Bennett, Matthew R2021-12-062021-122021-12-03December 2de Freitas Magalhaes, Barbara. "A Synthetic Intercellular Toggle Switch and its Implications on Pattern Formation." (2021) Diss., Rice University. <a href="https://hdl.handle.net/1911/111761">https://hdl.handle.net/1911/111761</a>.https://hdl.handle.net/1911/111761A genetic toggle switch is a circuit that possesses two stable mutually exclusive states of gene expression caused by cross-repression. It is widely found in natural processes, such as in development, given that cross-repression is important for refining pattern boundaries. Synthetically, many circuits that are applied to biotechnology are also based on toggle switches, such as many biosensors. However, single-cell synthetic genetic toggles are limited by individual noise fluctuations, and functions that do not cause metabolic load to the cell. Here, we propose the creation of an intercellular toggle switch, which is a synthetic toggle with quorum sensing (QS). QS is the mechanism through which bacteria communicate with each other, by producing signaling molecules that can affect gene expression in a density-dependent way. To overcome the main limitations of single-cell toggles, we aim to synchronize a population response, and enable circuit multicellularity through cell communication. Synthetic multicellular genetic circuits can perform complex functions that single cells cannot, and have the ability to better recreate naturally-occurring processes. In the first part, we describe the construction and characterization of a few QS toggle versions. We find that the dynamics of these toggle switches depend on their regulatory topologies, and their gene expression strength and leakiness. In the second part, we explore the aspects of one QS toggle version in a biofilm. We find that the QS toggle can form self-organized patterns when grown in a colony, by spatially segregating cells from different states. Comparatively, a non-QS (NQS) toggle colony does not show spatial-dependent pattern formation until three-dimensional aspects are investigated. NQS toggle shows a vertical segregation of states within colonies. It indicates that the addition of QS causes a directional shift in colony pattern behavior, and makes it more complex and dependent on growth, as shown with the mathematical model. These findings highlight the importance of spatial aspects to a synthetic circuit behavior. They also shed light into natural pattern formation mechanisms, and contribute to the development of synthetic self-organized multicellular systems in bacteria.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.toggle switchquorum sensingspatial patternsThesis2021-12-06