Fig. 4

Growth profiles and protein secretion of both wildtype and hyperbranching (∆racA) backgrounds obtained from shake flask cultivations after repeated DOX induction. The ∆racA (Tet-on-glaA, ∆glaA, ∆racA; MF22.4) and its parental strain (Tet-on-glaA, ∆glaA; MF19.5) were pre-grown for 18 h as described in Fig. 3; glucoamylase production was then induced with 20 µg/mL DOX, as well as after additional 24 and 48 h; samples were collected 3 h post-induction with DOX to obtain time points 3 h, 24 + 3 h and 48 + 3 h, after which (a) biomass yield (dry weight), (b) total protein secretion and residual glucose (c) were determined as reported in Fig. 3. d, e Glucoamylase secretion was quantified by dot blot analysis using a monoclonal antibody. Each sample was spotted twice (technical replicate) and signal intensities quantified with ImageJ. Lowest row of the dot blot includes a dilution of glucoamylase as standard (0.01–1.00 mg/mL glucoamylase). Note the clonal variance between the four biological replicates, which is a general phenomenon in culture samples taken from shake flask cultivations. We decided to discarded clone 4 from both strains since we observed an inverse trend in comparison with the other three clones with regard to glucoamylase secretion (i.e. for clones 1–3 less extracellular GlaA was observed at time point 48 + 3 h than at 24 + 3 h, whereas for clone 4 the opposite was true). Results are thus calculated from the first three biological replicates. Average signal intensity of the technical duplicates was determined, and values were normalized by biomass yield and total protein secretion before calculating median values and quartiles, and plotting on box plots. Lowest median value (i.e. median for parental strain at 48 + 3 h) was set arbitrarily as 1. Significance values were calculated with 2-tailed (bar charts) or 1-tailed (box plots) t-test with independent variables (*p < 0.05, **p < 0.01)