The Edinburgh iGEM 2015 team is participating in the second year of the iGEM InterLab study along with 82 other teams. The 2015 InterLab study focuses on obtaining consistent fluorescence data for three genetic devices expressing GFP. The devices consist of a constitutive promoter with either high (J23101), medium (J23106) or low (J23117) promoter strengths, fused to GFP (I13504).
The promoters and GFP from the Biobrick distribution kit were transformed into E.coli Top10 cells then digested to have complementary sticky ends and ligated resulting in the final devices. The devices were validated by restriction digest and sequence data. Each promoter fused to GFP was transformed once more grown in cultures overnight and then measurements of fluorescence were taken.
More detailed information regarding the cloning process can be found below and on our InterLab lab notebook page.
The protocol supplied in the iGEM 2015 InterLab Protocol form was followed with a few minor changes.
Each device and the two controls were streaked out onto LB agar plates supplemented with chloramphenicol and incubated at 37°C for 22 hours. 3 colonies from each plate were inoculated into 50ml Falcon tubes containing 10ml of LB media and chloramphenicol. The tubes were incubated upright on a platform shaker in a room kept at 37°C for 16 hours.
The positive control used was the recommended BBa_I20270, a chloramphenicol resistant GFP expression device in pSB1C3.
The negative control used consisted of cells transformed with BioBrick number BBa_R0040. This part consists of the promoter pTetR and should therefore have very low fluorescence
The cells were grown on LB agar supplemented with 1000x chloramphenicol.
A colony of cells was inoculated into 10ml of liquid LB with 10µl of 1000x chloramphenicol. The cultures were grown in a 50ml falcon tube, upright on shaking platform at 275rpm in 37°C room overnight.
For each device three colonies were inoculated to act as the three biological replicates.
After the overnight incubation period of 16 hours the liquid cultures were removed from the 37°C room. 200µl from each culture was added to a 96-well plate. The initial optical density of each sample was measured at 600nm using a plate reader. All samples were then diluted appropriately so as to have an OD600 value within 5% of 0.5. Two additional technical replicates for each of the biological replicates were set up at this point. The OD600 was measured again to ensure all samples had an optical density within 5% of 0.5 before the fluorescence measurements were taken.
To measure fluorescence, the cells were excited at a wavelength of 485nm and emitted light at 538nm as measured.
The values obtained for fluorescence were in relative fluorescence units.
A SpectaMax M5 plate reader was used to obtain the values of fluorescence.
The background absorbance of 6 wells containing 200µl of LB was measured. The average of these readings was then subtracted from the fluorescence values obtained for the devices.
The data is in relative fluorescent units.
Mean = 5645.097
Standard Deviation = 489.884
Mean = 2900.405
Standard Deviation = 437.946
Mean = -19.261
Standard Deviation = 6.956
2nd Replicate = 5202.079
3rd replicate = 5187.619
2nd replicate = 6008.646
3rd replicate = 6135.483
2nd replicate =6634.865
3rd replicate = 6254.715
Mean = 5817.633
Standard deviation = 541.009
2nd replicate = 3694.877
3rd replicate = 3606.107
2nd replicate = 3036.241
3rd replicate = 3140.1
2nd replicate = 2871.642
3rd replicate = 2801.428
Mean = 3094.623
Standard deviation = 396.837
2nd replicate = 7.859
3rd replicate = 0.659
2nd replicate = 28.499
3rd replicate = 11.4
2nd replicate = -17.823
3rd replicate = -6.295
Mean = -3.720
Standard deviation = 17.489