We have previously constructed a recombinant bacterium expressing a modified lipopolysaccharide (LPS) mimicking the Shiga toxin receptor, which binds toxin with high avidity. This involved cloning Neisseria galactosyl transferase genes (lgtC and lgtE) in pK184 in a derivative of Escherichia coli R1 (CWG308). Such constructs have considerable potential for prevention of disease caused by Shiga toxin-producing E. coli (STEC). However, neither the E. coli host strain nor the expression plasmid is suitable for human use, because the former is derived from a clinical isolate and the latter contains a kanamycin-resistance gene. We have constructed, as a prelude to human trials, a nonpathogenic E. coli K-12 C600 derivative with deletions in waaO and waaB, such that it has the same LPS core structure as CWG308. We also deleted the thyA gene from this strain, rendering it thymine dependent. The kanamycin-resistance gene was also deleted from pK184 and was replaced with Salmonella typhimurium thyA. Neisseria lgtCE was then cloned into this plasmid and transformed into C600 Delta waaOB Delta thyA. The plasmid was stably maintained, and the construct produced a modified LPS and neutralized Stx1 and Stx2c. Moreover, mice challenged with an otherwise fatal dose of STEC were completely protected by oral administration of the novel construct.