To allow the comparison of consistency in measurement between all participating teams all over the world the interlab study takes place as a benchmark. It is organized by the iGEM's measurement committee and serves the idea to improve measurement techniques in the field of synthetic biology. Therefore, all participating teams will perform the same biochemical experiments, the measurement of GFP and OD600 of the DH5α E. coli cells of samples provides by the iGEM's headquarter.
Description of the experiment:
The following devices were available used for the experiments:
Positive control
Negative control
Test Device 1: J23101 + I13504
Test Device 2: J23106 + I13504
Test Device 3: J23117 + I13504
Test Device 4: J23101.BCD2.E0040.B0015
Test Device 5: J23106.BCD2.E0040.B0015
Test Device 6: J23117.BCD2.E0040.B0015
The samples were transformed into the DH5 α E. coli strain by the following transformation protocol:
Thaw cells on ice
Add DNA and mix
Incubate on ice for 1 h
Heat-shock 1 min 4 °C
Incubate 4 min on ice
Add 850 µl 37 °C Luria-Bertani media (LB media) to cells
Incubate 1 h 37 °C 900 rpm
Centrifuge 4500 rpm 2 min
Remove 850 µl liquid
Resuspend cells in remaining liquid
Grease cell suspension on plates with cAMP resistance
Incubate overnight 37 °C
The cells were applied to plates with a chloramphenicol (cAMP) antibiotic with a concentration of 25 µg/ml and incubated over night at 37 °C.
After 16 - 18 hours of incubation two positive colonies for each transformation were transferred to 5 ml liquid media with a chloramphenicol antibiotic with a concentration of 25 µg/ml. The cells were incubated over night at 37 °C at 220 rpm.
The densities of the cultures were measured at OD600 The Tecan Genios Microplate reader was used for the measurement. The samples were diluted to an OD600 = 0.02 in 12 ml LB media with chloramphenicol concentration of 25 µg/ml. The samples were incubated at 37 °C and 220 rpm. Samples of 500 µl were taken after 0, 2, 4 and 6 hours and stored on ice for further experiments. 100 µl of each sample was given into a 96-well plate as in the following pattern:
Figure 1: Loading pattern of 96-well-plates for fluorescence and OD600 measurement.
For the measurement of the OD600 and the GFP fluorescence the Tecan Genios Microplate reader was used. The measurement of the OD600 transparent 96-well-plates were used and for the measurement of the fluorescence black 96-well-plates were used. The reader was calibrated with the following settings.
For the OD600 settings LUDOX-S40 was used as a single setting point. The LUDOX was provided by the iGEM headquarter and enables to obtain a ratiometric conversion factor for a standard OD600 measurement. Four samples of 100 % LUDOX-S40 were measured and as a comparison four samples of 100 % H2O.
For the measurement of the fluorescence a dilution series of fluorescein in four replicates was measured in a 96-well plate. Each well contained 100 µM. The starting concentration was 50 µM. The samples were diluted to: 25; 12.5; 6.25; 3.12; 1.56; 0.78; 0.39; 0.20; 0.1; 0.049; 0 µM. By the measurement of this defined concentrations a standard curve was created. By comparing the fluorescence of the samples to this standard curve, the concentrations of the samples were determined.
The measurements of the calibrations and the samples were performed under the same conditions with an excitation laser of 485 nm and an emission filter of 530/30.
Results
The following data shown in table 1 was received from the LUDOX-S40 calibration.
Table 1: Data for calibration for OD600 measurement performed with LUDOX-S40.
The following data shown in table 2 was received for the fluorescein standard.
Table 2: Data for calibration for fluorescence measurement performed with fluorescein.
Figure 2: Fluorescein standard curve for fluorescence measurement.
Figure 3: Fluorescein standard curve for fluorescence measurement in reciprocal presentation. The data points are shown in blue and the standard curve is shown in red, described by the formula.
With the aid of the calibration curves the following quotients were calculated:
With the aid of the calibration the samples were measured and delivered the following results in tables 3 - 8.
Measurement of OD600
Time point = 0 h
Table 3: OD600 Measurement of the samples after 0 h.
Time point = 2 h
Table 4: OD600 Measurement of the samples after 2 h.
Time point = 4 h
Table 5: OD600 Measurement of the samples after 4 h.
Time point = 6 h
Table 6: OD600 Measurement of the samples after 6 h.
Measurement of Fluorescence
Time point = 0 h
Table 7: Fluorescence Measurement of the samples after 0 h.
Time point = 2 h
Table 8: Fluorescence Measurement of the samples after 2 h.
Time point = 4 h
Table 9: Fluorescence Measurement of the samples after 4 h.
Time point = 6 h
Table 10: Fluorescence Measurement of the samples after 6 h.
The data from this measurement was processed in the following way. For both colonies the mean of all samples for each point of measurement was calculated after subtracting the blank measurement (LB + cAMP). This data is shown in the tables 11-14.
Table 11: Mean of all samples of OD600.
Table 12: Mean of all samples of Fluorescence.
From the mean of all samples the graphical analysis in the figures 4 and 5 were created.
Figure 4: Graphical analysis of OD600 of all samples.
From the mean of all samples the graphical analysis in the figures 4 and 5 were created.
Figure 5: Graphical analysis of fluorescence of all samples.
By using the standard curve from figure 3 y = 1587,5x0,9263 the concentration of the fluorescing product can be determined. The concentrations can be reviewed in table 13.
Table 13: Calculation of the concentration of the fluorescing protein of all samples by using the standard curve.
Discussion
In the graphical analysis it can be seen, that all measurements of the fluorescence are similar for the two colonies for each samples. This indicates representative colonies. Besides, it can be seen in figure 5 that Device 1 produces most of the fluorescing molecule. By using the standard curve in figure 3 the concentration of the fluorescing molecule can be determined, which can be reviewed in table 13. The highest production of Device 1 after 6 h is 3.191 µM. Furthermore, Device 2 and 4 also produce more product than the other devises. Their highest production is 2.188 µM for Device 2 and 1.148 µM for Device 4. The other Devices produce significantly less product. The highest production of Device 3 is 0.188, for Device 5 it is 0.420 and for Device 6 it is 0.18.
In conclusion, Device 1 produces most fluorescing product. Device 2 as well as Device 4 produce not as much product as Device 1, but still more than the Devices 3, 5 and 6.
