Application of an optimized electroporation procedure for replacement of the polyhydroxyalkanoate synthase I gene in Nocardia corallina.
To develop a system for gene replacement in Nocardia corallina, a protocol for electroporation was optimized by systematic alterations of growth conditions, field strength, time constant and the electroporation buffer. Transformation efficiencies of 0.5 x 10(6) - 3 x 10(6) transformants/microgram plasmid DNA were obtained routinely. The gene encoding the polyhydroxyalkanoate (PHA) synthase I of N. corallina was cloned and interrupted by insertion of a kanamycin-resistance gene. The resulting plasmid was introduced into N. corallina by electroporation to inactivate the wild-type gene by homologous recombination. Kanamycin-resistant clones were screened by Southern hybridization for the absence of the wild-type gene and analyzed for PHA accumulation.
Valentin
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Department of Biology, James Madison University, Harrisonburg, VA 22807, USA.
Dennis
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D000217:Acyltransferases / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D006880:Hydroxy Acids / Q000378:metabolism; D007612:Kanamycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009615:Nocardia / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols / Q000494:pharmacology; D014169:Transformation, Bacterial; D014631:Valerates / Q000378:metabolism
D017931:DNA Primers; D006880:Hydroxy Acids; D014631:Valerates; D011092:Polyethylene Glycols; D007612:Kanamycin; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D005947:Glucose