RRC ID 33381
Author Kast P.
Title pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones.
Journal Gene
Abstract 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.
Volume 138(1-2)
Pages 109-14
Published 1994-1-28
DOI 10.1016/0378-1119(94)90790-0
PII 0378-1119(94)90790-0
PMID 8125286
MeSH Alleles Base Sequence Cloning, Molecular / methods* Escherichia coli / metabolism* Fenclonine / metabolism Genetic Vectors* Molecular Sequence Data Phenylalanine-tRNA Ligase / biosynthesis Phenylalanine-tRNA Ligase / genetics* Phenylalanine-tRNA Ligase / metabolism Plasmids* Recombinant Fusion Proteins / biosynthesis Recombinant Proteins / biosynthesis* Restriction Mapping Substrate Specificity beta-Galactosidase / biosynthesis
IF 2.984
Times Cited 70
Prokaryotes E. coli pKSS