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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 275appropriate selective plates based on the purpose of one’s screen. If the secondbait is to be used mainly to increase the specificity of the primary screen, platethe mating on lysine selection plates only (and hold leucine testing for later). Ifone wishes to screen two independent baits, plate on two separate sets of lysineand leucine selection plates. Finally, if one is trying to identify proteins thatinteract with both baits, plating a fraction of the mating on lysine selectionplates, and another fraction on double selection (Leu − Lys − ) plates is suggested.1. On the day the screen is scheduled, thaw an aliquot of the mated transformants.Dilute 100 µL into 10 mL of Gal-Raff/CM Ura − His − Trp − liquid dropout medium,and incubate with shaking at 30°C for 5 h to allow yeast to begin active growth,and to induce the galactose-dependent expression of library proteins. If the frozenculture was not previously titered, plate serial dilutions onto Glu/CM Ura − His −Trp − plates.2. After 5–6 h of incubation, measure the OD 600of the culture. On the assumption thata culture at OD 600nm = 1 contains approx 1 × 10 7 cells/mL, plate 10 6 cells on five100-mm plates with the appropriate auxotrophic selection medium. Plate 10 7 cells oneach of five additional plates with the same medium. Generally, the plating of 10 6cells/plate yields the best result. Although, plating cells at 10 7 cells/plate densitygreatly reduces the number of plates that must be processed, allowing one to morethoroughly saturate the library, it may or may not contribute to cross-feeding betweenyeast, resulting in spurious background growth. Therefore, initially plating matedyeast at two different cell densities and comparing the results is recommended.3. Place the plates in a 30°C incubator for up to 6 d, inspecting cell growth regularly(see Note 21). Depending on the individual bait used, good candidates for positiveinteractors will generally produce LYS + colonies over this time period, with themost common appearance of colonies at days 3–5. LEU + colonies typically format days 2–4 (see Note 22).4. Observe the plates on a daily basis. On the first day that colonies are visible byeye, mark their location on the plate with dots of a given color. Monitor theappearance of the colonies over 5 d. Each day, mark further colonies arising withdifferent colors. At day 4 or 5, streak colonies in a microtiter plate format onto asolid master plate (Glu/CM Ura − His − Trp − ), in which colonies are groupedaccording to the day on which they appeared (see Note 23). If many apparent positivesappear, it might be necessary to pick separate master plates for coloniesobtained on day 2, on day 3, and on day 4, respectively: be sure to include controls(step 5) on these plates.It is important to compare selection plates seeded with lower and higher densities.A “lawn” should not be evident on either class, and the number of coloniesshould be roughly proportional to the seeding density. If no background growthappears on the more densely seeded plates, this strategy is successful as a means ofmore efficiently screening more of the library. If one gets disproportionally morecolonies on the more densely seeded plates (especially sitting on a thin lawn), thisis probably background owing to cross-feeding. In this case, take another aliquot of