Team:Toulouse-INSA-UPS/InterLab
INTERLAB
A fundamental notion in science is the acquisition of reliable and reproducible measurements. Synthetic biology does not escape the rule, however, having reproducible measurements in different labs is sometimes difficult. The primary purpose of the Interlab study is to reduce the variability in synthetic biology measurements.
With the Interlab study, the iGEM Measurement Committee developed a protocol for measuring the fluorescence of Green Fluorescent Protein (GFP), one of the most common marker used in synthetic biology.
Last year, the Interlab study showed that by calibrating the fluorescence measurement with a known concentration of fluorescent molecules, the variability in measurements between labs was reduced.
This year, the goal of the Interlab study is to normalize the fluorescence data with respect to the number of cells in the sample in order to obtain values corresponding to fluorescence per cell. After measuring the fluorescence, the absorbance of at 600 nm is also measured. In addition, a count of colony-forming units is performed to determine the correlation between optical density (OD) measurement and the number of cells.
The Interlabâs problematic this year is the following: âCan we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?â
In the next sections, we will present the manipulations we have done and the results we obtained and then conclude on our Interlab experience.
Manipulations and results
The protocols we used were all provided by the Measurement Committee and can be found in the iGEM website. You can find all the results we obtained in this downloadable excel sheet.
Calibration
Before starting the study, we had to calibrate our plate reader. To achieve this, we carried out three calibrations: an OD600 reference point, a particle standard curve and a fluorescence standard curve.
- OD600 reference point
- Particle standard curve
- Fluorescein standard curve
Because every plate reader is different and because absorbance measurements are volume dependent, we needed to normalize the measurements of our plate reader. To do so, we made an OD600 reference point. It will provide us with a conversion factor used to compare our results with those of the other teams. LUDOX CL-X (45% colloidal silica suspension) was used for this experiment.
Here, we wanted to convert absorbance at 600nm to an estimated number of cells. To do so, the cells were replaced by silica beads. Those beads have the same size and optical characteristics as the cells. The micro-particles we used have a defined concentration. We measured the absorbance of a dilution series and plotted absorbance against particle concentration.
Each plate reader gives fluorescence values in arbitrary units. These arbitrary units vary a lot between the different plate readers so it is impossible to directly compare directly fluorescence values from one plate reader to another. Because we want to be able to compare fluorescence values between each team, we need to create a standard fluorescence curve. To do so, we made a dilution series of a fluorescent molecule of known concentration. Ideally, this molecule should be GFP protein because we will use it for the rest of the experiments. However, it is very difficult to have a standardized amount of GFP, so we used fluorescein, another fluorescent molecule with the same fluorescence properties as GFP.
Cell measurement protocols
There are two axes in the cell measurement protocols. The first one is the measurement of GFP fluorescence and OD at 600nm. The second one is the UFC count. For both, we needed to transform E. coli K-12 DH5-alpha strain with the following plasmids:
- Negative control: BBa_R0040
- Positive control: BBa_I20270
- Test Device 1: BBa_J364000
- Test Device 2: BBa_J364001
- Test Device 3: BBa_J364002
- Test Device 4: BBa_J364007
- Test Device 5: BBa_J364008
- Test Device 6: BBa_J364009
All of these plasmids contain a GFP gene, the expression of which is controlled by different promoters. In order to check the competency of the cells, we also used BBa_J04450. This plasmid expresses a RFP gene.
Before transforming the strain, we had to prepare competent cells and test transformation efficiency. After competent cells were obtained, we transformed them with the plasmids listed above. Then, we selected two clones per plasmid and used them for the rest of the manipulations. We happened to observe different phenotypes among clones transformed with the same plasmid. In these cases, two clones with the different phenotypes were taken for the measurements.
For the first part, all the clones were cultivated in liquid medium and we measured the OD at 600nm and the fluorescence at times 0 and 6 hours of the cultures. For the second part, the strains transformed with the control plasmids were cultivated and diluted in order to perform surface cell counting.
Concerning the cell counting, we needed to have a relationship between the number of cells and their absorbance. To do so, we counted colony forming units (CFU) from a diluted culture of known absorbance. We used for this experiment colonies from two positive controls and two negative controls. After the count, we obtained a cell concentration corresponding to an OD at 600nm.
The Interlab experience
Working for Interlab was a great experience. We managed to take part in a larger project than that of our team and it is always a real pleasure to work with other people from the entire world to accomplish the same goal. We also think that the work of the Measurement Committee is crucial. Indeed, the reproducibility of the measurements is a big issue in science, especially in biology. Being able to tackle this problem to improve this aspect of science change was a great opportunity.
We sincerely hope that the results of all the teams will help the Measurement Committee to succeed in their study and that it will help biology experiments to be more reproducible.
No dogs were harmed over the course of this iGEM project.
The whole Toulouse INSA-UPS team wants to thank our sponsors, especially:
And many more. For futher information about our sponsors, please consult our Sponsors page.
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