Figure 2.

Scheme of in-frame markerless deletion method to construct the srf gene cluster mutants in B amyloliquefaciens Ba01. Single crossover recombinants were obtained by culturing the transformants at restrictive temperature (37°C) for plasmid pRY1 integration into Ba01 genome and using mls medium for selection. To evict the plasmid from Ba01 genome after homologous recombination, the strain was incubated overnight in 3 ml of LB broth at a permissive temperature (25°C) for plasmid replication. The cell cultures were diluted and plated on LB agar at 37°C for 24 h The colonies were patched on mls medium to select mls-sensitive colonies. The srf gene cluster mutants were checked by PCR with primers JC1783/1784.