Team:IIT Delhi/Oscillations

Oscillations: Sinusoids and Squares

                                                                                                                                                                                                                 

Having constructed our square wave circuit via cloning and synthesis, and having obtained Murray et al’s 5n1 plasmid (from addgene), we then tested out our system in microfluidic chambers, and obtained time lapse images, placed under a fluorescence microscope.

Cells containing the square wave circuit were first grown overnight in LB medium containing 100 ug/ml of antibiotic (ampicillin). The next day, the culture was diluted 1:100 in LB containing the appropriate concentration of ampicillin, and loaded into the microfluidic chamber (refer to microfluidic section for design specifications of the chamber). Cells caught at one end of the T junction were imaged, and a single cell among them was isolated. Flow rate ensured that as the cell divided, the daughter cells flowed out along with the fresh media flowing past the straight part of the channel, without taking the cells in the perpendicular part of the channel along with it.

Cells were also diluted, and induced by adding a minimal concentration of anhydrotetracycline (aTc < 0.1 nM). This barely induced the system, and set the oscillations into motion. The cells were loaded into 96 well plates with 4 replicates for each part, and the time evolution of GFP was seen. The data showed a good correlation with a square wave of the same time period imposed on it (shown in red in the graph).

Figure – Plate reader data showing square wave oscillations when a culture of the square wave generator was induced using a minimal concentration of aTc. One single time period of the square wave was captured.

The oscillations obtained in the microfluidic chamber are shown below –



Finally, the system was operated at various temperatures, to study the temperature robustness. No noticeable change in the qualitative nature of the oscillations was seen. Also, growth curves at three different temperatures were plotted, and not much difference in the growth rates was seen.