A schematic figure over the construction of the suicide plasmid and its incorporation into and it’s the removal from the genome.

(A) Amplification of the mutated wbeT gene the upstream DNA was generated to be fused together in a primerless PCR. (B) The fused sequenced were used in another primerless PCR together with the kanamycin resistant gene flaked by FRT–sites and the downstream sequenced of wbeT gene. (C) The result of the second primerless PCR used to be incorporated into the pMT–suicide–sacB plasmid for integration into the genome of O1 V. cholerae. (D) Primer orientation of the wbe region before and (E) after gene manipulation.