Transparent flat bottom 96 well plate

Circularly permuted iRFP yielding significant fluorescence over background were transformed into E. coli XL1 Blue using heat shock, and grown on LB-agar plates containing 50 µg/mL kanamycin at 37°C. After overnight growth, three or more colonies derived from each vector were used to inoculate 3 mL LB cultures in 15 mL Falcon tubes containing 50 µg/mL kanamycin. After 16 hour growth at 37°C and 250 rpm, 1 mL of cells were harvested by centrifugation, resuspended in 1 mL LB medium, and used to inoculate a fresh 4 mL LB culture containing 0.5 mM IPTG, 80 µM BV, and 50 µg/mL kanamycin. Cells were grown for 5 hours at the indicated temperatures while shaking at 250 rpm in the dark, washed with 25% glycerol (1 mL), and resuspended in 25% glycerol (1 mL) to a similar density. Aliquots (200 µL) of each resuspended sample were transferred to four wells in transparent flat-bottom 96-well plates (Corning), and whole cell absorbance (600 nm) and fluorescence (λ_ex = 690 nm, λ_em = 700–800 nm) was acquired using a Tecan M1000 plate reader. Emission data was normalized to absorbance in each well, and data reported represent the average of measurements performed on samples derived from three or more colonies. A vector that expresses full-length iRFP (pBAD/His-B-iRFP) and circularized P4 lacking the iRFP gene were used as positive and negative controls, respectively.