International InterLab Measurement Study

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

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.