摘要 |
PURPOSE:To provide a vector plasmid which can be inserted into thermophilic bacteria belonging to Bacillus genus and stably maintained and replicated in said bacterial cell, by separating from bacteria collected from a high-temperature natural environment. CONSTITUTION:A specimen collected from a high-temperature environment is placed into an L medium, and cultured at 55 deg.C for 4hr under shaking. The cultivation liquid is spread on an agar L medium containing kanamycin or tetracycline, and maintained at 55 deg.C for 20hr. Bacterial cells are fished from the grown colonies, subjectd to the pure culture, and then cultured in the L medium. The bacterial cells are collected and treated with lysozyme-SDS to effect the bacteriolysis. The liquid is centrifuged to obtain the supernatant liquid, which is subjected to the equilibrium density gradient centrifugation using CsCl-EdBr to fractionate and collect the plasmid DNA. The transformation is carried out with the plasmid DNA using the competent cells of Bacillus subtilis as the receptor bacteria, and the objective vector plasmid PTB19 which is resistant to kanamycin and tetracycline is obtained by selecting with kanamycin or tetracycline. |