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Reproducibility of results in different laboratories is crucial to facilitate science. We participated in the interlab study to support the iGEM community and provide them with data for the interlab comparison of measurement results.
Material
We applied the protocol for plate reader[link] that was provided by the iGEM HQ.
This year a positive and negative control additionally to six other plasmids containing GFP (green fluorescent protein) genes with different Anderson promoters were transformed into Escherichia coli K-12 DH5α. The following devices were tested:
Negative control BBa_R0040: TetR repressible promoter
Positive control BBa_I20270: GFP with weak RBS (ribosomal binding site)
Test Device 1 BBa_J364000
Test Device 2 BBa_J364001
Test Device 3 BBa_J364002
Test Device 4 BBa_J364007
Test Device 5 BBa_J364008
Test Device 6 BBa_J364009
All devices were transformed into Escherichia coli K-12 DH5α using 5 µL from the distribution plates. Colonies were picked and colony PCR was conducted to ensure whether transformation with the correct insert was successful. Afterwards the colonies were incubated overnight in 5 mL LB medium and 25 µg mL-1 chloramphenicol.
For measurements two cultures of each plate were picked and grown overnight in 5 mL LB media with 25 µg mL-1 chloramphenicol at 37 °C.
The bacteria cultures were diluted to OD600 of 0.02 in a total volume of 12 mL LB with 25 µg mL-1 chloramphenicol in 50 mL falcon tubes covered in foil and incubated in darkness for 6 hours at 37 °C and 220 rpm. 500 µL samples were taken after 0 and 6 hours of incubation and stored on ice until the measurement. Absorbance was measured by OD600, fluorescence by excitation at 485 nm and emission at 530 nm.
To determine the amount of colony forming units (CFU) per mL the overnight culture of the negative and positive control were diluted to OD600 0.1 in LB medium with 25 µg mL-1 chloramphenicol in a total volume of 1 mL. The culture was further diluted in LB medium with 25 µg mL-1 chloramphenicol in five dilutions steps according to figure 1. Dilution steps three, four and five were put on LB plates containing 25 µg mL-1 chloramphenicol and incubated overnight.
Figure 1:Dilution steps for measuring the CFU per mL of the negative and positive control. Figure from iGEM Headquarter
Material
Incubator: We used the GFL Type 30/32 incubator at 37 °C and 220 rpm. We covered the glass front with paper to make sure the samples grew in darkness.
Cuvette measurement: For the first OD600 measurement of the overnight cultures we used an Eppendorf 6131 BioPhotometer.
Plate reader: Our plate reader model was the Tecan Infinite M200 (Roche). The excitation wavelength was set to 485 nm and emission wavelength was detected at 530 nm [1].
96 well plate: We used the 96 well microplates in black (polystyrene) from Greiner Bio-One for our measurements with the plate reader.
E. coli strains
E. coli K-12 DH5α
Results
In order to quantify the results of the Interlab Study two standard measurements have been carried out. The first standard curve (Figure 1) shows measurements with silica beads. Figure 2 shows the standard curve with Fluorescein.
Figure 2:Fluorescein Standard curve. Figure 3:Particle Standard curve.
Cultures were treated as described in the [link]protocol. The overnight cultures were diluted to OD600 0.02 and cultivated at 37 °C and 220 rpm for six hours. Samples were taken after 0h and 6h. The OD600 and fluorescence of all samples were measured. The OD600 after 0h and 6h can be seen in figure 4. The fluorescence of the samples after 0h and 6h can be seen in figure 5.
Figure 4: OD600 after 0h and 6h of the negative control, positive control and the six test devices. Figure 5: Fluorescence after 0h and 6h of the negative control, positive control and the six test devices.
Furthermore the colony forming units per 0.1 OD600 of the positive and the negative control were determined. Overnight cultures were diluted to an OD600 of 0.1. The samples were diluted as seen in figure 1. Dilutions 3,4 and 5 were put on LB Agar + Chloramphenicol plates. The CFU of the positive and the negative control is illustrated in figure 6.
Figure 6: Colony Forming Units (CFU) of the Negative and the Positive Control. Dilution 3,4 and 5 of both cultures were considered.
