2.4. Supernatant Generation for Bacterial Growth Assay and SDS-PAGE

To generate control yeast and bacterial supernatants, overnight cultures were diluted to an optical density of 1 and 100 μL was added to 10 mL of YNB + glucose and grown for 48 h. To generate induced yeast samples, yeast cultures were prepared as before; however, after 24 h a 1 mL suspension of an avian pathogenic E. coli (APEC) overnight culture was diluted to OD 5, centrifuged and resuspended in 100 μL PBS, and then the total volume was added to the growing yeast culture. This APEC-supplemented culture was incubated for a further 24 h. After 48 h in total, cultures were centrifuged and the supernatant filter sterilised (0.22 μm). To assess the supernatant effect on the APEC growth rate, an overnight culture of bacteria was diluted to OD 1 and 100 μL was inoculated in 9 mL SMB + 1 mL of each respective filter-sterilised supernatant. Cultures were incubated at 25 °C with 200 rpm agitation, and the optical density of each culture was tracked.

The supernatants of strains Q1 and F3 were assayed for their effects on bacterial growth rate, as well as via crude protein secretome analysis using SDS-PAGE. Supernatants were analysed from yeasts grown separately for 48 h, as well as cultures where APEC was dosed in after 24 h to elicit an induced response and grown for a further 24 h. For SDS-PAGE analysis, 5 mL of filter-sterilised supernatant was precipitated with methanol, centrifuged and allowed to dry. Proteins were resuspended in 1× NuPAGE LDS sample buffer (ThermoFisher, Oslo, Norway) and run at 200 V for 30 min in a 4 to 12% Bis-Tris mini-gel (ThermoFisher, Oslo, Norway). Gels were then stained with SimplyBlue SafeStain (ThermoFisher, Oslo, Norway) and imaged.