TRANSFORMATION OF PSEUDOMONAS-PUTIDA BY ELECTROPORATION

The optimum electrotransformation conditions were determined for Pseudomonas putida PpY101 with plasmid pSUP104 (9.5kb) and pSR134 (18.6kb). Field strength was a very important parameter for electrotransformation efficiency. Optimum efficiencies (1.1x10(5) transformants/mu g DNA) with pSUP104 and pSR134 were obtained at a field strength of 12.5 kV/cm, a time constant of about 4.5 ms (resistance setting of 200 Omega), a supercoiled DNA concentration of 100ng/ml, and a cell concentration of 10(9)/ml. Because the efficiency obtained is high enough, electrotransformation is useful for the direct cloning of P. putida PpY101. No significant relationship between plasmid size and electrotransformation efficiency was observed. These efficiencies were about 4.5 times higher than those using the MgCl2, method. Under these conditions, electrotransformation efficiencies of relaxed plasmid DNA treated with topoisomerase I and that linearized by EcoRI digestion were high.