The main goal in iGEM is to develop a globally open community and collaboration in the synthetic biology field. Therefore, in order to achieve that goal, a standard should be created for all the labs around the globe. The interest of achieving this standard generated the InterLab initiative.
Not all the labs from across the world use the same protocol for their experiments. Thus, attempting to unify the measurement procedures and for the possibility of comparing results from different teams, iGEM invited all the 2017 teams to participate in the InterLab Study.
The participating teams are provided with the same materials and they are requested to follow the same protocols and rules. The results from the teams will be used to build and consolidate a standardized protocol that aims improvement of the synthetic biology standardization and thereby, improve science worldwide.
The chosen methodology by the iGEM's measurement committee was the measurement procedure for green fluorescent protein (GFP). This marker is one of the most used in synthetic biology, so most of the labs should be equipped and prepared to measure it. The GFP measurement procedure is being developed for over the last three years, and this year's goal is to obtain a protocol that anyone with a plate reader can use.
To help with this study, iGEM AQA UNES followed the protocols and we sent iGEM's measurement committee the obtained results.
materials and methods
devices and controls
We were provided with eight plasmids (listed below); six test devices, one positive control and one negative control. All tests and controls have the pSB1C3 plasmid as backbone and as requested by iGEM's protocol, they were all transformed into E. coli DH5α cells, also following iGEM's transformation protocol.
Positive Control (BBa_I20270): well 20B
Negative Control (BBa_R0040): well 20D
Test Device 1 (BBa_J364000): well 20F
Test Device 2 (BBa_J364001): well 20H
Test Device 3 (BBa_J364002): well 20J
Test Device 4 (BBa_J364003): well 20L
Test Device 5 (BBa_J364004): well 20N
Test Device 6 (BBa_J364005): well 20P
plate reader
Tecan Infinite M200 PRO.
OD600 absorbance
Wavelenghts: 600nm
Number of Flashes: 10
Settle time: 0ms
Plate: Costar 96 Flat Bottom Transparent Polysterene
Fluorescence measurements
Mode: Fluorescence Top Reading
Excitation Wavelenght: 485nm
Emission Wavelenght: 535nm
Gain: 52
Number of Flashes: 10
Settle time: 0ms
Z-Position: 16.9mm
For the experiments, we followed the protocols here.
results
0D600 calibration
Fluorescein standard curve
*built by the arithmetic mean of the four replicates
OD600 analysis
Fluorescence reading
*built by the arithmetic mean of the four replicates
our challenges
The InterLab study took us longer than we expected. First of all we have to re-do the protocols, we suspected that 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 exhibits 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).
