Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

In order to test the activation response due to exposure to sound waves, a plasmid containing a putative promotor along with the red fluorescent protein sequence mRFP1 was first assembled for each sequence. This was achieved using the New England Biolab (NEB) Hifi DNA Assembly Master Mix; following these protocols, the plasmid constructed was believed to contain a chloramphenicol resistance gene backbone and an insert with the putative promotor sequence, a ribosome binding site, the mRFP1 gene, and a terminator. Each plasmid was then transformed into NEB 5-alpha, a strain of E. coli known for its transformation efficiency, and incubated on chloramphenicol plates to determine if the cells showed antibiotic resistance; this was our first indication if the plasmids were successfully transformed. Finally, the plasmids were purified from the growing cultures to verify the assembled plasmid sequences; there have been complications with the sequencing, but the plasmids are continuing to be analyzed. To run the experiment, ___ uL of a solution of transformed NEB 5-alpha cells, containing one of the plasmids, were placed in a single well of a 96 well plate and diluted ___ folds after each well. A speaker was held on a stand ___ inches above the plate playing the desired frequency and left in an incubator for six hours to expose the cells to sound during the growth period. After the incubation, plates were read to measure and calculate the if any red fluorescence was expressed. It was hypothesized that through our plasmid construction and testing, those putative promotors that were activated by sound would express mRFP1 and indicate higher readings of fluorescence compared to a control group of cells that were incubated with no sound. (Press here to view the protocols)