Characterization of OptoINVRT Systems.

OptoINVRT5 and OptoINVRT7, controlling GFP expression, were transformed in YEZ25 (CENPK.2–1C, gal80-Δ) to make YEZ230–5 (EZ-L400) and YEZ230C (EZ-L437C), respectively. OptoINVRT circuits were characterized in strains YEZ100 (OptoINVRT1), YEZ101 (OptoINVRT2), YEZ102 (OptoINVRT3), YEZ230–5 (OptoINVRT5), and YEZ230C (OptoINVRT7), using YEZ140 and YEZ186 as controls. We monitored GFP expression as previously described,⁵ except in this case we exposed cells to full light, complete darkness, or light pulses of 8 s ON/72 s OFF.

We tested P_GAL1-M and P_GAL1-S in an S288C background, Y202 (S288C, gal80Δ, pdc1Δ, pdc5Δ, and pdc6Δ, containing pJLA121-PDC1⁰²⁰²), with strains YEZ210 (EZ-L437) and YEZ209 (EZ-L436), (Supplementary Table S2), using YEZ94 and YEZ171 as controls. The new promoters were tested in a CEN.PK2–1C background with YEZ229 (EZ-L436) and YEZ230 (EZ-L437), using YEZ140 and YEZ186 as controls (Supplementary Table S2). We monitored GFP expression, as previously described.⁵

To investigate the effects of OptoINVRT circuits on cell growth, we grew cultures of OptoINVRT1 (YEZ100), OptoINVRT2 (YEZ101), OptoINVRT3 (YEZ102), and OptoINVRT7 (YEZ230C), as well as a control strain (YEZ140), overnight in SC-his + 2% glucose under blue light (485 nm). The next day, cultures were diluted in triplicates into fresh media to an OD₆₀₀ of 0.1, and grown under full 485 nm light or in the darkness at 200 rpm and 30 °C. OD₆₀₀ values were taken 10 h after inoculation, then taken every 2–3 h until stationary phase was reached.