Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of <i>E. coli</i> in Drinking Water

Modern genome editing methods permit the flexible modification of organisms at the genome level. However, bacteriophages, despite their small genomes, pose unique challenges due to the need to edit during their infection cycle, then select/screen for the modified genomes against a background of the wild type phage. Direct genome synthesis enabled by High-Complexity Golden Gate Assembly (HC-GGA) offers an alternative approach that permits rapid, accurate, and flexible genome modification. Here, we demonstrate HC-GGA's bacteriophage engineering potential, particularly in addressing the public health challenge of detecting hazardous pathogens and nonpathogenic bacteria as indicators of fecal contamination (indicator organisms) in water supplies. A bacteriophage-based biosensor was developed by recoding the genome to enable <i>in vivo</i> incorporation of the alkyne-modified noncanonical amino acid L-homopropargylglycine into the capsid. The modification enabled a bio-orthogonal cycloaddition reaction with azide-conjugated magnetic nanoparticles resulting in magnetized phages which were able to bind, capture, and concentrate their host <i>E. coli</i>. In parallel, the engineered phage expressed luciferase during infection, allowing detection of <i>E. coli</i> at concentrations below 10 CFU per 100 mL in drinking water samples. The approach significantly reduces assay time and cost associated with such assays, particularly in field-based applications, thereby illustrating the practical benefits of synthetic biology in environmental monitoring and public health initiatives.