Figure 2. Large-Scale Secondary Screens to Retest Candidates.

(A) Schematic overview and summary of large-scale testing approach to verify screen hits.

(B) Construct specificity in candidates with increased toxicity. Candidate deletion strains that showed increased toxicity in the screen were re-arrayed in duplicate columns in 96-well plates. One column was transformed with the construct coding for galactose-inducible PD-YFP and the second column was transformed with a plasmid coding for galactose-inducible YFP. To induce overexpression of YFP or PD-YFP, cells were pinned onto galactose (inducing) medium and onto glucose (non-inducing) medium as a control. Effects in deletion strains were then compared to the parental BY4741 strain (BYwt) of the deletion library (YGDS).

(C) Strategy to retest suppressors of toxicity. We used the strategy of SGA [45] to combine two copies of the galactose-inducible PD-YFP with our candidate deletions that suppressed toxicity. The donor strain ([RNQ ⁺]) was mated to the candidate gene deletion strains by pinning onto plates containing the appropriate complete synthetic medium (SD). After overnight incubation, we replica-plated them four times onto medium selective for diploids. Diploids were then sporulated in liquid medium in 96-well plates at 23 °C for 6 d. Sporulation cultures were pinned and replica-plated three times onto selection plates for spores that combine the desired features. Overexpression of PD-YFP was achieved by spotting onto galactose medium, or glucose-based medium as a control.