Kast P
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum/LFV, Zürich, Switzerland.
Gene. 1994 Jan 28;138(1-2):109-14. doi: 10.1016/0378-1119(94)90790-0.
A new small plasmid vector (pKSS) for the direct selection of insert-containing plasmid clones is presented. The selection strategy is based on the acquired sensitivity of Escherichia coli cells to p-chloro-phenylalanine (p-Cl-Phe) if they carry a pheS allele encoding a phenylalanyl-tRNA synthetase alpha subunit with relaxed substrate specificity. This pheS allele is present on pKSS. Insertion into, or replacement of, the plasmidial pheS gene by a cloned fragment enables transformed pheS wild-type cells to survive on agar plates containing p-Cl-Phe plus ampicillin. This host strain-independent positive selection of recombinant clones proved to be highly efficient (> 99%) and did not require purification of the vector fragment prior to cloning. The high-copy-number vector pKSS offers a multitude of restriction sites and all of the features for analysis of cloned fragments that stem from the cloning vector pBluescript (Stratagene, La Jolla, CA, USA). Thus, pKSS represents a valuable alternative to previously reported positive-selection vectors; it should prove particularly useful for cloning when expecting a high fraction of cells transformed with non-recombinant vector, and for construction of DNA libraries.
本文介绍了一种用于直接筛选含插入片段质粒克隆的新型小质粒载体（pKSS）。筛选策略基于这样一个事实：如果大肠杆菌细胞携带一个编码具有宽松底物特异性的苯丙氨酰 - tRNA合成酶α亚基的pheS等位基因，那么这些细胞对对氯苯丙氨酸（p-Cl-Phe）会产生获得性敏感性。这个pheS等位基因存在于pKSS上。通过克隆片段插入或替换质粒的pheS基因，可使转化的pheS野生型细胞在含有p-Cl-Phe加氨苄青霉素的琼脂平板上存活。事实证明，这种不依赖宿主菌株的重组克隆阳性筛选效率很高（>99%），并且在克隆前不需要对载体片段进行纯化。高拷贝数载体pKSS提供了大量的限制性酶切位点以及所有源自克隆载体pBluescript（美国加利福尼亚州拉霍亚的Stratagene公司）的用于分析克隆片段的特性。因此，pKSS是先前报道的阳性选择载体的一种有价值的替代物；当预期有很大一部分细胞用非重组载体转化时，它对于克隆以及构建DNA文库应该会特别有用。
