Spatiotemporal Characterization of Stochastic Bacterial Growth in Biofilm Environment
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Research on bacteria in their biofilm form is limited by the ability to artificially culture bacterial biofilms in a system that permits the visualization of individual cells. The experiments comprising this thesis research are on-going investigations of bacterial culture systems engineered to provide an environment that mimics biofilms while enabling real-time microscopy. Specifically, the microfluidic systems developed and assessed as part of this thesis permit the visualization of individual bacteria cells within consortia growing within a narrow space provided by a microfluidic device. This research demonstrates the versatility of these microfluidic systems across potentially high-throughput microbiological experiments utilizing genetically engineered Escherichia coli. Before demonstrating the efficacy of these systems, the development of the field of synthetic biology over the past half century is reviewed, focusing on synthetic genetic circuits and their applications (Chapter 2). The first and main microfluidic device explored in this research was developed to mimic the nutrient-deficient conditions within biofilms by forcing media to enter the culture area through a narrow, torturous channel. The microfluidic channel was thin enough (0.97 μm) to prevent the motility of 1-μm-wide E. coli cells, enabling visualization of individual cells. The bacteria cultured in the device contained either a simple Plux-driven quorum sensing receiver (Chapters 3 and 5) or a LacI- and TetR-driven genetic toggle switch (Chapter 4). Under the culture conditions, the quorum sensing reporter signal was detected even without addition of the signaling molecule (Chapter 3). The genetic toggle switch was stable when the system began in the high-LacI expression state, but after 5 days of culture, >5% of high-TetR expression cells began to consistently express the high-LacI state (Chapter 4). This system was also employed to track lineages of cells using real-time microscopy, which successfully characterized the inheritance of aberrant, enlarged cell phenotypes under stress (Chapter 5). Another microfluidic device, a droplet bioreactor, was also developed to culture small numbers of cells in an aqueous bubble suspended in oil (Chapter 6). Quorum sensing receiver cells were cultured in this device, demonstrating that it is well suited for testing the effects of compounds on biofilms within water-in-oil droplets.
- Doctoral Dissertations