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Experiments

Plasmid Construction

Each plasmid was constructed using two gBlocks to form the insert and a chloramphenicol backbone. The first gBlock contains a biobrick prefix, the putative promotor, and the first 25 base pairs of the mRFP1 gene sequence. The second gBlock contains the full mRFP1 gene sequence, the BBa_B0015 Double Terminator, and the biobrick suffix. Using the New England Biolab (NEB) Hifi DNA Assembly Master Mix protocols, the biobrick prefix and suffix allowed one end of both gBlocks to anneal to their respective sides of the Chloramphenicol backbone and the mRFP1 gene to anneal the two gBlock segments together. This process finalized the construction of our test devices and were then transformed into NEB 5-alpha cells for testing.

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. The experiment was run on a 96-well plate with triplicates of each test cell with 2 different dilutions for a total of 6 wells for each test cell. Three wells contained test cells at the stationary phase and the other three wells contained cells diluted from the stationary phase by a factor of 10. A speaker was held on a stand 1 inch 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 using the Synergy H1 Microplate reader from Biotek. The excitation-emission wavelengths for mRFP are 584 and 607nm but we decided to broaden the wavelength to 575 and 610nm in order to reduce wavelength interference. The plate reader allowed us to look at the Fluorescence level and Optical density. We Use a ratio of Relative Fluorescence Units to Optical density in order to assess the level of fluorescence of the cells. 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. A PDF of all our protocols will be added to the during the wiki thaw.