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
) or filter paper
- Each DB-X fusion is tested for autoactivation capability on -His +3AT medium. Those that test positive for autoactivation are eliminated from further screening.
- 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.
- 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.
- 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.
- 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.
- Full-length clones based on selected ISTs are prepared as DB and AD fusions
for retesting according the binary Y2H described above.