Versatile mercury-resistant cloning and expression vectors

• Published in: Gene Vol. 39; no. 2; pp. 293 - 297
• Main Authors: Diane Gambill, B., Summers, Anne O.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1985; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Biological Transport; Biotechnology; Cloning, Molecular - methods; Drug Resistance, Microbial; Fundamental and applied biological sciences. Psychology; Galactokinase - genetics; galK fusions; Gene Expression Regulation; Genetic engineering; Genetic technics; Genetic Vectors; IncQ; Mercury - toxicity; Methods. Procedures. Technologies; Operon; Organomercury Compounds - toxicity; plasmid P15A; Recombinant DNA; Replicon; Vectors (cloning, transfer, expression). Insertion sequences and transposons; ss; Km; Su; Recombinant DNA; Ap; IncQ; R; plasmid P15A; s; kb; GalK; Sm; and Tc; galK fusions; Pseudomonadales; Escherichia coli; Neisseriaceae; Resistance; Antibiotic; Acinetobacter calcoaceticus; Bacteria; Pseudomonadaceae; Molecular cloning; Escherichieae; Mercury; Pseudomonas putida; Vector; Enterobacteriaceae; Replicon
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(85)90326-9

Cloning vectors have been constructed employing two diverse replicons, IncQ and P15A. Both vectors confer resistance to kanamycin (Km) and mercuric ions (Hg 2+). One of these vectors, pDG105, is a broad-host-range, nonconjugative, oligocopy IncQ plasmid, which is capable of transforming Escherichia coli, Acinetobacter calcoaceticus, and Pseudomonas putida. The second vector, pDG106, is a narrow-host-range, multicopy cloning vector compatible with pBR322. Both vectors contain unique cloning sites in the Km-resistance gene for HindIII, SmaI, and XhoI, as well as unique EcoRI and ScaI sites in the mer operon. Cloning into the EcoRI site in the mer operon results in the mercury “supersensitive” phenotype, easily detectable by replica plating. Insertion of the galK gene into the EcoRI site in the mer operon results in Hg 2+ -inducible galactokinase activity, demonstrating the application of these plasmids as regulated expression vectors.