Team:BioIQS-Barcelona/InterLab
Wet Lab | InterLab
Have a look!Overview
This is the first edition of iGEM BioIQS-Barcelona to take part in the InterLab Study. The InterLab Study is an essential part of the iGEM competition and it is increasingly being taken part by more institutions each year. The goal of this study is to evaluate the variability and reproducibility of the measurements taken among different labs that have different equipment by using the same protocol.
Why?
As a team, we were interested in participating in this study because we were curious in expectation to see whether the measures taken in our lab would be the same that the ones taken in the other side of the globe; and of course, because we love doing science.
Although it was not directly related to our project, this study allowed us not to underestimate the value of replicates and to be more aware of the adequate controls to be used in the experiments.
Interlab Topic
This year, the InterLab study is focused on trying to reduce the lab-to-lab variability in fluorescence measurements by normalizing to colony-forming units (CFU) instead of optical density (OD). Fluorescence measurements are important in synthetic biology due to the use of the Green Fluorescent Protein (GFP) as a standard reporter. One source of variability is the number of cells in the sample. This year’s InterLab study will check if counting CFU helps to reduce this variability better than using OD as a standard. Reducing the variability in fluorescence measurements between laboratories would bring synthetic biology closer to an engineering discipline by facilitating standardization.
All the protocols to be followed were provided by iGEM and can be found here. For cell transformation, electrocompetent cells were used and transformed following the electroporation protocol. Measurements were taken with SpectraMax M2 plate reader with the following settings:
- For Absorbance reads:
- Wavelength: 600nm
- For Fluorescence reads:
- Excitation wavelength: 485 nm
- Emission wavelength: from 500nm to 540nm at 5nm step
- PMT Gain: Automatic
- Flashes per reading: 6
- Shake: 5s before reading
Results
All the replicates gave the same result and thus, we confirm its reproducibility.
It is observed in the graph that the standard curve follows a line at a reasonable reliability. Lower values seem to be less reliable probably due to the sensitivity of the plate reader.
Fluorescein standard curve seems to be correct since it gives a straight line in both linear and log scale.
Overall, raw measures give similar results between replicates although there are some inconsistencies between colonies.
The colonies obtained in this experiment were lower than expected. We believe that this could be caused by a misunderstanding with the protocol so that the cultures were overdiluted prior to spreading them into the Petri plates.
Overall, the experience with the InterLab was satisfactory. The protocol provided was very clear throughout the entire process, but we had a little confusion with the colony forming units part since we had a misunderstanding with the diagram.