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Related Experiment Videos

A small mobilizable IncP group plasmid vector packageable into bacteriophage lambda capsids in vitro.

G Selvaraj, V N Iyer

    Plasmid
    |January 1, 1985
    PubMed
    Summary

    Researchers developed a small, mobilizable cosmid vector for broad-host-range gene transfer in gram-negative bacteria. This efficient cloning tool facilitates genetic manipulation across diverse bacterial species.

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    Area of Science:

    • Molecular Biology
    • Microbiology
    • Genetics Engineering

    Background:

    • IncP plasmids are crucial for horizontal gene transfer in bacteria.
    • Cosmids are valuable vectors for cloning large DNA fragments.
    • Efficient gene transfer systems are needed for diverse bacterial species.

    Purpose of the Study:

    • To construct a novel, small, mobilizable cosmid vector derived from an IncP plasmid.
    • To incorporate the RK2 origin of transfer (oriT) and bacteriophage lambda packaging sequences.
    • To create a versatile tool for genetic manipulation in gram-negative bacteria.

    Main Methods:

    • Cloning the oriT region of RK2 and lambda phage packaging sequences into an IncP plasmid derivative.
    • Characterizing the resulting 13 kb cosmid vector for unique restriction sites (EcoRI, XhoI, HindIII, SalI).

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  • Assessing the mobilizability of the cosmid vector from Escherichia coli to various gram-negative bacteria.

    • Main Results:

    • A 13 kb mobilizable cosmid vector was successfully constructed.
    • The vector contains unique restriction sites, with some located within antibiotic resistance genes.
    • Efficient mobilization from E. coli to diverse gram-negative bacteria was achieved (0.8-10 per 100 donors).
    • The developed vector is among the smallest wide host-range cosmids reported.

    Conclusions:

    • The new cosmid vector offers a compact and efficient system for broad-host-range gene transfer.
    • Its small size and mobilizability make it a valuable tool for genetic studies in diverse bacteria.
    • An additional vector lacking the packaging sequence but including a BglII site was also developed.