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Related Experiment Videos

A novel phosphate-regulated expression vector in Escherichia coli.

T Z Su1, H Schweizer, D L Oxender

  • 1Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.

Gene
|May 31, 1990
PubMed
Summary

A novel Escherichia coli expression vector, pPH3, utilizes the phosphate-controlled ugp promoter (pugp) for strong gene expression. This system offers tunable gene regulation, ideal for controlled protein production and large-scale fermentation.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Microbial Genetics

Background:

  • The ugp promoter (pugp) regulates the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.
  • Development of versatile expression vectors is crucial for recombinant protein production and genetic studies.

Purpose of the Study:

  • To construct and characterize a novel Escherichia coli expression vector, pPH3, utilizing the pugp.
  • To evaluate the efficiency and regulatory properties of the pugp for controlled gene expression.

Main Methods:

  • Cloning of the pugp into a pBR322 derivative (pTER5) to create the pPH3 expression vector.
  • Utilizing reporter genes, xylE and lacZ, to measure promoter activity and enzyme expression (catechol-2,3-dioxygenase and beta-galactosidase).

Related Experiment Videos

  • Assessing gene expression levels under varying conditions of inorganic phosphate (Pi) and glucose concentrations.

    • Main Results:

    • The pPH3 vector demonstrated strong, yet tightly regulated, gene expression.
    • Expression was repressed by high inorganic phosphate and glucose levels, with maximal induction at limiting Pi.
    • Maximal beta-galactosidase synthesis reached approximately 80% of the strong ptac promoter, and induced catechol-2,3-dioxygenase comprised about 50% of total cellular protein.

    Conclusions:

    • The pugp is a valuable tool for developing expression vectors that enable strong, controlled gene expression in E. coli.
    • The phosphate-controlled nature of this vector makes it suitable for large-scale fermentation processes using Pi-limiting conditions.