Zhang, Qiuge2024-07-242024-07-242022-05https://hdl.handle.net/11299/264380University of Minnesota Ph.D. dissertation. May 2022. Major: Chemical Engineering. Advisors: Casim Sarkar, Samira Azarin. 1 computer file (PDF); x, 136 pages.Bacteria can be engineered as vehicles for context-dependent protein production, enabling the diagnosis and treatment of numerous diseases. However, translation to clinical applications still requires concurrent efforts to enhance tunability, specificity, efficacy, and safety. Our work contributes to three such areas. First, we performed model-guided engineering of DNA sequences with predictable site-specific recombination (SSR) rates, demonstrating that recombinase attachment sites with predictable SSR rates could be used to achieve kinetic control in gene circuit design. This high-throughput, data-driven method enhanced our understanding of recombinase function and expanded the synthetic biology toolbox, and it can enable rational tuning of the response dynamics of bacterial diagnostics and the pharmacokinetic profile of bacterial therapeutics. Second, we engineered Escherichia coli to sense extracellular proteins. A cell-surface sensor is crucial to enable broad diagnostic and therapeutic applications of bacteria; however, Gram-negative bacteria such as E. coli can only detect small molecules that cross the cell envelope. We proposed a novel strategy that leverages protein-specific attenuation of maltodextrin uptake via engineered LamB porins to modulate intracellular maltose signaling, thereby linking the extracellular protein concentration to the cell response. We then demonstrated its modularity to detect different target proteins and its tunability to alter the dose-response curves. Endowing E. coli with this ability to sense extracellular proteins would enable detection of clinically relevant proteins both in vitro and in vivo. Finally, E. coli strain Nissle 1917 (EcN) is a versatile probiotic that is safe for human consumption; however, its use in diagnostic and therapeutic oral delivery applications is hindered by the low pH and high concentration of bile salts in the gastrointestinal tract that reduce EcN viability. We performed adaptive laboratory evolution on EcN to select for mutated populations with greater tolerance to pH and bile and we identified specific mutations that contribute to their enhanced viability in the presence of these chemical stressors. The developed strains may not only enhance the bioavailability of orally administered EcN but also modulate their interactions with intestinal cells, suggesting potential applications to improve gut health. In summary, through biomolecular engineering, genetic circuit design, mechanistic and data-driven modeling, and strain development, our research has contributed to improved understanding and engineering of bacterial properties that should accelerate their translation to diagnostic and therapeutic applications.enBacterial TherapySynthetic BiologyThesis or Dissertation