Patent ID: 8067239

Claim:
A method of producing an attenuated pathogenic microorganism, comprising: (a) introducing a vector into a pathogenic microorganism, the vector comprising (i) a λ exo and a λ bet nucleotide sequences encoding bacteriophage λ Red recombinase; (ii) a λ gam nucleotide sequence encoding bacteriophage anti-RecBCD; (iii) a Ptac promoter sequence operably linked to the nucleotide sequence of (i) and (ii); and (iv) a nucleotide sequence encoding Lad operably linked to its native promoter; and (v) at least one origin of replication sequence which confers low copy number on the vector; (b) introducing a substrate into the pathogenic microorganism, wherein the substrate comprises recombination segments comprising nucleotide sequences homologous to a gene required for pathogenicity or surrounding native sequences; and (c) culturing the microorganism under conditions such that recombination between the substrate and the gene sequences or surrounding native sequences occurs; such that the gene required for pathogenicity is mutated, thereby producing an attenuated pathogenic microorganism, and wherein the pathogenic microorganism is a strain of Escherichia coli which is pathogenic to humans, animals, or plants.