A. James Clark School of Engineering
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Item CONTEXTUALIZATION OF THE E. COLI LSR SYSTEM: RELATIVE ORTHOLOGY, RELATIVE QS ACTIVITY, AND EMERGENT BEHAVIOR(2015) Quan, David Nathan; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Within bacterial consortia there exist innumerable combinatorial circumstances, some of which may tip the scale toward pathogenicity, some of which may favor asymptomatic phenotypes. Indeed, the lines and intersections between commensal, pathogenic, and opportunistic bacteria are not always clean. As a foothold to mediate pathogenicity arising from consortia, many have puzzled at communication between bacteria. Primary among such considerations is quorum sensing (QS). Analogous to autocrine signaling in multicellular organisms, QS is a self-signaling process involving small molecules. Generally, QS activation is believed to have pleiotropic effects, and has been associated with numerous pathogenic phenotypes. The research herein focuses on autoinducer-2 (AI-2) based QS signaling transduced through the Lsr system. Produced by over 80 species of bacteria, AI-2 is believed to be an interspecies signaling molecule. Outside of the marine bacteria genera Vibrio and Marinomonas, the only known AI-2 based QS transduction pathway is the Lsr system. We sought to deepen the characterization of the Lsr system in contexts outside of the batch cultures in which it was originally defined. First, we interrogated E. coli K-12 W3110 Lsr system orthologs relative to the same strain's lac system. Both systems are induced by the molecule which they import and catabolize. We searched for homologs by focusing on the gene order along a genome, as gene arrangement can bear signaling consequences for autoregulatory circuits. We found that the Lsr system signal was phylogenetically dispersed if not particularly deep, especially outside of Enterobacteriales and Pasteurellaceaes, indicating that the system has generally been conferred horizontally. This contrasts with the lac system, whose signal is strong but limited to a select group of highly related enterobacteria. We then modeled the Lsr system with ODEs, revealing bimodality in silico, bolstering preliminary experimental evidence. This bifurcated expression was seen to depend upon nongenetic heterogeneity, which we modeled as a variation of a single compound parameter, basal, representing the basal rate of AI-2 flux into the cell through a low flux pathway. Moreover, in our finite difference-agent based models, bimodal expression could not arise from spatial stochasticity alone. This lies in contrast with the canonical LuxIR QS system, which employs an intercellular positive feedback loop to activate the entire population. We examined the consequences of this contrast, by modeling both systems under conditions of colony growth using finite difference-agent based methods. We additionally investigated the confluence of Lsr signaling with chemotactic sensitivity to AI-2, which has been demonstrated in E. coli. Finally, the consequences of bimodality in interspecies interactions were assessed by posing two populations containing different Lsr systems against each other. While few natural consortia consist of only two interacting bacteria, these studies indicate that AI-2 based Lsr signaling may mediate a multitude of transitional intraspecies and interspecies bacterial dynamics, the specifics of which will vary with the context and the homologs involved.Item Design and Implementation of Microfluidic Systems for Bacterial Biofilm Monitoring and Manipulation(2014) Meyer, Mariana; Ghodssi, Reza; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms - pathogenic matrices formed through bacterial communication and subsequent extracellular matrix secretion - characterize the majority of clinical bacterial infections. Biofilms exhibit increased resistance to conventional antibiotics, necessitating development of alternative treatments. Standard microbiological methods for studying biofilms often rely on in vitro systems with involved instrumentation for biofilm quantification, or destroy the biofilm in the process of characterization. Additionally, biofilm formation is sensitive to many growth parameters, and can exhibit a large degree of variability between repeated experiments. This dissertation presents the development of systems designed to address these challenges through integration of continuous biofilm monitoring in a microfluidic platform, and through creation of a microfluidic platform for multiple assays performed on one biofilm formed in a single channel. The microsystems developed in this work provide building blocks for developing controlled, high throughput testbeds for development and evaluation of drugs targeting bacterial biofilms. The first platform developed relied on optical density monitoring as a means for evaluating biofilm formation. This method was noninvasive, as it used an external light source and array of photodiodes to evaluate biofilms by the amount of light transmitted through the microfluidic channel where they were grown. The optical density biofilm measurement method and microfluidic platform were used to evaluate the dependence of biofilm formation on quorum sensing, an autoinducer-mediated intercellular communication process. This system was also used in the first demonstration of biofilm inhibition and reduction by two different autoinducer-2 analogs. The second microfluidic system developed addressed the challenge of variability in biofilm formation. Biofilms formed in a single microfluidic channel were partitioned by hydraulically actuated valves into three separate segments, which were then treated as representatives of the original biofilm in further experiments. A novel photoresist passivation process was developed in order to create the multi-depth channels needed to accommodate both valve actuation and biofilm formation. Biofilms grown in the device were uniform throughout, providing reliable experimental controls within the system. Biofilm partitioning was demonstrated by exposing three segments of one biofilm to varying detergent concentrations.Item Biological Nanofactories: Altering Cellular Response via Localized Synthesis and Delivery(2008-11-19) Fernandes, Rohan; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Conventional research in targeted delivery of molecules-of-interest involves either packaging of the molecules-of-interest within a delivery mechanism or pre-synthesis of an inactive prodrug that is converted to the molecule-of-interest in the vicinity of the targeted area. Biological nanofactories provide an alternative approach to targeted delivery by locally synthesizing and delivering the molecules-of-interest at surface of the targeted cells. The machinery for synthesis and delivery is derived from the targeted cells themselves. Biological nanofactories are nano-dimensioned and are comprised of multiple functional modules. At the most basic level, a biological nanofactory consists of a cell targeting module and a synthesis module. When deployed, a biological nanofactory binds to the targeted cell surface and locally synthesizes and delivers molecules-of-interest thus altering the response of the targeted cells. In this dissertation, biological nanofactories for the localized synthesis and delivery of the 'universal' quorum sensing signaling molecule autoinducer-2 are demonstrated. Quorum sensing is process by which bacterial co-ordinate their activities at a population level through the production, release, sensing and uptake of signaling autoinducers and plays a role in diverse bacterial phenomena such as bacterial pathogenicity, biofilm formation and bioluminescence. Two types of biological nanofactories; magnetic nanofactories and antibody nanofactories are presented in this dissertation as demonstrations of the biological nanofactory approach to targeted delivery. Magnetic nanofactories consist of the AI-2 biosynthesis enzymes attached to functionalized chitosan-mag nanoparticles. Assembly of these nanofactories involves synthesis of the chitosan-mag nanoparticles and subsequent assembly of the AI-2 pathway enzymes onto the particles. Antibody nanofactories consist of the AI-2 biosynthesis enzymes self assembled onto the targeting antibody. Assembly of these nanofactories involves creation of a fusion protein that attaches to the targeting antibody. When added to cultures of quorum sensing bacteria, the nanofactories bind to the surface of the targeted cells via the targeting module and locally synthesize and deliver AI-2 there via the synthesis module. The cells sense and uptake the AI-2 and alter their natural response. Prospects of using biological nanofactories to alter the native response of targeted cells to a 'desired' state, especially with respect to down-regulating undesirable co-ordinated bacterial response, are envisioned.Item Rewiring Quorum Sensing Circuitry for Recombinant Protein Production in E. coi(2007-08-08) Tsao, Chen-Yu; Bentley, William E.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The global objective of this research is to rewire the circuitry of bacterial quorum sensing to facilitate recombinant protein production in bacteria. Previous research has shown that the activity of AI-2, the putative "universal" bacterial autoinducer, decreased in culture fluids when several proteins were overexpressed in E. coli W3110, suggesting bacteria communicate or possibly potentiate the "metabolic burden" associated with protein overexpression. Additionally, conditioned medium obtained from LuxS+ and LuxS- strains was added to these cultures, resulting in a 2-4 fold increase in specific yield for both chloramphenicol acetyltransferase (CAT) and organophosphorus hydrolase (OPH). These simple observations set the stage for examining the role of quorum sensing in recombinant protein expression systems and also suggested that "rewiring" the quorum sensing circuitry would lead to significant improvement of yield. In this dissertation, we have inserted luxS into expression vectors (IPTG inducible) which can co-synthesize target recombinant proteins (arabinose inducible) to accomplish the modulation of the metabolic landscape for protein synthesis via altered AI-2 signaling. Our results show significant enhancement in both protein yield and activity, and reveal a strong linkage between bacterial cell communication and cellular processes involved in synthesis and folding of recombinant proteins in E. coli. Second, we have attempted to rewire the native quorum sensing signaling circuitry and couple it to the widely-used T7 expression system for constructing an autoinducible recombinant protein expression platform. We demonstrate, for the first time, true autoinduction of recombinant proteins in E. coli or, in fact, any expression system. That is, our results showed that GFPuv, CAT, and LacZ were all expressed using this innovative system without cell growth monitoring or inducer addition.