FIGURE 1.

Scheme of the oriC-specific recombineering method. Recombineering strains harbor sacB and cat genes replacing all of the oriC sequence, inducible genes encoding the lambda RED system, and a plasmid origin of replication (R1ori) linked to a kanamycin resistance determinant. The strain also has a deletion in the dnaA gene. For insertion of oriC mutants into the chromosome, a PCR fragment carrying the desired mutation is electroporated to transform cells in which the RED system was induced. Recombination results in replacement of the sacB and cat genes with the mutated oriC. Successful recombineering confers the ability to grow in the presence of sucrose, and sensitivity to chloramphenicol.