To compare performance of strains, we resuspended yeast colonies in 40 μl of 1 M sorbitol. Ten microliters of aliquots of the suspension were dropped on YPD agar plates containing different concentrations of caffeic acid and zeocin in three technical replicas. The plates were then incubated for 15 min at room temperature until the drops dried completely and further incubated for 24 hours at 30°C.
We imaged a “no-substrate” frame on Fusion-Pulse.7 (Vilber Lourmat) with 7-min (in the case of 10 μM caffeic acid) or 1-min exposure (in the case of 100 μM caffeic acid). We then applied 10 μl of caffeic acid [220 mM in 40% dimethyl sulfoxide (DMSO) in phosphate-buffered saline (PBS)] to yeast spots and imaged a time-lapse series on Fusion-Pulse.7 (Vilber Lourmat) every 5 min with 0.5-s exposure. Imaging continued for 6 to 8 hours at room temperature.
We analyzed images using the Fiji ImageJ distribution (version 1.53t) (48 (link)) and custom Python scripts (Python version 3.8). For luminescence quantification, mean values in the region of interest after background subtraction were used. Background subtraction was performed using the following formula: signal = signalraw − (backgroundmean − 3 * backgroundstd).
We imaged a “no-substrate” frame on Fusion-Pulse.7 (Vilber Lourmat) with 7-min (in the case of 10 μM caffeic acid) or 1-min exposure (in the case of 100 μM caffeic acid). We then applied 10 μl of caffeic acid [220 mM in 40% dimethyl sulfoxide (DMSO) in phosphate-buffered saline (PBS)] to yeast spots and imaged a time-lapse series on Fusion-Pulse.7 (Vilber Lourmat) every 5 min with 0.5-s exposure. Imaging continued for 6 to 8 hours at room temperature.
We analyzed images using the Fiji ImageJ distribution (version 1.53t) (48 (link)) and custom Python scripts (Python version 3.8). For luminescence quantification, mean values in the region of interest after background subtraction were used. Background subtraction was performed using the following formula: signal = signalraw − (backgroundmean − 3 * backgroundstd).