Ribonuclease-charged vector for facile direct cloning with positive selection

S. M. Deyev, S. A. Yazynin, D. A. Kuznetsov, M. Jukovich, R. W. Hartley

    Research output: Contribution to journalArticle

    12 Citations (Scopus)

    Abstract

    Plasmid vectors for positive selection of cloned inserts in Escherichia coli were devised, based on an expression plasmid (pMT416) for the bacterial ribonuclease barnase. In addition to the barnase gene under control of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which protects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. Having a unique SmaI/XmaI site in the barnase structural gene, pMT416 itself can be used as a selective vector: uncut or religated pMT416 will preclude growth while plasmids with inserts in the barnase gene will allow the cells to survive. The entire pUC polylinker was inserted into the barnase gene in place of the Val-36 codon. This insert of nineteen largely hydrophilic amino acids does not prevent the lethal effect of full expression of the gene. The resulting plasmid, pMT440, is a generally useful selective cloning vector representing the 'kill-the-rest' approach.

    Original languageEnglish
    Pages (from-to)379-382
    Number of pages4
    JournalMolecular and General Genetics
    Volume259
    Issue number4
    DOIs
    Publication statusPublished - 1998

    Keywords

    • Barnase
    • Molecular cloning
    • Polylinker

    ASJC Scopus subject areas

    • Genetics

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