The B. subtilis SCK6 strain was inoculated into 3 ml of LB medium with 1 µg ml−1 erythromycin in a test tube. The cells were cultivated at 37°C with shaking at 200 r.p.m. overnight (∼12 h). The culture was then diluted to A600 at 1.0 in a fresh LB medium containing 1% (w/v) xylose and then grown for 2 h. The resulting cell culture was ready to be transformed or divided into aliquots and stocked at −80°C with 10% (v/v) glycerol.
One microlitre of the PCR product containing plasmid multimers was mixed with 100 µl of the supercompetent cells in a plastic test tube and cultivated at 37°C with shaking at 200 r.p.m. for 90 min. An aliquot of the transformed cells was diluted by 103‐ to 104‐fold for estimating the transformation efficiency. To facilitate the further segregation of different clones in the same cell (Shafikhani et al., 1997 (link)), the rest of the transformed cells were diluted into 10 ml of M9 minimal medium containing 5 µg ml−1 chloramphenicol and grown at 37°C for 14 h. The cell cultures were then diluted by 20‐fold with the same medium and grown for an additional 8 h. The serial diluted transformants were spread on LBR plates containing 5 µg ml−1 chloramphenicol and incubated at 37°C for 20 h. Positive colonies that were surrounded with big and clear halo zones were selected for further characterization and DNA sequencing using the primer pair P9/P10.
One microlitre of the PCR product containing plasmid multimers was mixed with 100 µl of the supercompetent cells in a plastic test tube and cultivated at 37°C with shaking at 200 r.p.m. for 90 min. An aliquot of the transformed cells was diluted by 103‐ to 104‐fold for estimating the transformation efficiency. To facilitate the further segregation of different clones in the same cell (Shafikhani et al., 1997 (link)), the rest of the transformed cells were diluted into 10 ml of M9 minimal medium containing 5 µg ml−1 chloramphenicol and grown at 37°C for 14 h. The cell cultures were then diluted by 20‐fold with the same medium and grown for an additional 8 h. The serial diluted transformants were spread on LBR plates containing 5 µg ml−1 chloramphenicol and incubated at 37°C for 20 h. Positive colonies that were surrounded with big and clear halo zones were selected for further characterization and DNA sequencing using the primer pair P9/P10.
