Foodborne and waterborne illnesses are the most common causes of death in the developing world. Rapid pathogen detection is one technique used to prevent these infections. Portable biosensors that give visual readouts of bacterial concentration can address this challenge, but to be appropriate for the low resource areas that are disproportionately affected by these diseases they must meet many disparate criteria, including low cost, ease of use, high sensitivity, and high throughput. Bacteriophages, tuned by billions of years of evolution to efficiently recognize bacteria, are readily engineered using molecular biology techniques for use in biosensors that meet these stipulations. The separation and concentration of bacteria from their matrix remains the bottleneck to rapid detection. While we have previously demonstrated that the use of phage-based magnetic separation is superior to antibody-based separation of bacteria, there is still a need for a more efficient method to conjugate the phage to the magnetic particle. We are developing a T7 bacteriophage-based biosensor that uses site-specific artificial amino acid incorporation and bioorthogonal azide-alkyne cycloaddition to enable efficient conjugation to magnetic particles and thus allow the separation and detection of bacteria in liquid samples. To this end, we have synthesized the artificial alkyne-containing amino acid propargylglyoxy phenylalanine, incorporated it into T7 capsid proteins, and demonstrated its reactivity in vitro. This engineered protein will allow the production of phages that bind efficiently and specifically to azide functionalized magnetic beads, creating biosensors that effectively concentrate the bacteria in a sample. Previous research in the Nugen lab has shown that incorporation of the reporter enzyme nanoluciferase into the T7 genome allows for rapid quantitation of low levels of bacteria in solution. By performing the conjugation and separation as described above in combination with this reporter technology, we hope to enhance the limit of detection of the assay to the 10<sup>-2</sup> cfu/ml standard for drinking water safety while retaining a same-day timeframe. This research shows that artificial amino acids can be incorporated site-specifically into viral proteins during the infection cycle, a finding that can be applied in many contexts, including biotechnology and nanosensors. This technique of expanding a viral genetic code is also applicable in many synthetic biology contexts.<br/>

Engineering click-conjugated bacteriophages on magnetic nanoparticles for rapid detection of waterborne pathogens

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.