Difference between revisions of "Team:William and Mary/Speed Control"

To measure speed, we measured our pTet mScarlet-I pdt constructs along with our pLac mf-Lon construct into NEB 10-Beta cells. After growing them for 8 hours and diluting them to an OD of .005, we simultaneously induced each construct with .1mM IPTG and 50ng/mL ATC. Simultaneous induction was chosen as our preliminary math modelling suggested that a greater speed change would be observed when using simultaneous induction rather than having mf-Lon at a steady state concentration.

Before we took speed measurements we first tested a variety of different time course methods for use with flow cytometry. We tried cryogenic freezing, staggering cells, and fixation with 1% PFA for various lengths of time. However, in the end the most robust method was simply taking time points into PBS on ice and immediately FACSing them. We found that this method was robust across days, as well as robust to experimental conditions and other errors. We noted that fluorescence per cell did not change significantly after 20 minutes on ice, which represents the longest any sample sat on ice before being measured. All measurements were performed in NEB 10-Beta cells and M9 media, and dilutions were made to maintain cells in the midlog growth phase for the entirety of the experiment. A full protocol can be found here.

All data on this page represents the geometric means of at least three biological replicates (colonies) taken on the same day. Each biological replicate’s fluorescence was determined on the FL3 channel of an S3e cell sorter, and the geometric mean of at least 10,000 (typically 20,000) cells were used. Conversion to absolute units (MECY) was performed using spherotek rainbow calibration beads and Flowcal. Shaded region represents the geometric standard deviation of the biological replicates.

In our experiment we observed a robust tag dependent speed change during the course of our experiments. Figure 1 shows plots of normalized fluorescence over time. Steady state was defined as the point where the next two consecutive data points did not exhibit any increase in fluorescence. That data point was then used to normalize the previous values of that time course. Figure 2 shows these results without truncation, and as predicted by mathematical modeling we see that fluorescence starts to decay down to a second lower steady state. This result is because of the choice of simultaneous induction, and was later used to help create our IFFL circuits. Add more?