PBIP > Screening

Overview of Y2H binary screen

MaV103 (Mat a) and MaV203 (Mat a) (for -His, -Ura selection) or Y8800 (Mat a) and Y8930 (Mat a) (for -His, -Ade selection) yeast cells are transformed with DBdest and ADdest vectors, respectively, containing the DB and AD domains in fusion with members of the poplar biomass ORFeome (DB-X, AD-Y). Transformants are grown in the absence of leucine (-Leu, for DB-X) or tryptophan (-Trp, for AD-Y) (Walhout and Vidal, 2001). Yeast colony PCRs are performed using primers for vector sequences flanking the inserted gene to identify single colonies for the binary screen. Fresh DB-X and AD-Y yeast cells are cultured in liquid YPD overnight, then mixed by ratio of 1:1:8 (DB:AD:YPD) into 96-well plates and allowed to mate for 20 hours. Mated cells are spotted onto -Leu -Trp selective plates, grown for 2 days, cloth-cleaned (with velveteen), then grown for another 2 days. Cells from each mating event are resuspended in liquid -Leu -Trp and spotted onto -Leu -Trp (LacZ assay), -Leu -Trp -His +3AT and -Leu -Trp -Ura or -Ade selective plates for 2 days, after which plates are cloth-cleaned and allowed to re-grow for 2 to 4 days before scoring for reporter activation. As an alternative to cleaning with velveteen, concentrations of mated cells may be adjusted according to OD at A600, spotted in a dilution series (10x and 100x diluted) and growth monitored on selective plates for 2 to 4 days. For LacZ reporter activity LacZ is assayed by colorimetric detection of b-galactosidase according to the X-gal agarose overlay method developed by Herskowitz lab (http://biochemistry.ucsf.edu/labs/herskowitz/xgalagar.html) or filter paper overlay.

Overview of Y2H cDNA library screen

  1. Each DB-X fusion is tested for autoactivation capability on -His +3AT medium. Those that test positive for autoactivation are eliminated from further screening.
  2. Bait proteins (DB-X) and the AD fusion prey library (AD-prey) are co-transformed into yeast (Y8800) and plated on -His, +3AT medium from which obvious fast-growing colonies are selected.
  3. Putative positives are retested for activity with -His +3AT; -Ade, using dilutions as described for binary screens for 2 to 4 days before scoring for reporter activation.
  4. To eliminate multiple plasmids from putative positives, colonies that activate the His and Ade reporter are streaked on -His +3AT plates and single colonies are selected for re-streaking. This process is repeated at least once, or until colony PCR results in amplification of a single band from the AD-prey plasmid.
  5. Single-band PCR products are sequenced to identify the putative interactor and determine whether the sequence is in frame with the AD domain. In-frame sequences are designated as interaction sequence tags (ISTs) and posted on the project website.
  6. Full-length clones based on selected ISTs are prepared as DB and AD fusions for retesting according the binary Y2H described above.