Since iGEM is an international competition with many teams from all over the world, it is inevitably coping with the problem of unreproducible scientific measurement results. Multiple teams are measuring fluorescence differently, in different units and are evaluating the results non-identically. Therefore, the InterLab Study aims to develop a standardized protocol for consistent fluorescence measurements of GFP.
Previous studies showed that variability in measurements can be reduced by measuring the GFP expression in absolute fluorescence units calibrated against known fluorescent molecule concentrations. Measurements of cell populations still show high variability caused by the number of cells in the sample. Therefore, it is necessary to determine the mean expression level of GFP per cell to eliminate a source of variability.
This year’s question to be answered was: “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?”
Our team followed the standardized procedure guiding us through transformation, inoculation and measurement process. For more information: Fifth International InterLab Measurement Study
Below, this year's results are shown.
Several measurements of LUDOX-CL-X and H2O were analyzed spectrometricly. The results are shown in table 1 and figure 1.
|Corrected Absorbance 600||0.0169|
Defining the correction factor for upcoming experiments allows the conversion of Absorbance 600 from a plate reader into OD600.
Particle Standard Curve
The absorbance of monodisperse silica microspheres in four replicates in a dilution series were measured in standard modes in our Tecan plate reader (infinite 200Pro).
Due to the similarity in size and shape of the microspheres to cells and that there is a known number of particles per volume, the absorbance at 600 nm can be converted to an estimated number of cells for later experiments. The generated standard curve for absorbance of different numbers of particles is shown in figures 2 and 3 in linear and log-scales.
Fluorescein Standard Curve
The fluorescence of fluorescein in four replicates in a dilution series were measured in standard modes in our Tecan plate reader (infinite 200Pro).
The Generated standard curve of fluorescence for different fluorescein concentrations is shown in figures 4 and 5 in linear and log-scales.
Fluorescence and absorbance were measured from six different devices, positive and negative control which are built in pSB1C3 containing chloramphenicol resistance and transformed into E. coli K-12 DH5-alpha. Samples were taken after 0 and 6 hours. ♝
The raw data is shown in table 4 and 5.
µM Fluorescein per OD after 0 and 6 hours
Molecules of Equivalent Fluorescence Level (MEFL) per particle
In figure 6 you can see, the positive control showed similar data to cells transformed with devices 1 and 2 and had the highest fluorescence per OD. Devices 4 and 5 showed negative results, but differed from the negative control, which shows positive data.
In our cell measurements the data varies widely from colony to colony (Tables 4 and 5). Together with the results of all other participating teams the data will hopefully not vary this widely anymore.
The positive control of our performed cell measurement showed similar data to cells transformed with devices 1 and 2. These exhibited the highest fluorescence per OD of all the other devices. In contrast the devices 4 and 5 showed negative results, but differed from the negative control.
During measurements we differed the gains up to 50 in our plate reader settings. We noticed a much higher fluorescence data which could influence the reproducibility from laboratory to laboratory.
As suggestions for improvement we recommend to put all the information needed for performing the InterLab study in one big file and to be more precise regarding the settings for the plate reader. This may help to develop a standardized protocol for consistent fluorescence measurements all around the world.