Team:JMU Wuerzburg/InterLab

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Introducton

We successfully participated in the fifth International InterLaboratory Measurement Study in synthetic biology 2018! Due to the high importance of the reproducibility and consistency of experiments in every field of science the InterLab Study is a great opportunity to ensure these characteristics / attributes in different laboratories.

The fifth InterLab Study for iGEM 2018 follows the purpose to reduce variability in fluorescence measurements in different labs by replacing normalization in OD with absolute cell count or colony-forming units (CFUs). We followed a detailed protocol and used the provided data analysis form that yields absolute units for measurements of GFP in a plate reader. This would enable researchers to compare results more accurately to standardize protocols so that experiments yield reproduceable results when repeated.

During the past years the Measurement Committee developed a robust measurement procedure as a part of the iGEM competition.

Materials and Methods

Devices:

All test devices contain GFP (except from the negative control) and the vector pSB1C3 with a chloramphenicol resistance.

Microorganism: Escherichia coli K-12 DH5⍺ strains , E. coli XL-1 Blue

Plate Reader Settings:



We followed the protocol¹ provided by iGEM.

Comments

Since the first approaches of cell transfection were unsuccessful, we concluded incompetence of our E. coli K-12 DH5α cells. Apart from the protocol we implemented then for our measurements E. coli XL-1 Blue competent cells instead of the E. coli K-12 DH5α strain.

Results and Discussion

Over the six hours of incubation the cells grew well (Figure 1). Device 1 and 5 had a significant lower growth rate than the other device. At time point 0 h the devices should have had the same Abs₆₀₀, but potentially because of contamination our data differed from the expected values.

Figure 1:

The graphic shows the absorbance for the negative control, the positive control and all test devices at time point zero and after six hours.

In figure 2 fluorescence is observable which indicates the success of transformation. While device 6 emitted quite low amounts of fluorescence, device 4 had the highest reading. Noticeable device 3 showed no significant fluorescence at all, but since the cells grew quite well the transformation certainly failed here.

Figure 2:

The graphic shows the fluorescence for the negative control, the positive control and all test devices at time point zero and after six hours. The graphic excludes values smaller zero.

In most of the devices the fluorescence measurements were too high at time point 0h compared to the ones at time point 6h with respect to the OD₆₀₀.

Figure 3:

The graphic shows the fluorescence with respect to the OD600 for the negative control, the positive control and all test devices at time point zero and after six hours. The graphic excludes values smaller zero.

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List of References

¹ https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf