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Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria

Nature Protocols volume 4, pages 1262–1273 (2009)Cite this article

Abstract

Many techniques have been developed for studying inducible gene expression, but all of them are multicomponent systems consisting of cis-acting elements at the DNA or RNA level, trans-acting regulator proteins and/or small molecules as inducers. RNA thermometers are the only known single-component regulators of gene expression. They consist of a temperature-sensitive secondary structure in the 5′ untranslated region of the mRNA, which contains the ribosome-binding site. The ribosome-binding site can be masked or unmasked by a simple temperature shift, thereby repressing or inducing translation. Recently, we and others have designed synthetic RNA thermometers that are considerably simpler than naturally occurring thermometers and can be exploited as convenient on/off switches of gene expression. In this protocol, we describe the construction and use of synthetic RNA thermometers. We provide guidelines for the in silico design of thermometer-controlled mRNA leaders and for their experimental testing and optimization; the entire procedure can be completed in 2–3 weeks.

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Figure 1: Examples of a natural and two synthetic RNA thermometers.
Figure 2: Modular design of synthetic RNA thermometers.
Figure 3: A quick comparison of RNA thermometer constructs by temperature-controlled GFP (green fluorescent protein) expression in E. coli.
Figure 4: Workflow for design, construction and experimental testing of synthetic RNA thermometers.
Figure 5: Optimization of the switch temperature (melting temperature) of synthetic RNA thermometers.

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Acknowledgements

We thank D. Karcher (Max Planck Institute of Molecular Plant Physiology) for helpful discussion and comments on the paper.

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Authors and Affiliations

  1. Max-Planck Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg, Potsdam-Golm, Germany

    Juliane Neupert & Ralph Bock

Authors
  1. Juliane Neupert
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  2. Ralph Bock
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Contributions

J.N. and R.B. jointly prepared the paper.

Corresponding author

Correspondence to Ralph Bock.

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Neupert, J., Bock, R. Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria. Nat Protoc 4, 1262–1273 (2009). https://doi.org/10.1038/nprot.2009.112

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