Fig. 2
Gene replacement via allelic exchange at the ca_c1502, upp and ca_c3535 loci. PCR confirmation of the different double-crossover deletion mutants using external primers annealing to the chromosome upstream and downstream of each deletion cassette used in the different pREP plasmids: a Δca_c1502 deletion mutants with CAC-0 + CAC-5 primers, b Δupp deletion mutants with UPP-0 + UPP-5 primers, c Δca_c3535 deletion mutants with RM-0 + RM-5 primers (lanes 1, 2 and 3). For each experiment, lanes 2 and 3 refer to before and after excision of the MLSR marker by the FLP recombinase, respectively, giving finally an unmarked deletion mutant (Δ-unm). The previous unmarked deletions in the final Δca_c1502 Δupp Δca_c3535 were confirmed with CAC-0 + CAC-5 (c, lane 4) and UPP-0 + UPP-5 (c, lane 5) primers. Lane M, 1 kb DNA ladder (0.5–10 kb) (NEB)