Team:Cologne-Duesseldorf/InterLab

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Interlab study

The feasibility of reproducing reliable biological data remains a key obstacle in modern science. Even though green fluorescent protein (GFP) is the most commonly used fluorescence protein in biological investigation, different working groups frequently generate different data for similar experiments. For that reason, iGEMs interlab study provides a simple GFP fluorescence measurement protocol in order to compare obtained data from different laboratories from all over the world.

Our measurements were performed using the plate reader protocol provided by the iGEM headquarter. The parts which were used are located in the pSB1C3 plasmid backbone and include six distinct test plasmids containing the GFP gene with different ribosomal binding sites and varying Anderson promoters (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All parts were transformed to E.coli DH5-α.

We used 1 $µ$L of each resuspended part from distribution kit plate 6. Two colonies of each plasmid were picked and incubated in 5 mL LB containing 0.25 $µ$g / mL chloramphenicol. Bacteria cultures were incubated over night at 37°C. The following day, cultures were diluted to an OD₆₀₀ value of 0.02 dissolved in 12 mL LB medium containing 0.25 $µ$g / mL chloramphenicol using the dilution calculation sheet provided by the iGEM headquarter. Subsequently, the cultures were incubated at 37°C and 220 rpm whereby 500 $µ$L of each culture was taken at 0, 2, 4 and 6 hours of incubation in order to measure optical density at 600 nm and GFP fluorescence.

Previously, calibration protocols were performed in order to generate the OD₆₀₀ reference point and the fluorescein fluorescence standard curve. On the one hand, LUDOX-S40 was used as a single point reference to calculate a ratiometric conversion factor to transform our absorbance data into a standard OD₆₀₀ measurement. The absorbance at 600 nm of 100 $µ$l LUDOX and 100 $µ$L H2O were detected as displayed in figure 1. We used the same type of 96-well plate and plate reader settings in our cell measurement protocol in order to avoid potential calculation errors.

Figure 1: LUDOX measurement at 600 nm. Obtained mean value was used as ratiometric conversion factor for eventual optical density measurement

On the other hand, the fluorescein standard curve was generated through a dilution series of four fluorescein replicates. Initially, a 2x fluorescein solution of 100 $µ$M was prepared in 1x PBS. Subsequently, the 2x fluorescein stock solution (100 $µ$M) was diluted to 50 $µ$M. Afterwards, serial dilution was prepared in a 96-well plate. Measuring fluorescein with the standard GFP detection settings enables the correction of our cell based reading to an equivalent fluorescein concentration. Moreover, this facilitates the conversion of our obtained cell measurement data to an actual GFP concentration. Figure 2 shows our generated fluorescein standard curve. As mentioned above, the same type of 96-well plate was used for subsequent cell measurement analysis, as well as the same plate reader adjustment.

Figure 2: Calculated fluorescein standard curve for the calculation of fluorescence analysis in cell measurement performance. Serial dilution was prepared utilizing 4 replicates for each fluorescein concentration. GFP excitation of 485 nm and emission of 530 nm was used for analysis.

After the preparation of the OD₆₀₀ reference point and the fluorescein standard curve, cell measurement analysis was performed as previously described. Calculated data of OD₆₀₀ and GFP fluorescence reading measurement are depicted in figure 3 and 4.

Figure 3: Optical density at 600 nm development during the six-hour interval. In addition to the test devices 1-6, positive and negative control was quantified. Data presented as mean ±SEM.

Figure 4: Fluorescence measurement during the six hours of cultivation of test devices 1-6 as well as positive and negative control, respectively. GFP excitation of 485 nm and emission of 530 nm was used for analysis. Data presented as mean ±SEM.

Conclusively, the generated data varies from our expected results, since we suggested a proportional increase in optical density at 600 nm with GFP fluorescence. Despite the fact that the optical density at 600 nm increases in every culture within six hours of incubation (Figure 3), calculated fluorescence data did not reveal marked increase in most of the tested devices (Figure 4). Merely the fluorescence signal of test device 1 appears to rise within the cultivation although its OD₆₀₀ value increases very slowly in comparison to the other cultures (Figure 3, Figure 4).