Negative control: BBa_R0040
Positive control: BBa_I20270
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 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 device were 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.
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 .
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 DH5α
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.
|Negative Control||Dilution 3 [CFU x mL-1]||Dilution 4 [CFU x mL-1]||Dilution 5 [CFU x mL-1]|
|Colony 1||1,10 x 108||8,48 x 107||1,12 x 108|
|7,38 x 107||6,56 x 107||8,00 x 107|
|1,01 x 108||1,00 x 108||1,28 x 108|
|Colony 2||9,15 x 107||1,05 x 108||9,60 x 107|
|9,78 x 107||9,52 x 107||1,20 x 108|
|1,03 x 108||9,84 x 107||1,36 x 108|
|Positive Control||Colony 1||9,47 x 107||1,05 x 108||9,60 x 107|
|1,15 x 108||1,15 x 108||8,80 x 107|
|7,84 x 107||8,72 x 107||6,40 x 107|
|Colony 2||9,22 x 107||8,96 x 107||7,20 x 107|
|8,54 x 107||1,14 x 108||9,60 x 107|
|8,48 x 107||9,68 x 107||1,04 x 108|