pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones.
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.
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Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum/LFV, Zürich, Switzerland.
D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D010134:Fenclonine / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010652:Phenylalanine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000096:biosynthesis
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D010652:Phenylalanine-tRNA Ligase; D010134:Fenclonine