Abstract
Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial. This protocol outlines how to use computational predictions and synthetic biology approaches to design, construct, and validate synthetic sRNA functionality for their application in bacteria. The computational tool, SEEDling, matches the optimal seed region with the user-selected sRNA scaffold for repression of target mRNAs. The synthetic sRNAs are assembled using Golden Gate cloning and their functionality is subsequently validated. The protocol uses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli. Since AcrA is part of a multidrug efflux pump, acrA repression can be revealed by assessing oxacillin susceptibility in a phenotypic screen. However, in case target repression does not result in a screenable phenotype, an alternative validation of synthetic sRNA functionality based on a fluorescence reporter is described.
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Acknowledgement and Statement
This work was supported by the Max Planck Society within the framework of the MaxGENESYS project (DS), the European Union (NextGenerationEU) via the European Regional Development Fund (ERDF) by the state Hesse within the project “biotechnological production of reactive peptides from waste streams as lead structures for drug development” (DS), and an Exploration Grant from the Boehringer Ingelheim Foundation (BAB). We are grateful to all laboratory members for extensive discussions on synthetic sRNAs in particular Cedric Brinkman for the development of SEEDling. All material is available from the corresponding author upon request.
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Brück, M., Berghoff, B.A., Schindler, D. (2024). In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria. In: Braman, J.C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 2760. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3658-9_27
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DOI: https://doi.org/10.1007/978-1-0716-3658-9_27
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