Single-cell dynamics of mammalian gene regulation

Observation of single-cell behavior has become increasingly important to our understanding of complex biological systems given the highly dynamic nature of underlying regulatory processes which is often obscured by population-level observation. The focus of this dissertation is to explore gene regulatory dynamics in individual mammalian cells with the use of microfluidic technology and synthetic biology. First, I develop a novel microfluidic device platform which enables dynamic stimulation of mammalian cells during long-term perfusion culture. The device incorporates an innovative cell-loading method which uses a temporary on-chip vacuum to capture cells in culture regions isolated from detrimental effects of fluid-induced shear stress. Second, I use this device to characterize the single-cell NFkappaB response to dynamic stimulation with TNFalpha. The induction of cells with a linear temporal gradient compared to a step impulse reveals significant differences in timing variability and amplitude of the response for the two modes of stimulation. Finally, I follow a synthetic biology approach to construct artificial negative and positive transcriptional feedback gene circuits in mammalian cells and I explore the potential of this architecture to generate oscillatory expression behavior.