Fig. 3

The transformation efficiency of P. aeruginosa PAO1 with pBSP11Tc^R under various electroporation conditions. The maximum efficiency was achieved at 2.0 kV using a 1 mm gap cuvette at 4 °C or at 2.2 kV using 2 mm cuvette at 23 °C. The field strength applied at 4 °C in a 1 mm gap cuvette is 20 kV/cm. Since the membrane permeation voltage is doubled at 4 °C as compared to room temperature [17], the field strength at 23 °C in a 2 mm cuvette (11 kV/cm) is equivalent to 22 kV/cm at 4 °C. Therefore, the optimum field strength is determined to be 20–22 kV/cm (4 °C equivalent). Since the 2 mm gap cuvette is less prone to arcing, electroporation at 2.2 kV using a 2 mm gap cuvette at room temperature was used to transform the suicide pEX18Tc allelic exchange construct into P. aeruginosa PAO1. Pulse durations of 4.0 to 4.5 ms were used in most experiments