Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella

A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of “locked” recombinant phages that carry foreign DNA but which cannot propagate themselves in nature.