Synthetic Phages for Identifying and Enumerating Strains (SPIES) of bacterial pathogens

According to the Centers for Disease Control and Prevention, foodborne pathogenic bacteria cause 1.35 million illnesses, 26,500 hospitalizations, and 420 deaths in the United States each year. To address this challenge, there is a strong demand for novel biosensing technologies that can accurately count specific bacteria from food products. Bacteriophages, the widely existing viruses that naturally infect and kill bacteria, have evolved to target specific types of bacteria. This project aims to re-engineer bacteriophages to serve as biosensors for the precise enumeration of pathogenic bacteria. Leveraging advancements in synthetic biology and paper-based sensors, the proposed research will develop Synthetic Phages for Identifying and Enumerating Strains (SPIES) of pathogenic bacteria. SPIES incorporates synthetic gene circuits into the phage genome, allowing precise control over cell breakdown and reporter gene expression levels. These elements are essential for achieving high sensitivity and specificity in targeting pathogenic strains. To expand the impact of this research, the project will integrate teaching and outreach activities focused on promoting diversity and inclusion, improving retention rates, and providing hands-on experiences for K-12 students. This project not only aims to advance scientific knowledge but also contributes to the national interest by promoting the technology advancement in a real-world context, enhancing public health and safety, and supporting educational and societal welfare. Current methods for detecting and counting pathogenic bacteria, such as culture-based methods, genotyping tests, and existing phage-based sensors, encounter accuracy challenges and necessitate trained personnel, specialized laboratory equipment, and time-consuming processes. The proposed SPIES technology aims to overcome these obstacles by developing synthetic phages that can express reporter genes in direct response to specific bacteria. This will be achieved by integrating toehold riboswitches into the phage genome, enabling translational-level regulation of the reporter gene, which will be activated upon detection of mRNA molecules specific to the target bacteria. Additionally, the synthetic phage will utilize a transcriptional repressor to suppress the expression of phage genes associated with host cell lysis, thereby facilitating the quantification process. By incorporating these genetic-level regulations, the engineered phage can accurately identify pathogenic Shiga toxin-producing Escherichia coli and distinguish highly virulent serotypes, such as E. coli O157. A paper-based sensing platform will be employed to store the synthetic phages, carry out phage infection, and count the infected bacterial cells. Through the integration of these innovative strategies, the SPIES technology introduces a novel bacterium sensing paradigm. It offers rapid assay time, cost-effective sensing, high specificity, and the remarkable capacity to directly count single cells with minimal user interventions. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.