Natural cellular networks are very good at processing diverse inputs, generating complicated responses, and confounding researchers with their complexities. As an alternative to traditional cellular engineering approaches, the field of synthetic biology attempts to avoid the complexities of natural systems by focusing on the bottom-up construction of artificial cellular circuits. By rationally building up circuit complexity, synthetic biologists hope to both create novel systems capable of programming unique cellular responses, and gain insights into the design principles of natural systems. Circuits that allow for the programming of intercellular responses are of particular interest, and researchers have focused on the use of bacterial communication mechanisms (quorum sensing) to construct such circuits. At their most basic, quorum-sensing systems are composed of three main components, making them amenable to genetic manipulation. These components, however, have properties that have been finely tuned through evolution to function in very specific ways, and repurposing them for our own uses requires methods to overcome their naturally evolved properties. This thesis details our work in the construction and engineering of synthetic circuits based on components of the LuxI-LuxR quorum-sensing system. Using these components, we demonstrate methods for altering both the sensitivity and the form of the quorum-sensing response through the creation of three unique systems: an ultrasensitive positive feedback loop, a logical AND gate, and a coupled feedback loop oscillator. Construction and tuning of each circuit's properties were achieved through a mixture of rational and evolutionary approaches, with particular emphasis on the directed evolution of the LuxR transcriptional activator. Mathematical modeling was also used during the construction of the more complex circuits to predict the properties that were essential to their functionalities. With the construction and characterization of these circuits, we have provided both well-defined modules that can be used in the construction of more complex systems, and developed methods that will allow for the creation and engineering of additional synthetic circuits.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:open_access_dissertations-1304 |
| Date | 01 September 2010 |
| Creators | Sayut, Daniel Jon |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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