I propose the use of the tandem fluorescent timer protein as a reporter of dynamic gene regulation. The tandem fluorescent timer is a fusion of two fluorophores with different maturation kinetics whose fluorescence ratio is a reporter of protein age. Traditional approaches to live single-cell tracking of dynamic gene expression involve the use of destabilized fluorescent reporters. The reduced stability of these reporters improve performance but also result in reduced signal and an increased signal to noise ratio. I first develop a platform to test reporter performance by designing and implementing an inducible synthetic network orthogonally in S. cerevisiae cells and by developing a microfluidics-enabled live cell-tracking pipeline. To test the performance of different reporters, I develop an algorithm to decode the underlying regulatory dynamic signal of a fluorescence profile. I then simulate the fluorescence output of my platform under dynamic regulatory signaling to demonstrate the potential reporter performance of a stable timer protein. Finally, I conduct live cell-tracking experiments of yeast cells expressing the timer under a periodic signal to test in vivo performance of the tandem fluorescent timer. I demonstrate that compared to a traditional stable fluorescent reporter, the tandem fluorescent timer is more accurate when tracking faster periodic signals and it is more robust to global fluctuations.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/39021 |
| Date | 02 April 2019 |
| Creators | Salem, Danny |
| Contributors | Kaern, Mads |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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