Team:Bielefeld-CeBiTec/InterLab

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Interlab Study

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.

Protocol

We applied the protocol for plate reader 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.**

Cultures were treated as described in the 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 0 h and 6 h. The OD₆₀₀ and fluorescence of all samples were measured. The OD₆₀₀ after 0 h and 6 h can be seen in figure 4. The fluorescence of the samples after 0 h and 6 h can be seen in figure 5.
In both figures it becomes clear that the growth of the colony seems to be independent from the fluorescence. The negative control shows of course the smallest fluorescence. The highest fluorescence has device 1 followed by device 4. Device 6 and 4 show the smallest fluorescence of the devices. The highest OD₆₀₀ after 6 hours has the negative control and device 3. Device 5 reveals the slowest growth after 6 hours but does not show the smallest fluorescence.

**Figure 4: OD₆₀₀ after 0 h and 6 h 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.**

Negative Control	Dilution 3	Dilution 4	Dilution 5
Colony 1	Jill	Smith	50
	Eve	Jackson	94
	John	Doe	80
	John	Doe	80
	John	Doe	80
	John	Doe	80