Transformation of Pseudomonas putida by electroporation.
The optimum electrotransformation conditions were determined for Pseudomonas putida PpY101 with plasmid pSUP104 (9.5 kb) and pSR134 (18.6 kb). Field strength was a very important parameter for electrotransformation efficiency. Optimum efficiencies (1.1 x 10(5) transformants/micrograms 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 ohms), a supercoiled DNA concentration of 100 ng/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.
Iwasaki
K
K
Regional and Community Environment Division, National Institute for Environmental Studies, Ibaraki, Japan.
Uchiyama
H
H
Yagi
O
O
Kurabayashi
T
T
Ishizuka
K
K
Takamura
Y
Y
D001709:Biotechnology; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D008627:Mercuric Chloride / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics*; D014170:Transformation, Genetic
D008627:Mercuric Chloride