The Fourth International Interlaboratory Measurement Study aims to establish a plate reader-based measurement protocol that could be used by various teams all around the world to measure fluorescence (GFP) of 8 standard devices. The primary goal of this collective study is to see how close the numbers are when fluorescence is measured by different iGEM teams around the world using the same protocol.
Methods and Procedures:
Calibration: We used LUDOX-S40 as a single point reference to obtain a conversion factor to transform our absorbance data into a standard OD600 measurement.
Fluorescein Fluorescence Standard Curve: We prepared a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate using the plate reader, thereby generating a standard curve of fluorescence for fluorescein concentration. Using the standard curve, we can then correct our cell reading to an equivalent fluorescein concentration and convert it to a GFP concentration.
We transformed E.coli DH5 α with these following plasmids (provided in the Kit Plate 7 in the 2017 Distribution Kit):
● Positive control
● Negative control
● Test Device 1: J23101+I13504
● Test Device 2: J23106+I13504
● Test Device 3: J23117+I13504
● Test Device 4: J23101.BCD2.E0040.B0015
● Test Device 5: J23106.BCD2.E0040.B0015
● Test Device 6: J23117.BCD2.E0040.B0015
We picked two colonies from each of the plate and inoculate it on 5mL LB medium + Chloramphenicol. We grew these cells overnight for 18 hours at 37°C at 220 rpm. We diluted the cultures to a target OD600 of 0.02 (using the dilution calculator) in 12mL LB + chloramphenicol and incubate them at 37°C and 220 rpm. We took out 500µL samples of the cultures at 0, 2, 4, and 6 hours of incubation and measured their OD values and fluorescence.
The data can be found here