Pyronic Sensor Expression in E. coli

The four variants of the pyruvate sensor were transformed into competent E. coli BL21 (DE3). A single colony was inoculated into 100 ml of LB medium with 100 mg/ml ampicillin (no IPTG) and shaken in the dark for 2–3 days. Cells were collected by centrifugation at 5000 rpm (4°C) for 10 min and disrupted by sonication (Hielscher Ultrasound Technology) in 5 mL of Tris-HCl buffer pH 8.0. A cell-free extract was obtained by centrifugation at 10,000 rpm (4°C) for 1 hour and subsequent filtering of the supernatant (0.45 µm). Proteins were purified using a nickel resin (His Bin® from Novagen) as recommended by the manufacturer. Eluted proteins were incubated overnight at 4°C, quantified using the Biuret method and stored at −20°C in 20% glycerol. The variant that showed the largest change in fluorescence ratio, which we named Pyronic (Pyruvate Optical Nano-Indicator from CECs), was cloned into pcDNA3.1(-) for expression in eukaryotic cells using the restriction sites BamHI and HindIII.