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Expression using the T7 RNA polymerase/promoter system.

S Tabor1

  • 1Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
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This protocol details gene expression using bacteriophage T7 RNA polymerase, a highly efficient enzyme. Rifampicin addition ensures exclusive T7-driven gene expression in E. coli, enabling targeted protein production.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Expression Systems

Background:

  • Bacteriophage T7 RNA polymerase offers advantages over E. coli RNA polymerase for gene expression.
  • T7 RNA polymerase exhibits high activity, selective promoter recognition, and resistance to inhibitors like rifampicin.
  • Existing methods may lack specificity or efficiency in controlling gene expression.

Purpose of the Study:

  • To describe a method for efficient and specific gene expression using T7 RNA polymerase.
  • To outline a protocol for controlling gene expression in E. coli via T7 RNA polymerase.
  • To enable targeted production and labeling of specific gene products.

Main Methods:

  • Utilizing two plasmids in E. coli: one for gene expression under a T7 promoter (pT7), and another for T7 RNA polymerase under a heat-inducible promoter.

Related Experiment Videos

  • Inducing T7 RNA polymerase production via heat shock.
  • Employing rifampicin to inhibit endogenous E. coli RNA polymerase, ensuring exclusive pT7-driven transcription.
  • Optional: Performing gene expression in minimal medium with [35S]methionine for protein labeling.

    • Main Results:

    • Successful exclusive expression of genes controlled by the T7 promoter (pT7) after heat induction.
    • High transcription rates and potential for large RNA molecules due to T7 RNA polymerase activity.
    • Effective inhibition of E. coli RNA polymerase by rifampicin, leading to specific gene expression.

    Conclusions:

    • The described system provides a robust method for controlled and efficient gene expression in E. coli.
    • This approach is valuable for producing specific proteins and facilitates isotopic labeling.
    • The use of T7 RNA polymerase and rifampicin offers a powerful tool in molecular biology and biotechnology.