Team:Amazonas Brazil/InterLab
InterLab study emerges as a precious guide to help us to develop all Synbio concepts including the major principle for our project: the standardization principle. Interlab is a large-scale interlaboratory study made by iGEM's Measurement Committee to obtain reliable measurement protocols for Green Fluorescent Protein (GFP) aiming that anyone with a plate reader can replicate in their lab! GFP was chosen because it is one of the most used in synthetic biology labs, so it will be easier to measure it around the world. The 2017 InterLab study aims to provide a trustworth plate reader-based measurement protocol suitable throughout the world. So, The question which begs to be answered is:
"How close can the numbers be when fluorescence is measured all around the world?"
Without excitation, our team decided to dive into this great opportunity! The InterLab protocol involved cell growth evaluation by optical density and fluorescence analyses of transformed cells for 0, 2, 4 and 6 hours. Before the cell culture set up and evaluation, we performed the standard methodologies for optical density and fluorescence emission. To obtain a standard OD600 measurement from absorbance data, the LUDOX-S40 ere used as a reference. Unfortunately, our LUDOX tube was frozen. To solve this issue, we contact the AQA-Unesp team that kindly provided one of their extra LUDOX tubes. Furthermore, the fluorescence standard curve were achieved based on dilution series of fluorescein, which as LUDOX-S40 was provides in the Kit Plate in the 2017 Distribution Kit. All of 8 devices: Positive control, Negative control, Test 1 (BBa_J364000), Test 2 (BBa_J364001), Test 3 (BBa_J364002), Test 4 (BBa_J364003), Test 5 (BBa_J364004) and Test 6 (BBa_J364005) were transformed and cultured in LB (Luria Bertani) media containing 25 mg/ml chloramphenicol. Two colonies were picked from each plate and cultured into 5 ml cultures for 18 hours, at 37 ºC using 220 rpm condition. The overnight culture were diluted to OD600 of 0.02 in 12 ml LB medium. The next step was performed cell measurement protocol and fluorescence emission resulting at the end at 4 plates one of each time point: 0, 2, 4 and 6 of growing time. For this experimental groups, we used the same plate reader instrument, a Hidex Chameleon V Multilabel Microplate Reader and sample condition as volume performed at standard protocols described above.
The InterLab experience was an amazing opportunity to empower our freshly Igemers to focus on objectives, standardization and controlled conditions needed in an experimental protocol. Finally, the results discussion was taken to present the importance of reliable and repeatable measurement to validate an biological experiment.
