A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.

R. Burioni, P. Plaisant, M. L. Riccio, G. M. Rossolini, G. Satta

Research output: Contribution to journalArticlepeer-review

Abstract

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.

Original languageEnglish
Pages (from-to)201-206
Number of pages6
JournalNew Microbiologica
Volume18
Issue number2
Publication statusPublished - Apr 1995

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

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