Team:Freiburg/InterLab
Interlab Study
Introduction
Reproduction of experimental data remains one of the big challenges in synthetic biology. In order to overcome this problem, three years ago the iGEM's measurement committee started to develop a robust measurement procedure for green fluorescent protein (GFP), in the form of the Interlab Study.
This year’s Interlab Study tried to determine the absolute fluorescence units of eight plasmids either in a plate reader or by FACS analysis. These set of plasmids consisted of one positive control, one negative control and 6 test devices containing different ribosome binding sites (RBS) and promoters of different strengths.
For a more detailed description visit the homepage of the iGEM's measurement committee.
Materials and Methods
All steps of the OD600 reference point calibration, the fluorescein fluorescence standard curve and the cell measurements were performed according to the protocol provided by iGEM's measurement committee. For the measurements Corning 96-well flat white polystyrol plates and a Synergy H4 from Biotek plate reader were used. Competent E. coli K-12 DH5-alpha cells were produced using the Zymo Research Mix & Go E. coli Transformation Kit.
Calibration
OD600 Reference Point
Because plate readers do not have standardized path length, the first step of the InterLab study was to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This was achieved by using LUDOX-HS40 as a single point reference. After data analysis a ratiometric conversion factor of 3.4 was calculated. The following table shows the results of the OD600 reference point calibration.
Table 1: Results of the OD600 reference point calibration.
| Replicate | LUDOX-S40 | H2O |
|---|---|---|
| 1 | 0.043 | 0.035 |
| 2 | 0.049 | 0.036 |
| 3 | 0.047 | 0.035 |
| 4 | 0.052 | 0.035 |
| Arith. Mean | 0.04775 | 0.03525 |
| Corrected Abs600 | 0.0125 | |
| Reference OD600 | 0.0425 | |
| OD600/Abs600 | 3.4 |
Fluorescein Fluorescence Standard Curve
In the second step, a standard curve of fluorescence intensity dependent on the fluorescein concentration had to be determined to convert cell-based readings to an equivalent fluorescein concentration. Afterwards, the fluorescein concentration can be converted into the GFP concentration. Results for the fluorescein fluorescence standard curve are depicted in figure 1 and 2.
Fig. 1: Fluorescein Fluorescence Standard Curve on a linear scale
Fig. 2: Fluorescein Fluorescence Standard Curve on a log scale
Cell Measurements
After the calibration steps were successfully established, the test devices were transformed into E. coli K12 DH5-alpha and characterized. The results of this characterization are shown in figure 3.
Results
Fig. 3: Fluorescence quantification of test devices.
Plate reader measurements of transformed E. coli K12 DH5-alpha converted to µM Fluorescein/OD600.
Discussion
Expecting an increase in the fluorescence intensity over the time for the different test devices, it was difficult to evaluate the obtained data. Repetition with the initial conditions showed no effect. After looking for possible error sources, chloramphenicol stock solutions were exchanged. Additionally, new competent E. coli K12 DH5-alpha cells were produced. Afterwards the InterLab study was performed for a last time but still no differences were observable In conclusion, no meaningful data could be obtained. Since both positive and negative control did not perform as expected, the problem could not be identified.
