InterLab

Every year, the Measurement Committee tries to analyze the causes of difference in values of fluorescence measurements obtained in laboratories around the year. Particularly in this year, the main goal of the Fifth International InterLaboratory Measurement Study is to investigate if the normalization of fluorescence measurements to the absolute cell count can contribute to the reduction of lab-to-lab variability in results. Our team, NU_Kazakhstan 2018, used the Varioskan LUX Multimode Microplate Reader to measure the fluorescence of DH 5 𝛼 cells transformed with the GFP inserted in pSB1C3 plasmid with different promoters (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009, plus negative BBa_R0040 and positive BBa_I20270 controls).

Protocol

We strictly followed the instructions from the protocol provided by iGEM

Plate reader configuration:

Photometric

Wavelength: 600 nm

Bandwidth: 5 nm

Measurement time: 100 ms

Fluorometric

Excitation Wavelength: 485 nm

Emission Wavelength: 530 nm

Excitation bandwidth: 12 nm

Fluorescence reading: top optics

Measurement time: 100 ms

Results

OD600 reference point


Particle Standard Curve


Fluorescein Standard Curve



Plates for Colony Forming Units



Raw Plate Measurement

Conclusion

The data from measurements shows that test device 4 (BBa_J364007) has the promoter with the highest fluorescence among the rest of the provided promoters. Its maximum value is 11.84 net fluorescein a.u. after 6 hours of incubation. The weakest promoter was found to be the test device 3 (max. value 0.48 net fluorescein a.u. at 6 hours). Samples transformed with test devices 1,5, and 6 have about similar results (approx. 2 net fluorescein a.u.). The fluorescence of samples transformed with test device 2 is slightly higher than these three. Its maximum value of fluorescence is 5.53 net fluorescein a.u.