Team:Aix-Marseille/InterLab
Interlab
Over the past years, iGEM has advanced the frontiers of science with the biggest interlaboratory study ever done in synthetic biology. These studies established a baseline for replicability of fluorescence measurements and identified likely key sources of error and have now been published as an open-access journal article in PLOS ONE.
The InterLab study has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. GFP was chosen as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein.
The fifth international InterLab study aims to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. This year, we helped in answering the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
Materials and methods
For this study we transformed six different plasmids, along with a negative and positive control into E. coli K-12 DH5-alpha. The plasmids, from here on referred to as devices, contain a constitutive promoter with low, medium or high strengths. The test devices express GFP (as a reporter in the pSB1C3 backbone). A plasmid containing a constitutive promoter and GFP sequence ( BBa_I20270 ) and a plasmid containing a TetR repressible promoter ( BBa_R0040 ) in the pSB1C3 backbone, were used as positive and negative controls. All plasmids were delivered to us in the iGEM 2018 distribution kit.
We made overnight cultures of two colonies of each transformation plate. The following day, the OD600 values of the overnight cultures were measured, diluted to a target OD600 of 0.02 and grown over 6 hours. Afterwards, the absorbance (600 nm) and fluorescence (ex. 485 – em. 520, gain = 50) of the growing cultures were measured in a 96 wells black plate in a plate reader (TECAN M200 Infinite) at 0, 3 and 6 hours.