Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item PROGRAMMING BACTERIAL CONSORTIA FOR AUTONOMOUS REGULATION AND COORDINATED ACTIVITY(2020) Stephens, Kristina; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The potential of genetically engineered microbes seems nearly infinite with applications ranging from human health to bioprocessing. However, metabolic burden and unbalanced use of cell resources are frequent challenges when engineering cells to carry out synthetic functions. To work around this challenge, engineers are attempting to use co-cultures or synthetic consortia wherein labor is divided amongst subpopulations that work together. This emerging strategy requires new tools to regulate the composition of subpopulations and to enable robust coordination between subpopulations. Here, we investigated and rewired a native cell-cell communication process, quorum sensing, in order to develop tools to regulate co-cultures. We developed modules for signal regulated cell growth rate and cell-cell communication in bacteria, and we used these modules to construct co-cultures with autonomous composition control. Specifically, we developed a “controller” strain for signal modulated cell growth rate by using quorum sensing signals to regulate levels of HPr, a protein involved in sugar transport. We developed a second “translator” strain that detects the universal quorum sensing signal AI-2 and translates it into a species-specific AI-1 signal. The composition of the resulting co-culture adjusts autonomously in response to AI-2. Importantly, we developed a simple mathematical model based on individual monocultures that predicts behavior of the co-culture. Then, we used our model to explore in silico alternate construct designs operating in varied environments. To complement the co-culture model, which explores behavior due to interactions between strains but does not encompass information about the genetic circuits underlying the quorum sensing process, we then developed a gene circuit model of a dual-input synthetic AI-2 quorum sensing system. Finally, we demonstrate that the strategies developed in our co-culture platform can be used to engineer co-cultures where the culture composition is controlled electrically. We also show that these strategies can be used to change the culture composition of a synthetic co-culture where each population is working together to produce pyocyanin, thereby changing the rate of pyocyanin production in the co-culture. The techniques developed here may enable further use of co-cultures or synthetic consortia by synthetic biologists and metabolic engineers for varied applications.Item A MATHEMATICAL MODEL TO STUDY THE ROLE OF THE LSR INTERGENIC REGION IN MEDIATION OF AUTOINDUCER-2 QUORUM SENSING IN ESCHERICHIA COLI(2013) Graff, Steven Meyer; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quorum sensing (QS) is a process that allows bacteria to communicate with each other to coordinate collective behavior in response to changes in environmental conditions. Their ability to mediate biofilm formation of biofilms and antibiotic resistance has created challenges on healthcare systems, and an impetus for us to understand QS systems. QS mediated by autoinducer-2 is likely to be the most common of these mechanisms. Recent work has elaborated on the LuxS-regulated (Lsr) system which can mediate and process AI-2 to QS-dependent behaviors, particularly regulatory elements including the lsr intergenic region and the repressor LsrR, the so-called QS"switch". In this thesis, we present a simulation of an example lsr-QS-system to elucidate the role of the lsr intergenic region binding site interactions and how this model integrates with recent literature on LsrR's protein structure to provide further details on the mechanisms of how the switch may operate in real systems.Item New Sensing Modalities for Bacterial and Environmental Phenomena(2013) Betz, Jordan; Rubloff, Gary W; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Intercellular communication is a ubiquitous phenomenon across all domains of life, ranging from archaea to bacteria to eukarya. In bacteria, this is often achieved using small molecules that allow bacteria to sense and respond to environmental cues about the presence, identity, and number of neighboring bacteria. This confers survival and competitive advantages to bacteria by providing a coordinated, population-scale response to a given stimulus in the environment. This dissertation describes the development of a microfluidic system for immobilizing and culturing of cells that also enables control over the genetic composition of the bacteria and their subsequent response to environmental stimuli via a new nonviral nucleic acid delivery mechanism. This nonviral nucleic acid delivery occurs outside the parameter space of traditional nonviral nucleic acid delivery methods such as electroporation and chemical transformation. The bacteria are immobilized in an optically clear alginate hydrogel which simulates the physical and chemical environment normally experienced by bacteria in a biofilm. Complementing the microfluidic cell culture work, surface enhanced Raman spectroscopy (SERS), a label-free vibrational spectroscopic technique that lends itself well to use in aqueous systems, was used to detect bacterial signaling molecules. SERS was performed with three different examples of bacterial communication molecules: the universal quorum sensing molecule autoinducer-2 (AI-2), the species-specific Pseudomonas Quinolone Signal (PQS), and the stationary phase indicator molecule indole. SERS substrates were formed by galvanic displacement, a substrate fabrication method that can be adapted to many SERS applications. Taken together, these new sensing modalities represent a step toward developing systems that allow researchers to investigate, understand, and ultimately control a cell's response to its environment.