Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli 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 in vivo 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 E. coli. In parallel, the
engineered phage expressed luciferase during infection, allowing detection
of E. coli 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.