Figure 1.

Minicircle (MC) preparation. MCs containing a single vTopo cleavage site (CCCTT) were prepared by inserting an engineered DNA sequence between the attP and attB λ integration sites in plasmid pattD to give pMC454 (the attP and attB sites are in the direct orientation). The engineered plasmid sequence was devoid of any other pentapyrimidine sequences that might serve as good cleavage sites but also includes engineered nicking sites (Nb. BbvC1) and a zinc finger binding motif for future mechanistic explorations (see the Discussion). For the propagation and recovery of the MC plasmids, pMC454 was transformed into bacterial strain LZ54, which is lysogenic for λ bacteriophage, and integrase expression was induced by heat shock. The addition of norfloxacin results in decatenation of the circular products of the disintegration reaction, and MCs are purified by preparative agarose gel electrophoresis. The products of the integration reaction and purified MC^sp (454 bp) are shown. Gel electrophoresis was performed using a 1% agarose gel with visualization by EtBr staining.