The purpose of the iGEM 2018 Interlab Study was to create an accurate procedure, protocol, and analysis method to measure GFP through a plate reader. The goal of this Interlab Study was to find whether we could reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units instead of relying on optical density. The University of Texas at Austin team did this in the following manner:
- We first did 3 calibrations before doing any experimentation. One was with measuring the OD600, the second was a measuring the ABS600 of monodisperse silica beads with the plate reader, and the third was measuring a dilution series of fluorescein. These all created standard curves for their specific measurements that would be compared to the actual cells.
- After doing the calibrations, we had to make sure we used all the same settings and equipment and procedure for the experiment to make sure the data was consistent.
- We then had 6 devices, and a positive and negative control that were transformed into DH5α cells and plated. We used the transformation methods provided by iGEM.
- After colonies grew, we picked 2 from each plate and made 10 mL overnight cultures with LB and CRB. These were kept at 37°C and 220 rpm.
- We then made 1:10 dilutions of our overnight cultures and measured their Abs600. We then diluted the cultures further to a target Abs600 of 0.02 in a final volume of 12 mL LB. We put them in Eppendorf tubes and at 0 and 6 hours took 500 µL out and measured their Abs600. We also measured the fluorescence of each as well. This was done by pipetting in a 96 well plate the cultures.
- Here we used the 4 overnight cultures from the positive controls the two negative controls, 2 per each one. We then diluted them to a 1:8 dilution and took OD600 measurements.
- The goal was to have an OD600 of 0.1, so we did another dilution if necessary to have all samples read that. We then had 12 starting samples, 6 from either positive or negative control, and three from each test tube.
- We then made 3 different dilutions of each 12 samples, and plated those in a total of 36 plates.
- We let the plates incubate overnight and then counted the number of colonies that grew on each.
Problems That Arose
Some problems we had during the Interlab Study was how the protocol was worded. We often found ourselves emailing iGEM to understand what the protocol was asking us to do. For example when we were supposed to take measurements of Abs600 and the fluorescence of each 500 µL sample from O/N cultures, it was hard to decipher the order which they wanted us to do the calculations. Another place where the wording was puzzling was in the beginning of the colony counting protocol where it says to use overnight cultures from the positive and negative controls. We could assume it is talking about the last experiment, but with these kind of studies we feel the protocol should leave no unertainty. Overall, the biggest issue we found was that the wording was simply unclear. Other than that, Interlab Study went smoothly and we were able to submit our results to iGEM.