Background

For achieving reliable results there is a need for repetition of experiments. In this course, maybe the most ambitious aspect is the repetition of measurements in different labs: the usage of different strains, protocols and devices makes it hard to generate comparable results.

By now, fluorescent proteins such as GFP became useful and widely applied tools in synthetic biology. The comparison of fluorescence data from different labs is even more challenging as they might be reported in different ways or generated by various techniques. Therefore, the Measurement committee uses the framework of the international competition iGEM to gain experimental data from all over the world. 2017, the fourth International InterLab Measurement Study was set-up with the aim to develop a robust GFP measurement. We as the TU_Dresden Team also work with fluorescent proteins ourselves, therefore we definitely wanted to participate in the this years InterLab Study.

“How close can the numbers be when fluorescence is measured all around the world?” (iGEM Measurement Committee)

Materials and Methods

To generate a reliable and robust standard, certain requirements need to be fulfilled. They include following a precise protocol, the usage of competent Escherichia coli DH5-alpha cells and GFP measurement with a plate reader. For more detailed information, see https://2017.igem.org/Competition/InterLab_Study and the plate reader protocol. Plate Reader Protocol (provided by iGEM Measurement Committee)

Devices

The following eight devices were used, all composed in the backbone of pSB1C3 including an chloramphenicol resistance gen and a GFP reporter gene (except for the negative control). They differ in the combination of promoter and ribosome binding site (RBS) upstream of the reporter.

• 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)

Transformation of plasmids into E. coli DH5α

In a first step, the given plasmids (provided in the Measurement Kit) had to be transformed into E. coli DH5α cells. We used the provided Transformation protocol

Calibration

OD600 reference point

LUDOX-S40 is used as a single point reference to standardize the OD600 measurements and to avoid any influences from the used instrument.

Fluorescein fluorescence standard curve

Fluorescein was also provided in the measurement kit. We prepared a serial dilution and obtained the fluorescence and used the generated data as standard curve.

Cell Measurement

After transformation, two colonies were picked from each plate and used for overnight cultures in LB supplemented with chloramphenicol at 37 °C and 220 rpm. The next day, cell growth was measured via OD600 measurements, then cultures were diluted to a target OD600 of 0.02 in 12 ml LB + chloramphenicol and the assay was started. Subsequently, samples were taken at time points 0, 2, 4 and 6 hours of incubation and measured in a 96-well plate. Luminescence was recorded, using a Biotek Synergy Neo plate reader.

Results and Discussion

Results were reported in the provided Exel-sheet and sent to the Measurement committee. Click here to see our results.

The graph indicates that the highest fluorescence signal was measured after two hours of incubation for most test devices. Directly after starting the assay, the fluorescence was lowest. Regarding the differences between the used Test Devices, the following conclusions can be drawn:

• Comparing the different promoters used, Test Devices with BBa_J23101 (Test Devices 1 and 4) gave highest fluorescence signals, the signal was decreased when BBa_J23106 (Test Devices 2 and 5) was included and was lowest with BBa_J23117 (Test Devices 3 and 6).
• Comparing the different RBS used, BBa_B0034 as in Test Devices 1, 2 and 3 gave better results than BBa_J364100 in Test Devices 4, 5 and 6 when compared to the device with same promoters.
• To obtain highest signals, choosing a strong promoter has a higher impact on the fluorescence signal than the RBS.

Outlook

We are looking forward to seeing the InterLab Study presentation of the Measurement Committee at the Giant Jamboree and are curious about the outcomes of other teams and their differences to our results. In general, labs provide experimental data and try reproducibility within their research. Global reproducibility is an ambitious goal which might never be achieved. The InterLab Study is surely the first intention to tackle this approach.