Quantitative analysis of genetic expression responses to dynamic microenvironmental perturbation.

摘要

Dynamic environments are commonplace in the natural world, from fluctuations in nutrient sources that control metabolic rates, to radiative cycling that drives circadian rhythms, to mechanical stresses that reform vasculature. So, intuitively one would assume that the regulatory systems that control cellular behavior are acutely adapted to respond to such variable conditions in a robust and appropriate fashion. Yet, despite their potential to provide increased quantitative detail and insight to the natural behavior of cells, highly dynamic perturbations are rarely utilized in the analysis of cellular gene expression and regulation. Part of this stems from the lack of technologies that enable such studies. However, recent advances in microfluidic devices designed to address biologically relevant questions promise to fill this void. Moreover, recently discovered knowledge that the galactose metabolism in S. cerevisiae, and possibly similar pathways, are in fact rudimentary memory systems, strengthens the need for the ability to examine gene regulation under complex and dynamic stimulation. In this project, microfluidic technology was developed specifically for isolating, observing, and dynamically probing colonies of model host microbes. The devices created not only sustain cells under ideal growth conditions, but do so in a way that allows for long duration acquisition of highly resolved time evolved gene expression within single cells. Furthermore, these imaging capabilities were coupled to a novel microfluidic system that was able to produce precise and continuous concentration waveforms. The microfluidic platform was then utilized to explore the dynamic response profile of the galactose utilization pathway in S. cerevisiae under fluctuating nutrient conditions. Using experimental data, this study revealed that the pathway kinetics lead to low-pass information filtration. Further experimental investigation coupled with computational model simulations uncovered coupling to glucose metabolism that provides a globally robust response, despite galactose utilization impairment. These results emphasize both the utility of microfluidic device platforms in quantitative biological studies, and the importance of studies conducted in more natural environments for gaining a more detailed understanding of how gene systems result in complex behavior.