构建带有异源DNA序列的枯草芽孢杆菌克隆载体。

Construction of a Bacillus subtilis cloning vehicle with heterologous DNA sequence.

作者信息

Yoshimura K, Ikenaka Y, Murai M, Tanabe M, Seki T, Oshima Y

出版信息

Gene. 1983 Oct;24(2-3):255-63. doi: 10.1016/0378-1119(83)90086-0.

Abstract

A cloning vehicle, pFTB91, for the Bacillus subtilis host was constructed with DNA fragments heterologous to the host chromosome. It consists of three DNA fragments: (i) chromosomal DNA of Bacillus amyloliquefaciens which complements the leuA and ilvC mutations in B. subtilis; (ii) a B. amyloliquefaciens plasmid DNA that supplies an autonomously replicating function; and (iii) a HindIII fragment of Staphylococcus aureus plasmid pTP5 that carries gene tetr, conferring the TetR phenotype. It has sufficiently low DNA homology to prevent its integration into the host chromosome in recombination-competent cells of B. subtilis. It is 9.3 kb, and approx. 10 copies are present per chromosome. The SalI and KpnI sites in the ilvC+ and tetr genes, respectively, could be used for selection of recombinant plasmids by insertional inactivation. The plasmid has unique sites for EcoRI, PstI, and XbaI.

摘要

构建了一种用于枯草芽孢杆菌宿主的克隆载体pFTB91，其含有与宿主染色体异源的DNA片段。它由三个DNA片段组成：（i）解淀粉芽孢杆菌的染色体DNA，可互补枯草芽孢杆菌中的leuA和ilvC突变；（ii）提供自主复制功能的解淀粉芽孢杆菌质粒DNA；（iii）金黄色葡萄球菌质粒pTP5的HindIII片段，携带tetr基因，赋予TetR表型。它具有足够低的DNA同源性，可防止其在枯草芽孢杆菌的重组 competent细胞中整合到宿主染色体中。它为9.3 kb，每条染色体约有10个拷贝。ilvC+和tetr基因中的SalI和KpnI位点可分别用于通过插入失活筛选重组质粒。该质粒具有EcoRI、PstI和XbaI的独特位点。