Team:WPI-Worcester/Interlab
Interlab Study
Sequencing Data
All devices for the interlab study were constructed via Biobrick cloning. Sequencing confirmed successful construction of the following 3 devices:
While complete sequencing information could not be obtained on J23117+I12504 due to time constraints, the sequence of the correct promoter and the beginning of GFP are present in the sequencing we did obtain for this part. Based on this and the fact that experiments with this part yielded expected results, we can assume that the part is correct.
Protocol
Once we confirmed the sequences of each of the devices, we streaked LB agar plates supplemented with chloramphenicol with glycerol stocks of each device, including the positive and negative control. Then we picked 3 colonies from each plate, and prepared 5mL liquid culture cultures of each colony in a 15 mL conical tube containing LB supplemented with chloramphenicol. The liquid cultures were incubated for 18 hours in a New Brunswick I 24 Incubating Shaker. Following the 18 hour incubation period, we measured the OD600nm of each culture and diluted each culture to an OD of 0.5 We plated 100 μL of the dilutions in sets of four wells in a black, clear bottom 96-well plate according to the diagram below.
Finally, to measure the fluorescence and OD590nm of each well, we used a Wallac 1420 Multilabel Counter manufactured by Perkin Elmer. For each well, we divided the reading for fluorescence by the OD590nm. We averaged the fluorescence/OD590nm for each set of four wells to obtain an average for each biological replicate, and averaged the averages of the biological replicates for the negative control to obtain a standard zero. We subtracted the standard zero from the average of each biological replicate, the averaged the fluorescence/OD590nm for biological replicates of the same device to reach the results displayed below. Summary of Results
For the interlab study, we constructed three GFP expression devices consisting of a promoter from the Anderson constitutive promoter family and a GFP screening plasmid intermediate.These devices were BBa_J23101+ BBa_I13504, BBa_J23106+BBa_I13504, and BBa_J23117+BBa_I13504. For positive and negative controls, we used the parts BBa_I20270 and BBa_J23117 respectively. Based on the promoter strengths provided on the Anderson constitutive promoter family page, we expected the BBa_J23101 device to fluoresce the most, followed by the BBa_J23106 device, then the BBa_J23117 device. Our results confirmed this order of promoter strength. The measured fluorescence of the BBa_J23101 device was greater than that of the BBa_J23106 device. Both of these devices exhibited greater fluorescence than the positive control, BBa_I20270, which is regulated by a mutant of the low strength promoter BBa_J23114. Fluorescence in the device containing the low strength promoter BBa_J23117 was the lowest of the three devices, but was greater than that of the negative control.
Click HERE to see the complete fluorescence data!
