A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation.
A rapid microcentrifuge-based method is described for preparation of Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold increased transformation efficiencies over existing procedures. This increased efficiency now enables the use of transformation for all applications requiring DNA transfer. These include transfer of chromosomal mutations marked with antibiotic resistance genes between P. aeruginosa strains, which solves the riddle of not having an efficient and reliable transduction procedure for this bacterium. Not surprisingly, the method also allows for very efficient transformation with replicative plasmids, with transformation efficiencies ranging from 10(7) to >10(11) transformants per microgram of DNA. Lastly, with efficiencies of up to >10(3) transformants per microgram of DNA the method replaces in most instances conjugation for the transfer of non-replicative plasmids used in gene replacement, site-specific gene integration and transposon mutagenesis experiments.
Choi
Kyoung-Hee
KH
Department of Microbiology, Immunology and Pathology, Colorado State University, 1682 Campus Delivery, Fort Collins, CO 80523, USA.
Kumar
Ayush
A
Schweizer
Herbert P
HP
D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D014170:Transformation, Genetic
D004269:DNA, Bacterial