Team:USP-EEL-Brazil/InterLab
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 team to participate to complete this goal. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, because labs will not be able to reliably build upon others’ work.
This year, Interlab study became a requisite to reach bronze medal. This sense, every team received a kit with the same materials, with which they were oriented to follow the same protocols. Through the results obtained by each team, the samples will be compared, and then, the community could get trustable info. Analyzing the info, some adjustments will be made in the protocols or even new protocols can be created to improve the synthetic biology standardization.
The Interlab consist in making measures of the Green Fluorescent Protein (GFP) and use the values 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? ‘’
Materials and Methods
In order to make the experiments, we followed the method in the link below: (colocar o link)
The plasmids used are available in the iGEM kit:
Device Part Number Plate Location
Negative control BBa_R0040 Kit Plate 7 Well 2D
Positive control BBa_I20270 Kit Plate 7 Well 2B
Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
Test Device 6 BBa_J364009 Kit Plate 7 Well 2P
Equipment used for measurements: TECAN INFINITY M200 PRO Plate used for measurements: Corning® Costar Clear Polystyrene 96-Well Plates
Results
Interlab
https://2017.igem.org/Team:ColumbiaNYC/InterLab
https://2017.igem.org/Team:NUS_Singapore/InterLab