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Related Experiment Videos

A versatile low-copy-number cloning vector derived from plasmid F

J Shi1, D P Biek

  • 1Department of Microbiology and Immunology, University of Kentucky, Lexington 40536-0084, USA.

Gene
|October 16, 1995
PubMed
Summary

Researchers developed a new low-copy-number plasmid vector, pZC320, for stable cloning in Escherichia coli. This vector enables blue/white screening and T7-driven transcription for efficient gene expression studies.

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Area of Science:

  • Molecular Biology
  • Microbial Genetics

Background:

  • Plasmid vectors are essential tools in molecular biology for gene cloning and manipulation.
  • Stable maintenance of plasmids, especially at low copy numbers, is crucial for reproducible experimental results.
  • Existing vectors may have limitations in copy number, stability, or ease of insert screening.

Purpose of the Study:

  • To construct and characterize a novel low-copy-number (lcn) cloning vector based on the mini-F plasmid.
  • To incorporate features for stable plasmid maintenance and efficient insert detection.
  • To demonstrate the utility of the new vector for cloning specific gene sets.

Main Methods:

  • Construction of the pZC320 plasmid using the F plasmid's ori-2 replication unit and sop partition functions.
  • Incorporation of a multiple cloning site (MCS) within the lacZ alpha gene for blue/white screening.

Related Experiment Videos

  • Integration of a T7 promoter for inducible transcription of cloned inserts.
  • Cloning of regulated tetracycline-resistance genes from Tn10 into the pZC320 vector.

    • Main Results:

    • The pZC320 vector exhibits very low copy number and stable maintenance in Escherichia coli without selection.
    • The MCS provides 16 unique restriction sites for versatile cloning.
    • Blue/white screening using beta-galactosidase (beta Gal) activity allows for easy identification of clones with inserts.
    • Successful cloning of tetracycline-resistance genes demonstrated the vector's functionality.

    Conclusions:

    • The pZC320 vector is a robust tool for stable, low-copy-number cloning in Escherichia coli.
    • Its features facilitate efficient insert screening and expression analysis.
    • This vector advances the capabilities for studying genes that require controlled expression or low copy numbers for proper function.