Hansen L H, Sørensen S J, Jensen L B
Department of General microbiology, University of Copenhagen, Denmark.
Gene. 1997 Feb 28;186(2):167-73. doi: 10.1016/s0378-1119(96)00688-9.
A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac- phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the pi protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.
一个包含完整大肠杆菌乳糖操纵子（lacIPOZYA）的12千碱基对的PstI片段以单拷贝形式插入恶臭假单胞菌、荧光假单胞菌以及一株具有乳糖阴性（lac-）表型的大肠杆菌的染色体中。这是通过改进现有的mini-Tn5转座子递送系统得以实现的（德洛伦佐等人，1990年；埃雷罗等人，1990年），该系统能将克隆的DNA片段以单拷贝形式随机整合到受体细菌染色体的位点上。这带来了以下结果：（a）构建了两种有用的低拷贝数克隆载体，它们都带有mini-Tn5递送系统所需的两侧为NotI位点的广泛多克隆区域；（b）产生了表达π蛋白的大肠杆菌非溶原性菌株，因此能够将基于R6K的mini-Tn5载体维持并递送至其他大肠杆菌菌株；（c）成功地将大肠杆菌乳糖操纵子插入荧光假单胞菌染色体，使荧光假单胞菌具备利用乳糖生长的能力；（d）Southern印迹法的证据与mini-Tn5递送系统总是产生单拷贝插入的假设相矛盾。这些改进使得能够将编码高表达蛋白的大片段DNA插入包括大肠杆菌在内的多种革兰氏阴性细菌的染色体中。
