Figure 2

Fidelity and selection of replica plated H, E(P) and E(M) colonies secreting/over-secreting HSA and EAP as detected by the immuno-chromogenic assay. (Figure 2a) Concordance of colonies on inductive, selective and rich media. H (400 colonies/88 mm plate) were seeded on BMGYA plates (30°C, 48 hours), sequentially replicated onto (Figure 2a1) BMMYA-NC, (2a2) MDA and (2a3) BMGYA plates and incubated (30°C, 24 hours). (Figure 2a) The filter was lifted and immuno-chromogenically stained for exported HSA. Numbered white arrows (Figure 2a2, 2a3: 2–6) and open white boxes (Figure 2a2, 2a3: box 1) are matched to black arrows (Figure 2a1: 2–6) and box (Figure 2a1: box 1). (b) Selection of α-sec-EAP over-secretor mutants is independent of colony size and seeded strain on nitrocellulose membranes. Primary Screens (Figure 2b1) 400 colony forming units of mutagenized E(P) cells were seeded per 88 mm BMGYA plate and incubated (30°C, 24–36 hours). Colonies were sequentially replicated onto BMMYA-NC plates and BMGYA plates and incubated (30°C, 24 hours and 4°C, overnight). Membrane immobilized proteins were immuno-chromogenically stained for α-sec-EAP activity. Secondary Screens (Figure 2b2): Over-secretor candidates from the primary screen were identified by their intense immuno-chromogenic stain above host cell phosphatase as reference for α-sec-EAP activity. Seventy-three candidate colonies from the primary screen and non-mutagenized E(P) colonies as controls, streaked onto BMGYA plates placed on a 48 square grid and incubated (30°C, 20 hours). Colonies were replicated onto BMMYA-NC, MDA and BMGYA plates and incubated (30°C for 48, 24 and 24 hours, respectively). Immuno-chromogenicaly stained membranes, from BMMYA-NC plates confirmed elevated EAP staining by several of the candidate over-secretors relative to the P(E) strain. Three of these mutant E(M) candidates #9, #M32, #44 (Figure 2b2), were re-purified by streaking onto BMGYA plates, then sequentially replicated onto BMMYA-NC, MDA, and BMGYA followed by immuno-chromogenic staining. Purified single candidate colonies (2–4 replicates) (Figure 2b3), #9, #32 and #44) were inoculated for confirmation as over-secretors by Western Blot and Direct Staining with Gel Code Blue (GCB) staining of electrophoretically resolved proteins.