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 GENETICALLY ENGINEERED PROBIOTICS FOR DIAGNOSTICS AND DRUG DELIVERY: APPLICATIONS FOR CROHN’S DISEASE(2018) McKay, Ryan; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the history of medicine, therapies have evolved while their mode of delivery has remained largely static. Generally, the active ingredient is formulated with an excipient to confer stability, and is ultimately delivered orally or intravenously in most applications. Crohn’s disease (CD), an illness with increasing global prevalence characterized by chronic inflammation of the intestines, is commonly treated with intravenously administered biologics. When these medicines spread throughout the body, only a small percentage acts at the desired site and side effects often arise. Thus, a targeted system is desired to localize treatment at sites of colonic inflammation. There is an entire field dedicated to localized delivery that typically employs drug-laden particles or capsules that can respond to local chemical or physical cues. We believe that bacteria can be “programmed” to respond analogously, and ultimately synthesize and deliver therapeutics. Nitric oxide (NO) levels are elevated at sites of intestinal inflammation, and thus serves as a targeting molecule that can attract programmed bacteria via a process called pseudotaxis. This is achieved by rewiring the native motility circuits of bacteria to respond to high NO levels. Additionally, localized treatment is attained by an NO- specific response whereby the designed bacteria produce and secrete a human protein reported to reduce inflammation in CD patients. This system may improve CD treatment via: 1) site-specific targeting to minimize side effects and increase efficacy, 2) in situ synthesis of the therapeutic avoids payload loss in the digestive tract and manufacturing obstacles associated with biologics, 3) probiotics are reported to provide innate benefits to CD patients, and 4) oral delivery is preferred by patients versus intravenous. We have also developed probiotics that fluoresce in response to NO which may serve as an ingestible biosensor for CD. We believe these reporter probiotics can assist in the diagnosis of CD by utilizing visualization of bacteria in a stool sample to reduce the need for invasive colonoscopies and biopsies. Overall, we have developed a platform of probiotic cells that respond to NO with applications for Crohn’s disease in mind, translating to noninvasive methods for both the diagnosis and treatment of CD.Item TOWARDS A GENETICALLY-ENGINEERED BACTERIUM FOR GASTROINTESTINAL WOUND HEALING(2017) Virgile, Chelsea; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Society and physicians frequently associate the increase of antibiotic-resistant bacteria with the overuse of antibiotics. This proposes a question, “Why use antibiotics to fight bacteria and risk resistance, when one could engineer bacteria to target and kill infectious bacteria?” Bacteria are often thought of as ‘good’ bacteria (e.g., commensals, probiotics) or ‘bad’ bacteria (e.g., pathogens). Synthetic biology enables the augmentation of biosynthetic capabilities and retooling of regulatory structures in the creation of cells with unprecedented ability to make products. One can also, however, think of the cell as the product – a cell that operates in a noisy environment to execute non-native tasks. There have been several recent reports of the rewiring of bacterial cells to function as conveyors of therapeutics. The engineering and rewiring of the bacteria such as E. coli into ‘smart' bacteria potentially allows for a broad range of applications, from the treatment of wounds, the elimination of pathogenic strains, to the delivery of vaccines, particularly in the gastrointestinal (GI) tract. I have engineered smart bacteria as a therapeutic delivery vehicle for wound healing in the GI tract. The approach comprises synthetic biology and microfluidics for the creation of a biological ‘test track' for ensuring the appropriate design and testing of engineered bacteria. Bacterial motility was engineered for response to wound-generating signals such as hydrogen peroxide. Specifically, we have placed a motility enzyme CheZ under the control of the hydrogen-peroxide-responsive oxyR/S gene-promoter pair so that the ‘run’ in the tumble and run scheme of bacterial movement is externally regulated. These engineered cells exploit pseudotaxis for directional swimming towards hydrogen peroxide, a non-native signal. Additionally, the therapeutic enzyme transglutaminase plays an important role in the tissue clotting cascade. Microbial transglutaminase can crosslink fibrinogen, similar in function to human transglutaminases during the clotting cascade, but independently of calcium ions. This allows for a potentially faster, increased wound-healing response. By combining microbial transglutaminase expression with controlling motility and lysis expression using the OxyR/S system, the ‘smart’ bacteria can potentially swim towards and treat at the wound site with subsequent cell lysis. Ultimately, this strategy can lead to new bacterial therapies.Item Bioengineered conduits for directing digitized molecular-based information(2015) Terrell, Jessica Lynn; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular recognition is a prevalent quality in natural biological environments: molecules- small as well as macro- enable dynamic response by instilling functionality and communicating information about the system. The accession and interpretation of this rich molecular information leads to context about the system. Moreover, molecular complexity, both in terms of chemical structure and diversity, permits information to be represented with high capacity. Thus, an opportunity exists to assign molecules as chemical portrayals of natural, non-natural, and even non-biological data. Further, their associated upstream, downstream, and regulatory pathways could be commandeered for the purpose of data processing and transmission. This thesis emphasizes molecules that serve as units of information, the processing of which elucidates context. The project first strategizes a biocompatible assembly process that integrates biological componentry in an organized configuration for molecular transfer (e.g. from a cell to a receptor). Next, we have explored the use of DNA for its potential to store data in richer, digital forms. Binary data is embedded within a gene for storage inside a cell carrier and is selectively conveyed. Successively, a catalytic relay is developed to transduce similar data from sequence-based DNA storage to a delineated chemical cue that programs cellular phenotype. Finally, these cell populations are used as mobile information processing units that independently seek and collectively categorize the information, which is fed back as fluorescently ‘binned’ output. Every development demonstrates a transduction process of molecular data that involves input acquisition, refinement, and output interpretation. Overall, by equipping biomimetic networks with molecular-driven performance, their interactions serve as conduits of information flow.Item Bridging the biology-electronics communication gap with redox signaling(2015) Gordonov, Tanya; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Electronic and biological systems both have the ability to sense, respond to, and communicate relevant data. This dissertation aims to facilitate communication between the two and create bio-hybrid devices that can process the breadths of both electronic and biological information. We describe the development of novel methods that bridge this bi-directional communication gap through the use of electronically and biologically relevant redox molecules for controlled and quantitative information transfer. Additionally, we demonstrate that the incorporation of biological components onto microelectronic systems can open doors for improved capabilities in a variety of fields. First, we describe the original use of redox molecules to electronically control the activity of an enzyme on a chip. Using biofabrication techniques, we assembled HLPT, a fusion protein which generates the quorum sensing molecule autoinducer-2, on an electrodeposited chitosan film on top of an electrode. This allows the electrode to controllably oxidize the enzyme in situ through a redox mediator, acetosyringone. We successfully showed that activity decrease and bacterial quorum sensing response are proportional to the input charge. To engineer bio-electronic communication with cells, we first aimed for better characterizing an electronic method for measuring cell response. We engineered Escherichia coli (E.coli) cells to respond to autoinducer-2 by producing the β-galactosidase enzyme. We then investigated an existing electrochemical method for detecting β-galactosidase activity by measuring a redox-active product of the cleavage of the added substrate molecule PAPG. In our novel findings, the product, PAP, was found to be produced at a rate that correlated with the standard spectrophotometric method for measuring β-galactosidase, the Miller assay, in both whole live and lysed cells. Conversely, to translate electronic signals to something cells can understand, we used pyocyanin, a redox drug which oxidizes the E.coli SoxR protein and allows transcription from the soxS promoter. We utilized electronic control of ferricyanide, an electron acceptor, to amplify the production of a reporter from soxS. With this novel method, we show that production of reporter depends on the frequency and amplitude of electronic signals, and investigate the method’s metabolic effects. Overall, the work in this dissertation makes strides towards the greater goal of creating multi-functional bio-hybrid devices.