Bernard P, Gabant P, Bahassi E M, Couturier M
Département de Biologie Moléculaire, Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium.
Gene. 1994 Oct 11;148(1):71-4. doi: 10.1016/0378-1119(94)90235-6.
Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735-745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.
质粒pKIL18/19是阳性选择克隆载体，含有在lacP启动子控制下的活性细胞毒性ccdB基因。它们是高拷贝数pUC18/19质粒的衍生物，其中ccdB杀伤基因已在lacP MCS18和MCS19多克隆位点下游同框融合。当用此类载体转化大肠杆菌野生型gyrA+菌株时，ccdB基因产物会阻断细菌生长。然而，如果通过插入外源DNA片段使ccdB失活，这种重组质粒就不再干扰宿主的生存能力。重组克隆的阳性选择效率很高，并且实验操作被极大简化：将大肠杆菌转化子接种在丰富培养基上，只有含有重组质粒的细胞才能形成菌落。CcdB蛋白是一种有效的拓扑异构酶毒药，gyrA462突变赋予对CcdB的完全抗性[伯纳德和库蒂里耶，《分子生物学杂志》226 (1992) 735 - 745]。因此，pKIL18/19载体可以在gyrA462宿主中大量扩增和制备。与pUC载体一样，pKIL载体设计用于一般的克隆/测序程序。
