Team:Calgary/InterLab
INTERLAB
What is the InterLab Study?
The InterLab study is an international collaborative lab study administered by the iGEM Measurement Committee and was established several years ago to help create reliable and repeatable measurements in synthetic biology. Ultimately, the goal is to develop a robust measurement procedure for green fluorescent protein (GFP), which is one of the most commonly used markers in synthetic biology.
This year, the purpose of the study was to investigate if the lab-to-lab variability of GFP fluorescence measurements could be reduced by normalizing to absolute cell counts or colony-forming units (CFUs).
Methods
The parts listed below are from the 2018 iGEM Distribution Kit and were transformed into chemically competent E. coli DH5-α cells and were used for all cell measurements.
Normalizing fluorescence measurements to absolute cell counts:
E. coli cell concentrations were approximated by comparing the cell absorbance measurements to silica bead absorbance measurements. These silica beads are the same shape and size as an E. coli cell, therefore they scatter and absorb light in a similar way. Given that the bead concentrations were known, each cell absorbance measurement could be converted to a standard "equivalent concentration of beads".
Normalizing to colony-forming units (CFUs):
The number of CFUs grown after liquid media is poured onto a plate is an indication of the number of live cells that were in that media. Using this method with E. coli cells, a conversion factor from absorbance to CFU was computed.
The methods and protocols used for this study were distributed by the iGEM Measurement Committee and can be found here.
Results
Calibration
Before we began cell measurements, standard curves were created from the absorbance readings and fluorescent measurements of silica bead (particle) and fluorescein serial dilutions. This was done to standardize our absorbance readings and fluorescent measurements.
Figure 1. Particle standard curve
Figure 2. Log particle standard curve
Figure 3. Fluorescein standard curve
Figure 4. Log fluorescein standard curve
Cell Measurements
Figure 5. Net Abs600 of each device
Figure 6. Net fluorescein a.u. of each device
Figure 7. uM fluorescein / OD. This graph accounts for the amount of GFP produced and the number of cells present.
Figure 8. MEFL / Particle. This graph accounts for the measure of fluorescence per particle/single cell.