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Lambda phagemids and their transducing properties.

A A Melnikov, A P Tchernov, I Fodor

    Gene
    |April 1, 1984
    PubMed
    Summary
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    Researchers developed novel phagemid DNA constructs for efficient gene transfer in E. coli. These phagemids enable high-frequency transduction and stable plasmid maintenance, advancing genetic engineering tools.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • Recombinant DNA technology enables the construction of novel genetic elements.
    • Phagemids combine features of plasmids and bacteriophages for gene delivery.
    • Bacteriophage lambda systems are widely used in molecular biology.

    Purpose of the Study:

    • To construct and characterize two novel recombinant lambda DNA constructs, lambda gt::pMB9 and lambda NM::pBR322.
    • To evaluate the transfection efficiency and transduction capabilities of these phagemid DNAs in Escherichia coli.
    • To investigate the stability and maintenance of the phagemid DNA within host cells.

    Main Methods:

    • Construction of recombinant lambda DNA carrying pMB9 and pBR322 replicons.
    • Transfection of Escherichia coli cells with constructed phagemid DNAs.

    Related Experiment Videos

  • Selection of drug-resistant transductants.
  • Analysis of phage progeny and plasmid maintenance.
  • Determination of transduction efficiency under various conditions.

    • Main Results:

    • Both lambda gt::pMB9 and lambda NM::pBR322 phagemids successfully transfected E. coli, producing infectious phage particles.
    • Phagemid particles efficiently transduced E. coli, establishing stable, covalently closed circular plasmids.
    • Lambda NM::pBR322 demonstrated high transduction frequency (up to 1), enabling nonlethal plasmid establishment.
    • Lambda gt::pMB9 transduction efficiency varied (10^-7 to 10^-2) and was maximal in lambda-lysogenic E. coli.
    • Precise excision of pMB9 was observed in lambda gt::pMB9 transductants, but not pBR322 from lambda NM::pBR322.

    Conclusions:

    • The constructed phagemids serve as effective tools for gene transfer and stable plasmid maintenance in E. coli.
    • Transduction efficiency is influenced by factors such as phage repressor activity and host cell lysogeny.
    • Lambda NM::pBR322 exhibits superior transduction capabilities for establishing nonlethal plasmids.
    • Differential excision properties of the integrated replicons were observed.