in the next step (see Subheading 3.3.4.).1. Start a 30-mL Glu/CM Ura − His − liquid culture of the bait strain from the Glu/CMUra − His − master plate prepared in Subheading 3.2., step 5 (see Note 19). Growwith shaking at 30°C to mid- to late-log phase (OD 600nm = 1–2). See step 6 forparallel bait controls.2. Collect the cells by centrifuging at 1000g for 5 min at room temperature.Resuspend the cell pellet in 1 mL of sterile water and transfer to a sterile 1.5-mLmicrofuge tube. This will yield a yeast suspension of about 1 × 10 9 cells/mL.