Yeast Expression Modulation Protocol

Well-isolated single yeast colonies were picked from plates and incubated overnight in synthetic drop-out medium supplemented with 2% glucose at 30 °C. Twelve hours later, the 1-mL cell suspensions were diluted to the concentration 1 × 10⁶ cells per mL—concentration estimated using a Nexcelom Cellometer Vision cell counter (Nexcelom Bioscience)—in fresh medium supplemented with 2% galactose. Triplicate cultures were grown for 48 h in galactose medium with the appropriate concentration of doxycycline (Acros Organics) for each condition to allow gene expression levels to stabilize (32 (link), 33 (link)). Cell density was measured (Nexcelom Cellometer Vision) and resuspended to a concentration 1 × 10⁶ cells per mL every 12 or 24 h depending on the experimental condition to keep them in log growth phase.