Nicking of target plasmid by CisA

Three transformations were carried out as shown in Fig. 2A: the intact target plasmid into cells without the chromosomal gene for CisA, the target plasmid without initiation and termination sequences into cells with the chromosomal gene for CisA, and the intact target plasmid into cells with the chromosomal gene for CisA. Transformant colonies were selected on LB agar plates supplemented with chloramphenicol (15 ng/μl) and inoculated to liquid LB cultures with chloramphenicol. After overnight growth in 37°C shaken at 225 rounds per minute (the same growth conditions from here on unless specified otherwise), 1 ml of culture was purified and eluted to 40 μl of elution buffer, and 5 μl was used for electrophoresis (0.8% agarose gel, 110 V, 40 min; Bio-Rad Mini-Sub Cell GT Systems). An image was taken using UVP BioDoc-It Imaging System and analyzed using ImageJ to calculate fraction of the nicked target plasmid with the following equation: brightness of the band for nicked DNA (brightness of the band for nicked DNA + brightness of the band for supercoiled DNA + brightness of the band for linear DNA).