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Construction and properties of plasmid pKC30, a pBR322 derivative containing the pL-N region of phage lambda

R N Rao

    Gene
    |November 1, 1984
    PubMed

    Insights

    Phage lambda N gene expression in E. coli caused instability of the pKC30 plasmid. This instability was overcome by repressing pL transcription, introducing a nutL mutation, or adding an N-unresponsive terminator sequence.

    Area of Science:

    • Molecular Biology
    • Bacteriophage Genetics
    • Plasmid Biology

    Background:

    • The bacteriophage lambda N gene is crucial for its life cycle.
    • Plasmid pBR322 is a common cloning vector in Escherichia coli.
    • Expression of phage genes in E. coli can lead to plasmid instability.

    Purpose of the Study:

    • To investigate the effect of lambda N gene expression on plasmid pKC30 maintenance in E. coli.
    • To identify strategies for stabilizing pKC30 during lambda N gene expression.

    Main Methods:

    • Cloning the lambda N gene into plasmid pBR322 to create pKC30.
    • Culturing recombinant E. coli strains.
    • Assessing plasmid stability under different conditions.

    Main Results:

    • Lambda N-mediated pL transcription led to instability of the pKC30 plasmid.
    • Plasmid stability was restored by repressing pL transcription.
    • A nutL mutation or an "N-unresponsive terminator" sequence also stabilized pKC30.

    Conclusions:

    • Lambda N gene expression poses a challenge for plasmid maintenance in E. coli.
    • Strategies involving transcriptional control, specific mutations, or terminator sequences can stabilize plasmids during phage gene expression.

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