Team:TEC-Costa Rica/Interlab
Interlab
How close can the numbers be when fluorescence is measured all around the world?
This year’s Interlab study aims to establish standard units for the measurement of fluorescence, by establishing a comparison between the measurements of many different labs around the globe with previously measured standards.
Constructs
For this experiment, a series of five constructs were used, with three being a GFP (E0040) device with a constitutive promoter (J23101 - strong, J23106 - medium, and J23117 - weak), B0034 RBS, and B0015 terminator; and the other two the controls (I20270 - positive, and R0040 - negative).
Figure 1. Diagram of the constructs used for the experiment.
Equipment
The measurement was made on a BioTek Synergy H1 Multi-Mode Reader, in the National Center of Biotechnological Innovations.
Figure 2. Equipment used for measurement.
Materials and Methods
The experiments were made made on a transformed Escherichia coli, strain DH5α. The bacteria were grown on Luria-Bertani broth. The materials and protocols were supplied by igem HQ.
First, the OD600 measurement was standardized using the provided LUDOX to create a correction factor for the Abs600 measurement between the instruments.
Figure 3. OD600 calibration with LUDOX.
Then, the fluorescence value was corrected using a series of dilutions of the chromophore FITC as a standard. A curve was generated with the serial dilutions of the molecule which was measured with the plate reader with the GFP program.
Figure 4. FITC measurement standard curve.
After the bacteria were transformed and grown overnight, we set all the concentrations to an Abs600 of 0,02. These subcultures were kept in a shaker, and samples were taken every hour for 6 hours.
Figure 5. Abs600 of the overnight culture.
Results
In Figure 6., we show the data obtained from the growth of Escherichia coli. It can be seen that the device 1 has the lowest growth, while device 3 has the highest.
Figure 6. Growth of the bacteria vs time.
A curve for fluorescence was also generated, where it can be easily seen that the device 1 has the most fluorescence, followed by device 2, positive control, and negative control and device 3 in the bottom with no response.
Figure 7. Fluorescence of the constructs vs time.
Also, a ratio of fluorescence against absorbance was established in order to correct for the effect of the GFP production on the bacterial growth.
Figure 8. Fluorescence/Absorbance ratio.
