As with any discipline, reproducibility is critical for the advancement of synthetic biology. For the first time, our team participated in the InterLab study with the goal of ensuring reproducibility of results.
This year’s InterLab study had the goal of reducing variability in fluorescence measurements by measuring fluorescence relative to colony forming units (CFUs), instead of the traditional method of measuring optical density (OD). Our team participated by utilizing a plate reader to conduct fluorescence and absorbance measurements on the samples.
We followed the plate reader protocol released by the Measurement Committee. To calibrate, we measured the absorbance of the 45% LUDOX CL-X solution as well as a suspension of microspheres and a solution of fluorescein salt. The absorbance and fluorescence were measured on a Molecular Devices SpectraMax M2 plate reader. All measurements made were taken at room temperature, and absorbance measurements were made at 600 nm. The plate reader was equipped with a monochromator, and excitation and emission wavelengths for measuring fluorescence were measured at 485 and 525 nm, respectively.
All of the test devices were prepared using the protocol provided by iGEM and the Measurement Committee. They were grown overnight, and diluted to a standard OD in LB+CAM before measurement began. The cultures were grown for 6 hours in a rocking water bath at 37°C in 50mL centrifuge tubes covered in foil. Separate samples were prepared for growth on LB Agar + CAM plates, and were serially diluted to measure CFUs at 3 different concentrations. Finally, these colony counts were converted to CFU/mL.
Our raw data and results can be seen below. This spreadsheet was submitted for the InterLab study, and the final data was approved.
This year was the first time we had participated in the InterLab study, and we had an overwhelmingly positive experience. We generally found the protocol provided by the committee to be clear and easy to follow. We appreciated the ease of the data submission process, as well as the data entry process. Based on our experience this year, we look forward to participating in the InterLab study in future years!