title: Protoplast transformation of recalcitrant alkaliphilic Bacillus sp. with methylated plasmid DNA and a developed hard agar regeneration medium.
abstract: Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5) transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors: Chenghua
authors: C
authors: Gao
authors: Yanfen
authors: Y
authors: Xue
authors: Yanhe
authors: Y
authors: Ma
mesh_terms: D000362:Agar / Q000494:pharmacology; D001407:Bacillus / Q000187:drug effects / Q000235:genetics*; D003470:Culture Media / Q000494:pharmacology; D019175:DNA Methylation / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D005780:Gelatin; D010957:Plasmids / Q000235:genetics*; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism*; D012038:Regeneration / Q000187:drug effects; D013997:Time Factors; D014169:Transformation, Bacterial / Q000235:genetics*
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D005780:Gelatin; D000362:Agar