Fig. 8

The sketch displays the procedure for the deletion of the target gene on a chromosome using the dif/Xer recombination system. The shaded regions represented homology between the integration cassette and the genes flanking the target gene. zeo, the zeocin resistance gene zeo. The front (up-sequence) and back (down-sequence) regions flanking the target gene to be deleted were amplified from the chromosome of B. subtilis 168, and the zeo sequence was cloned from plasmid p7Z6. These three fragments were joined by PCR; the resulting PCR products were transformed into B. subtilis 168, and zeo ^r transformants were selected. After subculturing, Xer recombinants were selected