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Updated: Jul 7, 2026

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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New pSC101-derivative cloning vectors with elevated copy numbers.

James Peterson1, Gregory J Phillips

  • 1Department of Veterinary Microbiology, Veterinary Medical Research Institute, Iowa State University, 1802 University Boulevard, Building #6, Ames, IA 50011, USA.

Plasmid
|February 26, 2008
PubMed
Summary

New cloning vectors derived from pSC101 increase gene expression and allow for high-copy number replication. These vectors successfully suppressed temperature-sensitive mutations by overproducing 4.5S RNA.

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Area of Science:

  • Molecular Biology
  • Microbial Genetics

Background:

  • The pSC101 replicon is a low-copy-number plasmid system.
  • Modifications to replication initiator proteins can alter plasmid copy number.

Purpose of the Study:

  • To engineer novel pSC101-based cloning vectors with increased copy numbers.
  • To evaluate the utility of these vectors for gene expression and genetic assays.

Main Methods:

  • Mutagenesis of the RepA protein in the pSC101 replicon.
  • Construction and characterization of new cloning vectors.
  • Genetic assay involving suppression of temperature-sensitive ffh mutants via 4.5S RNA overproduction.

Main Results:

  • Mutations in RepA (e.g., E93) resulted in medium and high-copy-number plasmids (27 and ~240 copies/cell).

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  • The new vectors enable enhanced gene product expression and stable maintenance.
  • These vectors successfully suppressed temperature-sensitive ffh mutants through 4.5S RNA overproduction, unlike wild-type vectors.

    • Conclusions:

    • Novel pSC101-derivative cloning vectors with controllable, elevated copy numbers have been developed.
    • These vectors offer advantages for gene expression, genetic screening, and functional studies.
    • The engineered vectors are effective tools for manipulating gene expression to rescue conditional lethal phenotypes.