Synergy neo2 microplate reader

S1030 cells were transfomred by electroporation with two plasmids: (i) a constituitive N-terminal GFP-FRB fusion, and (ii) an arabionse inducible FKBP-C-terminal GFP fusion. The transformed cells were then plated onto agar plates (15 g/L in LB) with 50 μg/mL spectinomycin, 33 μg/mL chloramphenicol, and 10 mM glucose. Single colonies were then grown to saturation overnight at 37 °C, and then each well of a 96-well deep well plate containing 0.54 mL of LB with antibiotics, 10 mM glucose or 10 mM arabinose, and varying concentrations of rapamycin (0 nM, 1 nM, 10 nM, 0.1 μM, 1 μM, 20 μM, and 100 μM) was innoculated with 60 μL of the overnight culture. After growth with shaking at 37 °C for 3 h, 6 h, and 30 h, 150 μL of each culture was transferred to a 96-well deep well plate. The cultures were centrifuged (10 min, 25 ° C, 2000 rcf) and resuspended in 1 mL of PBS three times before suspending them in 150 μL of PBS and transferring them to a 96-well black wall, clear bottom plate (Costar), and GFP fluorescence (ex. 485/20 nm, em. 516/20 nm) and OD₆₀₀ was measured on a Synergy Neo2 Microplate Reader (BioTek). The data were analyzed by dividing the background-corrected GFP fluorescence values by the background-corrected OD₆₀₀ value. All values were then normalized to the 0 nM rapamycin (sample size n = 5 biological replicates for each condition).