Synthetic genetic clocks in E.coli and other species have been studied ever since the publication of the Repressilator (Elowitz and Leibler, Nature 2000).Dr. Alex Ninfa’s lab contributed to these studies by constructing a genetic clock based on the nitrogen assimilation control system in E. coli (Atkinson et al, Cell 2003). This clock showed oscillations at the population level, as opposed to other clocks that showed oscillations on single cells but no synchronized oscillations at the population level. Ninfa’s clock, however, displays damping in the amplitude of oscillations, until the oscillations are gone after 3 or 4 days.In this thesis, I describe the characterization of Ninfa;;s clock under a variety of conditions and in host cells differing in their genetic structure.I first developed instrumentation that facilitated the study of the synthetic genetic clock.Next, I characterized the behavior of Ninfa’s clock under several different environmental conditions (nutrients, temperature), and with genetic variations in the host cells, where the reporter gene and number of target sites for the clock activator and repressor were varied.I found that the period of oscillation is proportional to the growth rate of the cells, making the period of the clock a controllable parameter. I also found that the clock is very sensitive to variations in the number of target sites for its transcriptional activator. When studying the effect of host cell genetic variations on clock functions, I also found that the synchronization of individual cell’s clocks may be related to the acetate utilization/degradation pathway. Finally, I present theoretical work in mathematical models of synthetic clocks with three modules, aimed at finding the likelihood of sustained oscillations in these systems.
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Characterization and Analysis of Synthetic Genetic Clocks.
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