Team:Freiburg/InterLab
Interlab Study
Background
The aim of this year’s Interlab Study was to compare fluorescence measured in different labs using the exact same protocol, trying to exclude any deviations in the experimental procedure and to standardize all measurements so that they are no longer machine-dependent. Afterwards, it should be possible to evaluate if this approach is sufficient to generate a comparability between different measurements. The Interlab Study was divided into one part for standardization and one part for the cell measurements. The standardization part consisted of two experiments, firstly the OD600 calibration and secondly the standard curve of fluorescein.
Methods and Results
Single Point Reference
As absorbance is instrument-dependent, it was necessary to perform an OD600 calibration with LUDOX-S40 as a single point reference. Therefore, the absorbance at 600 nm of 100 µl LUDOX-S40 and of 100 µl H2O was measured with a spectrometer. Four replicates were done and the automatic path length correction was turned off. The respective results can be seen in the table below. After subtracting the values for H2O from the values for LUDOX-S40, a correction factor could be obtained which made the transformation of Abs600 measurements into OD600 measurements possible.
| 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 Standard Curve
For standardization, a dilution series of fluorescein was prepared. Fluorescein was diluted in PBS to a final concentration of 50 µM. For the standard curve, a 96 well plate was used. 100 µl PBS were added into well 2-12 of each row. Well 1 was filled with 200 µl of the diluted fluorescein (50 µM). For the dilution series, 100 µl were taken from well 1 and pipetted into well 2, then from well 2 into well 3. This step was repeated till the end of the row. Measuring the fluorescence (excitation at 485 nm, emission at 530/30 nm), a curve with an exponential increase could be obtained. The following graph shows the fluorescence intensity plotted against the fluorescein concentration.
Figure 1: Standardization curve of fluorescein.
To obtain the standardization, the fluorescence was plotted against the fluorescein concentration on a logarithmic scale.
Figure 2: Logarithmic scale of standardization curve.
Cell Measurements
E.coli (DH5-α) were transformed with each of the 8 plasmids provided by iGEM. The plasmids contained the following devices:
1. Positive Control
2. Negative Control
3. Test Device 1
4. Test Device 2
5. Test Device 3
6. Test Device 4
7. Test Device 5
8. Test Device 6
Every plasmid (except of the negative control) contained GFP as well as a chloramphenicol resistance. The bacteria were grown in LB medium with chloramphenicol (final concentration of 25 µg/ml). One day after the transformation, two colonies of each plate were picked and were grown overnight in 10 ml medium with chloramphenicol (37°C, 220 rpm). In the following, the respective first colonies are named 1.1, 2.1, etc. The second ones are named 1.2, 2.2, etc. The next day, OD600 of the bacteria was measured. As the final OD600 should be adjusted to 0.02, all samples had to be diluted in the respective amount of medium with chloramphenicol. Then, the bacteria were grown again at 37°C and 220 rpm. After 0, 2, 4 and 6 hours 0.5 ml of each sample were taken and put on ice. In the end, OD600 and the fluorescence of each sample for each time point was measured with the plate reader. Therefore, four replicates of each 100 µl were used. The values were converted from relative fluorescence units to [µM fluorescein/OD600], so they become comparable to the fluorescein standard curve measurements. Fig. 3 shows the fluorescence of each approach plotted against the time.
Figure 3: Relative fluorescence intensities of the bacteria, each containing a plasmids with one of the test devices.
For each device, the fluorescence goes against 0. As most of the initial values are lower than 0, it is likely that this is only background fluorescence. The bacteria do not seem to express GFP at all.
Conclusion
The expectation of this experiment was an increase in the fluorescence intensity over the time. For the different devices, curves with various intensities were expected. The fluorescence measurements from the Interlab Study cannot be compared at all because the whole experiment failed. There were several problems which prevented getting GFP expression. The low concentration of chloramphenicol was a problem which we initially missed. Additionally, the use of chloramphenicol-resistant bacteria hindered to get GFP expression. Nevertheless, these two problems did not seem to be the only ones. Also the repetition of the Interlab Study with new DH5-α and a final chloramphenicol concentration of 34 µg/ml could not produce a significant increase in the fluorescence intensity. The actual problem could not be identified. The results cannot become involved in the evaluation of the comparability of measurements. To evaluate the results of the Interlab Study as a whole, you would have to consider all data from many laboratories and statistically analyze them.
