Team:Wageningen UR/InterLab
InterLab
The iGEM InterLab study was set up 4 years ago to develop a robust measurement protocol for Green Fluorescent Protein (GFP), which will allow for accurate comparison of results between labs. Creating such a robust protocol requires fluorescence data from labs around the world, which is why all iGEM teams are encouraged to participate in this study and submit their results. To contribute to this study, the Wageningen iGEM team was proud to participate.
Details
Repetition of experiments is an essential process within all science-related fields. First and foremost, it is the main way of verifying experimental results. By repeating a scientific experiment, the impact of random variation can be reduced owing to the larger dataset giving a more representative view of the measured system. This allows for more accurate interpretation of results. However, repetition of measurements can be difficult due to variations in lab environments, equipment specifications, and human handling. This can lead to problems with the comparison of data obtained from similar experiments around the world.
To allow for more accurate interpretation of results in different labs, the iGEM Measurement Committee has been developing a robust measurement procedure in the InterLab study, specifically for the measurement of GFP. Fluorescence is an important tool in synthetic biology, as it acts as an easy reporter for engineered systems. By creating this robust procedure, the Committee hopes to solve the data variations in fluorescence data between labs. In the InterLab study, iGEM teams from around the world were asked to perform a standard GFP measurement on a set of bacterial 'devices' in their own labs. Data generated by these teams are then used to improve upon the measurement procedure.
We, of the Wageningen iGEM 2017, team were happy to participate in this study. On this page we will present the measurement protocol, as well as the results we obtained with it.
For this study, we had the choice of performing fluorescence measurements either with a plate reader or flow cytometer. As the use of the latter is restricted in our laboratory, we decided to adhere to the plate reader protocol. Using the BioTek’s Synergy Mx Monochromator-based Microplate Reader, we measured culture samples of the following 8 devices:
- Positive Control: BBa_I20270
- Negative Control: BBa_R0040
- Test Device 1: BBa_J364000
- Test Device 2: BBa_J364001
- Test Device 3: BBa_J364002
- Test Device 4: BBa_J364003
- Test Device 5: BBa_J364004
- Test Device 6: BBa_J364005
Successful transformation of these devices to E. coli DH5α cells resulted in the colonies that were used to inoculate 50 mL Greiner tubes containing 10 mL LB medium + Chloramphenicol. These liquid cultures were incubated at 37 degrees Celsius, during which samples were taken at timepoints 0, 2, 4 and 6 hours after inoculation. They were then used to measure fluorescence and OD600nm in the plate reader.
To make sense of this data, we first needed to make calibrations of both the OD600nm and fluorescence at 395nm/509nm (emission/excitation). For the former, we used LUDOX-HS40 and H2O, provided by iGEM; for the latter, we used fluorescein, also provided by iGEM.
| Table 1: OD600nm reference point. | |||
|---|---|---|---|
| LUDOX-HS40 | H20 | ||
| Replicate 1 | 0.122 | 0.087 | |
| Replicate 2 | 0.095 | 0.089 | |
| Replicate 3 | 0.098 | 0.088 | |
| Replicate 4 | 0.099 | 0.089 | |
| Arith. Mean | 0.1035 | 0.08825 | |
| Corrected Abs600 | 0.01525 | ||
| Reference Abs600 | 0.0425 | ||
| OD600/Abs600 | 2.786885246 | ||
Samples taken from the liquid cultures of the devices at the different time points were added to a black, clear bottomed 96 wells plate in order to measure them in the plate reader. Each well contained 100 μL of the sample following the scheme provided in the protocol:
Results
Measurement of the 96-wells plate in the BioTek’s Synergy Mx Monochromator-based Microplate Reader was performed at 27°C with a wavelength of 600nm for the OD and wavelengths of 395nm and 509nm for the fluorescence excitation and emission, respectively. Furthermore, path length correction was turned off in the absorbance measurement and a slit width of 0.09 mm with a gain of 75 was selected for the fluorescence measurement. Data obtained from these measurements was added to the calculation sheet provided by the iGEM Committee, resulting in data as presented in the final graph:
