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Revision as of 23:51, 16 October 2018
Introduction
IGEM is an international Synthetic Biology competition, which aims to set up standard protocols that work in any laboratory on the planet. To manage this task, Interlab Study was created, inviting all teams to participate by performing the same experiment: making measures of the Green Fluorescent Protein (GFP) to determine the feasible cell population.
In order to determine cell population, scientists usually use spectrophotometry to measure cells culture absorbance in 600 nm, and through it, the optical density (OD) can be obtained. So, theoretically, the higher cells number the higher OD. However, the values of OD obtained are under high variability, because of different calibration and precision between labs equipment, different glassware size, and others lab-to-lab factors. In addition, through this methodology, it isn’t possible to separate dead feasible cells.
In this sense, to respond to all the problems above, the methodology of measuring GFP is another way to determine feasible cells, once the dead ones will not express GFP. Through the GFP measurement methodology, the Measurement Committee wants our help to answer the following question:
‘’Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?‘’
In order to help on the achievement of this goal, Team USP-EEL-Brazil participeted on the Interlab Study, following the standart protocol and sendding the results to iGEM's Measurement committee.
Materials and Methods
For the experiments, we followed the InterLab's Plate Reader and CFU Protocol.
Equipment used for measurements: TECAN INFINITY M200 PRO
Plate used for measurements: Corning® Costar Clear Polystyrene 96-Well Plates
Results
The InterLab study took us longer than we expected. First of all, we have to re-do the protocols, we suspected that the cells we used were not DH5α as it was supposed to be. We had a partner lab from our university to lend it to us, after obtaining a new DH5α strain we could re-do the procedure and then we got positive fluorescence results. At first, we weren't able to notice any difference between the blank sample and the positive control samples. With the different new strain, we could notice de the difference. Also, we had problems transforming the devices from plate 7, they didn't grow or they didn't exhibit fluorescence, so we had to re-do all the transformations using the devices from plate 6. There were a few colonies that didn't grow well, and we believe that didn't help our final results where there are samples with negative fluorescences (less fluorescence than the blank).