Publication protocol
"Colonies of transformants were transferred into 0.5 mL of −Ura medium with 0.2 mg/mL adenine sulfate (designated as −Ura + Ade medium) with 2% (w/v) glucose in the 2 mL, 96‐well, round‐bottom, deep‐well plate (Ritter, Germany). The plate was sealed with AeraSealTM sealing film (Excel Scientific, CA) and shaken overnight (20–22 h) at 30°C at 1400 rpm on a Maximizer MBR‐022UP (TAITEC, Tokyo, Japan) plate shaker. The cells were diluted 65‐fold by introduction into 1 mL of −Ura + Ade medium with 0.1% (w/v) glucose and sub‐cultured for 7 h at 30°C with agitation at 1400 rpm. For induction, 2% (w/v) galactose was added and the cells were cultured for a further 22 h.
Cells were harvested by using a swing‐bucket rotor with microplate buckets at 3000g over the course of 30 min. The supernatant was removed by suction. The cell pellet was resuspended in 160 μL of the suspension buffer (50 mM Tris‐HCl, pH 7.5, 5 mM EDTA, 10% (v/v) glycerol, 0.12 M sorbitol, and complete protease inhibitor cocktail). The cell suspension (20 μL) was transferred into a low‐volume, 384‐well black plate (Greiner Bio‐One, Germany), and the whole‐cell GFP fluorescence was measured using the SpectraMax® Gemini plate reader (Molecular Devices, CA) at an emission wavelength of 525 nm using a 515 nm cutoff filter; the sample was excited at 490 nm.
The cells were disrupted on the same deep‐well plate by adding 100 μL of 0.5 mm glass beads or 0.5 mm zirconia–silica beads and shaking at 2500 rpm on a MicroMixer E‐36 (TAITEC, Tokyo, Japan) microplate mixer at 4°C. The suspensions with cell debris were transferred to 8‐strip PCR tubes or a 96‐well PCR plate and stored at −80°C until further use."
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