Comparative efficiency of the Ts-sacB system using zeo and kan disrupted allelic exchange substrates in M. chelonae ATCC 35752.

One to three transformants (T1, T2 and T3) were selected on plates upon transformation with the pPR27-derived plasmids, grown in 7H9-OADC broth at 30°C for 5 to 7 days, and finally plated onto 7H11-OADC containing Kan or Zeo and 10% sucrose at 37°C. The percentage of CFUs presenting the expected phenotype for allelic exchange mutants at the last selection step of the Ts-SacB procedure (sucrose resistant; KanR or ZeoR and XylE−) is indicated for each construct. Four to ten candidate mutants were analyzed by PCR in each case and the number of double crossover mutants identified is indicated in the last column.