Team:McMaster/InterLab
Overview
One of the biggest issues with scientific studies is inter-lab reproducibility. This is often due to differences in collecting and processing data. Within synthetic biology, fluorescence measurements are often used as a read-out for experimental data. This year’s interlab study focused on developing and validating a protocol that will yield absolute units for measurements of GFP in a plate reader. The committee aims to discover whether lab-to-lab fluorescence variability can be reduced by normalizing against absolute cell count or CFUs, as opposed to optical density (OD).
The first set of experiments involved converting between absorbance of cells to absorbance of a known concentration of beads. Silica beads modeled after E. coli cells were used to convert between absorbance measurements from a sample’s cell density into an equivalent concentration of beads. This measurement is more universal and comparable between labs.
The second set of experiments involved relating absorbance of a sample to the number of colony forming units (CFU).
By comparing both sets of experiments, the interlab study will be able to determine whether the two methods agree with each other, and whether either method can help reduce lab-to-lab variability in fluorescence measurements.
Methods
The official iGEM interlab protocol can be found here.
Results
Our raw data file that was submitted and accepted by the interlab committee can be downloaded.
OD600 Reference Point
The OD600/Abs600 based on 4 technical replicates was 4.278.
Particle Standard Curve
Fluorescein Standard Curve
The rest of our data can be found in our data Excel file.
Discussion
One problem was getting the standard curves to be perfectly 1:1.
As we were analyzing data, the biggest thing we noticed was that the CFU counts did not match up very well with the cell concentration determined through the silica beads method. Different options for reducing lab-to-lab variability need to be considered in the future.
Also, it would be beneficial if we were provided with relevant journal articles or textbooks to further explain the principles behind the interlab.