Team:TU-Eindhoven/Interlab

iGEM TU Eindhoven

Interlab Study

All iGEM teams were encouraged to participate in the 3rd Interlab Study. The goal of the Interlab Study is to be able to perform absolute fluorescence measurements instead of relative fluorescence measurements. This year iGEM teams from all over the world compared fluorescence data of test devices provided by iGEM HQ. For more information visit the iGEM Interlab website.

Test Devices

We were provided with five devices, all of these were built into the pSB1C3 backbone, which contains chloramphenicol resistance. There are three test devices, a positive control and a negative control.

  • Test Device 1: pSB1C3 backbone containing J23101 (promotor), B0034 (ribosome binding site), E0040 (GFP), B0015 (double terminater)
  • Test Device 2: pSB1C3 backbone containing J23106 (promotor), B0034 (ribosome binding site), E0040 (GFP), B0015 (double terminater)
  • Test Device 3: pSB1C3 backbone containing J23117 (promotor), B0034 (ribosome binding site), E0040 (GFP), B0015 (double terminater)
  • Positive Control Device: pSB1C3 backbone containing I20270 (GFP expression device)
  • Negative Control Device: pSB1C3 backbone containing R0040 (no GFP expression device)

Methods

To obtain reference values of the fluorescence measurement of our plate reader, iGEM HQ provided us with Fluorescein Isothiocyanate (FITC). With serial dilutions of this protein reference fluorescence measurements could be made.

To calibrate the OD600 (density/number of cells) we used water and a mono-dispersed silica suspension (LUDOX), also provided by the iGEM HQ. The OD600 difference between those two and a reference value from iGEM HQ yielded a correction factor for the OD600 that was used to correct the OD600 measurements of the test devices.

The host for each device was E. Coli strain BL21(DE3). The promotors in the vector were constitutive meaning no protein expression inducer was needed.

First the test devices were transformed in BL21(DE3), after overnight growing, two colonies were picked to grow small cultures. When the bacterial cultures of the test devices had grown overnight, they were diluted with LB medium to an OD600 of 0.02. Then every hour a sample was taken from each culture over a time span of six hours. From every sample the OD600 and fluorescence was measured. The samples were stored on ice before the measurements were done, so the measurement of the samples could be done simultaneously.

The Tecan Spark M10 plate reader was used for the measurements. All measurements were done with a volume of 100 µL per well. The measurements were corrected by subtracting them with a blank measurement, this blank was LB medium with chloramphenicol.

Results

Below are the results of the OD600 calibration with water and LUDOX and the fluorescence reference measurements using serial dilutions of FITC.

Figure 1: 0D600 calibration measurement with LUDOX and H2O.
Figure 2: Fluorescence reference point with serial dilution of FITC, measured at excitation wavelength 490 and emission wavelength 525.

From the fluorescence and OD600 data it can be deduced that the promotor of device 1 is the strongest promotor, because it shows the highest fluorescence. Followed by the promotor of device 2, with slightly lesser fluorescent. The weakest promoter was from device 3, it had the lowest fluorescence.

Figure 3: Corrected OD600 measurement for test devices, positive control and negative control.
Figure 4: Corrected fluorescence measurement for test devices, positive control and negative control.
Figure 5: Fluorescence with respect to OD600 for test devices, positive control and negative control.
  • Data OD600

    Table 3: OD600 data
     Table 4: OD600 data after blank substraction and correction

  • Data fluorescence

    Table 5: Fluorescence data
     Table 6: Fluorescence data after blank substraction

  • Data fluorescence/OD600

    Table 7: Fluorescence/OD600 data
     Table 8: Fluorescence/OD600 average and standard devation data

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