Figure 10.

The use of sacB as a counterselection marker in recombineering. The cat-sacB cassette is used as a template for PCR to generate an amplicon that has the cassette flanked by 50 bp of target homology. The recombineering event can either insert the cassette into the target gene, or replace sequences within the target gene with the cassette. After selection for chloramphenicol resistance and verification of sucrose sensitivity, the modified strain is electroporated with either a dsDNA substrate or an oligo that contains the desired mutation (red rectangle). The modified strain is selected by resistance to sucrose and screened for sensitivity to chloramphenicol.