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Related Experiment Videos

Drag&Drop cloning in yeast.

Gregor Jansen1, Cunle Wu, Babette Schade

  • 1Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6. gregor.jansen@mcgill.ca

Gene
|January 20, 2005
PubMed
Summary
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Researchers created pGREG vectors for yeast Saccharomyces cerevisiae, enabling efficient construction and expression of differentially tagged fusion proteins using Drag&Drop cloning. This system simplifies gene cloning and protein expression for various applications.

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • Protein Engineering

Background:

  • Developing efficient methods for constructing and expressing fusion proteins is crucial in molecular biology.
  • Existing cloning techniques can be time-consuming and require specific restriction sites.
  • The yeast Saccharomyces cerevisiae is a powerful model organism for genetic studies and protein expression.

Purpose of the Study:

  • To develop a versatile set of vectors (pGREG series) for efficient construction and expression of differentially tagged fusion proteins.
  • To enable Drag&Drop cloning and in vivo homologous recombination for simplified gene integration.
  • To facilitate rapid construction of a series of tagged proteins and expression of heterologous genes.

Main Methods:

  • Development of the pGREG vector series based on the pRS series with a kanR selection marker.

Related Experiment Videos

  • Utilizing in vivo homologous recombination for gene introduction into galactose-inducible expression vectors.
  • Employing common recombination sequences for PCR fragments, eliminating the need for specific restriction sites.
  • Using a selectable stuffer HIS3 gene that is replaced upon successful gene integration, with loss of selection identifying recombinants.

    • Main Results:

    • The pGREG vectors allow efficient, restriction site-independent cloning of genes into yeast expression vectors.
    • Successful gene integration leads to the loss of the HIS3 selection marker, enabling easy identification of recombinants.
    • The modular vector system permits rapid transfer of genes between vectors for constructing various tagged proteins.
    • The system supports efficient cloning and expression of heterologous genes, suitable for large-scale experiments.

    Conclusions:

    • The pGREG vector system provides an enhanced, user-friendly method for constructing and expressing differentially tagged fusion proteins in Saccharomyces cerevisiae.
    • This Drag&Drop cloning approach significantly simplifies gene manipulation, protein tagging, and library construction.
    • The developed vectors are valuable tools for diverse applications in yeast genetics, molecular biology, and high-throughput screening.