Yeast two-hybrid protocol (Proquest System) modified from Ramya

Preparation of competent Yeast and Samll Scale transformation:

  1. Single colony streak from glycerol stock of MaV203 from -80 freezer onto YPAD plate and incubate overnight at 30’C (this plate can be kept at 4’C for unto 4 weeks)
  1. Pick colonies from YPAD plate and dispense in 50 ul ultrapure water. Spread yeast cell suspension onto the center of YPAD plate (about 5cm in diameter). Prepare at least 2 plates for small scale transformation. Incubate overnight at 30’C (between 14 to 16 hours)
  1. Scrape the cells from the plate and disperse in 5 ml of ultrapure water. Inoculate appropriate amount of culture in 100 ml liquid YPAD so that the final OD at 600 nanometer is 0.1.
  1. Incubate at 30’C at 250 rpm until the OD600 is ~0.4 (take about 4 to 5 hours)
  1. Divide the culture into two 50 ml containers and centrifuge at 3000Xg (4650 rpm/SS-34 Rotor) for 5 min at room temperature.
  1. Remove supernatant, dissolve cell pellet in 10 ml 1X LiAc/TE

Stock 10X TE: 100 mM Tris-HCl (pH 7.5), 10 mM EDTA

Stock 10X LiAc: 1 M lithium acetate

  1. Centrifuge at 3000Xg for 5 min at room temperature.
  1. Remove supernatant, dissolve cell pellet in 175 ul 1X LiAc/TE and pool both suspensions together.
  1. Perform transformations

Transformation / Plansmid1 / Plasmid2 / selection / Purpose
1 / pDBLeu / none / SC-Leu / Transformation control
2 / none / none / SC-Leu and
SC-Leu-Trp / Transformation control
3 / pDBLeu / pPC86 / SC-Leu-Trp / Self-activation control
4 / pDBLeu-X / pPC86 / SC-Leu-Trp / Test self-activation
5 / pDBLeu-X / none / SC-Leu / Sequential transformation
6 / pDBLeu / pPC86-Y / SC-Leu-Trp / Test self-activation
7 / pDBLeu-X / pPC86-Y / SC-Leu-Trp / Interaction test X and Y
  1. For each transformation combine 50 ul competent cells with 5 ul ss carrier DNA, 100 ng of plasmid DNA (about 1 ul). Mix gently by pipetting up & down. Add 300 ul 1X LiAc /TE in PEG (stock PEG is 50% PEG-3350) then mix gently.
  1. Place in 30 ‘c waterbath for 30 min
  1. Heat shock for 15 min in 42’c waterbath
  1. Centrifuge at 6000-8000Xg for 20 to 30 seconds
  1. Discard supernatant and dissolve cell pellet in 300 ul ultrapure water.
  1. Plate 100 ul of undiluted transformation and 100 ul of 1:10 diluted transformation onto appropriate selection plates.
  1. Incubate at 30’C for 3-4 days until visible colonies appear.

Assessing self activation & determining lowest 3AT concentration to inhibiting growth:

  1. Preparing yeast control strains A-E onto SC-Leu-Trp plate from glycerol stock.
  1. Make a small streak using toothpick from 4 individual colonies from each transformation and two colonies of yeast control A-E.
  1. Incubate at 30’C for ~18 hours
  1. Replica plate onto 3AT (3-Amino-1,2,4-Triazole) plate with 10, 25, 50, 75, and 100 mM of 3AT using velveteen.
  1. Replica cleaning immediately after replica plate using autoclaved velveteen by applying pressure onto the plate until no traces of yeast cell is visible.
  1. Incubate at 30”c for 24 hours.
  1. Replica cleaning again and incubate at 30’C for 40-44 hours then check the results.

Screening cDNA library for interacting proteins:

  1. prepare MaV203 transformed with pDBLeu-X by small scale transformation
  1. Pick several colonies from the plate and disperse in 50 ul ultrapure water. Spread onto the center of SC-Leu plate (prepare 4-5 plates for library screening).
  1. Incubate overnight at 30’C
  1. Scrape yeast cell from SC-Leu plates and disperse in 10 ml distilled water. Inoculate appropriate amount of culture in 500 ml YPAD liquid to the final OD600 is ~ 0.1
  1. Incubate at 30’C at 250 rpm until OD600 reach 0.4 (4-5 hours)
  1. Divide the culture into two 250 ml containers and centrifuge at 3000Xg for 5 min at room temperature.
  1. Remove supernatant, dissolve cell pellet in 100 ml ultrapure water and centrifuge at 3000Xg for 5 min at room temperature.
  1. Remove supernatant, dissolve cell pellet in 50 ml 1X LiAc/TE and centrifuge at 3000Xg for 5 min at room temperature.
  1. Remove supernatant and resuspend the pellet in 1.25 ml 1X LiAc/TE and pool both suspensions together.
  1. Perform 25 transformations. Combine 2.5 ml competent yeast cell, 125 ul freshly boiled ss carrier DNA and 50 ul of cDNA library. Mix gently and add 1.5 ml of 1X LiAc/TE in PEG solution. Mix gently but thoroughly then aliquot 700 ul into 25 microcentrifuge tubes.
  1. Place in 30 ‘C waterbath for 30 min
  1. Heat shock in 42 ‘C waterbath for 15 min
  1. Centrifuge at 6000-8000Xg for 20 to 30 seconds
  1. Discard supernatant and disperse cell pellet in 400 ul ultrapure water.
  1. Plate the entire 400 ul onto SC-Leu-Trp +3AT (determine level) plates. Incubate at 30’C for 60 hours. Background of small colonies will appear after 60 hours incubation.
  1. Replica cleaning to reduce the visibility of yeast colonies and put back to incubate for 2 more days. After 2 days of incubation only bigger colonies should be selected for further analysis.
  1. Streak transformants that grow on the SC-Leu-Trp +3AT plates for single colony on SC-Leu-Trp plates and incubate at 30’C for 48 hours.
  1. Making master plate by patching two isolated colonies from yeast control strain A-E and four isolated colonies of each potential positive clone.
  1. Incubate at 30’C for 18 hours
  1. Replicate onto selection plates for positive interacter screening:
  1. SC-Leu-Trp +0.2% 5FOA
  2. SC-Leu-Trp-Ura
  3. SC-Leu-Trp-His +3AT
  4. YPAD plate fill with nitro cellulose membrane for X-gal assay
  1. Replica clean all the plates except YPAD plate for X-gal assay (using veletteer). Make sure that you clean them well so you cannot see any visible traces of yeast cell. Incubate at 30’C for 24 hours
  1. For YPAD plate perform X-gal assay (see protocol at the end of this protocol)
  1. Replica cleaning all plates again and incubate for 40 to 44 hours more at 30’c before checking the results.

X-gal assay for interacting protein in yeast:

  1. From the master plate containing yeast control strains A-E and potential clones, replica plate onto a membrane that has been placed on the surface of a YPAD agar plate. Make an asymmetric mark on the membrane to allow for realignment on the master plate.
  1. Incubate at 30’C for 18 to 24 hours.
  1. For each membrane, dissolve 10 mg X-Gal in 100 ul of N,N-dimethyl formamide (DMF). Prepare Z buffer [ 16.1 g Na2HPO4 7H2O (or 8.52 anhydrous), 5.5 g NaHPO4 H2O (or 4.8 g anhydrous), 0.75 g KCl, 0.246 g MgSO4 H2O (or 0.12 anhydrous), dissolved in 1 L autoclaved, distilled waterand adjusted to pH 7.0]. Combine 100 ul X-gal in DMF, 60 ul 2-mercaptoethanol and 10 ml Z buffer.
  1. Stack two round 125-mm Whatman 541 filter papers in a 15-cm Petri dish. Saturate with ~8 ml of the X-gal solution. Remove any air bubbles.
  1. Using forceps, carefully remove the membrane from the surface of the YPAD plate. Completely immerse the membrane in liquid nitrogen for 20-30 s. Place the frozen membrane on top of the soaked Whatman filters colony side up. Remove any air bubbles. Tip the plates slightly and remove excess X-gal solution.
  1. Cover and incubate at 37’C with the colonies facing up. Tip the plates at a slight angle to excess S-gal solution does not accumulate on the membrane. Monitor the appearance of blue color over a 24 hour period. Score final results at 24 hours.