Reliability and repeatability is an important aspect of science, however repeating a specific measurement across different laboratories accurately is a difficult process. This year, the iGEM InterLab aims to tackle this issue by allowing every participating team to take part in the fifth InterLab. Alongside developing a measurement procedure for GFP that can be used reliably across iGEM laboratories, the goal was to determine whether we can reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units instead of OD. This could potentially allow a way of better determining the fluorescence value by normalising against a known amount of cells in order to obtain fluorescence per cell which could be more reliable than obtaining fluorescence against light absorbance at 600nm.
The BioTroopers participated and obtained results for the InterLab study this year by taking the colony forming units (CFUs) approach. Using the protocol provided by iGEM, we obtained the 3 calibration LUDOX-CL-X, particle and fluorescence standard curves.
Once the calibration curves were obtained, the cell measurement biobricks in question were transformed into DH5alpha for the measurements.
The transformed DH5alpha E. coli were then cultured and using the InterLab protocol, the fluorescence of each device was measured as well as its optical density at 600 nm at time 0 hours and at time 6 hours. This will allow us to study the change in fluorescence over time in the devices. The raw data was processed and calibrated using the calibration curves that we had obtained previously to obtain results that can be interpreted.
As seen in figure 3, device 1 had the highest fluorescence reading of all devices, whilst device 3 had a lower fluorescence value than even the negative control. device 4 and 5 still had a significantly higher fluorescence value than the positive control, whilst device 2 matched the positive control, and device 6 was just under the positive control.
Using the InterLab protocol, the CFU analysis procedure was followed through, with the dilutions of the overnight culture of the negative and positive controls were carried out as stated and the diluents were plated overnight. the plates were taken out the next morning and the colonies that grew on dilution 4 results were counted for the CFU calculations.