Figure 1.

Method used to integrate deletion-units. (A) Multiple deletion strain at step N was used as the recipient for P1 transduction. A deletion-unit strain was used as the donor of deletions. (B) Integrated new deletion units were selected as to the chloramphenicol resistance of the marker cassette. Transduction of the most distal deletion from the marker cassette was confirmed by PCR. (C) The marker cassette including B. subtilis sacB was replaced by a DNA fragment without the marker cassette using a λ red recombination system or P1 transduction using a P1 phage prepared from a markerless-unit strain.¹⁹ A strain with additional deletions (strain at N + 1 step) was selected as to its sucrose resistance. Each circle represents an E. coli genome and wedges denote deletions. The white arrow represents the sacB_cat marker cassette.