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  In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria

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 J. C. Braman (Ed.), Synthetic Biology. Methods in Molecular Biology. Humana, New York, NY.

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Item Permalink: https://hdl.handle.net/21.11116/0000-000F-03BB-B Version Permalink: https://hdl.handle.net/21.11116/0000-000F-03BF-7
Genre: Book Chapter

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 Creators:
Brück, Michel1, 2, Author
Berghoff, Bork A.1, Author
Schindler, Daniel2, Author                 
Affiliations:
1external, ou_persistent22              
2Core Facility MPG MAXGenesys DNAfoundry, Max Planck Institute for Terrestrial Microbiology, Max Planck Society, ou_3266268              

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 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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Language(s): eng - English
 Dates: 2024-03-12
 Publication Status: Issued
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: URI: https://doi.org/10.1007/978-1-0716-3658-9_27
Other: Brück2024
DOI: 10.1007/978-1-0716-3658-9_27
 Degree: -

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Project name : -
Grant ID : -
Funding program : MaxGENESYS project
Funding organization : Max Planck Society
Project name : -
Grant ID : -
Funding program : European Regional Development Fund (ERDF)
Funding organization : European Union (NextGenerationEU)
Project name : project “biotechnological production of reactive peptides from waste streams as lead structures for drug development”
Grant ID : -
Funding program : -
Funding organization : the state Hesse
Project name : Exploration Grant
Grant ID : -
Funding program : -
Funding organization : Boehringer Ingelheim Foundation (BAB)

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Title: Synthetic Biology. Methods in Molecular Biology
Source Genre: Book
 Creator(s):
Braman, J. C., Editor
Affiliations:
-
Publ. Info: Humana, New York, NY.
Pages: - Volume / Issue: 2760 Sequence Number: - Start / End Page: - Identifier: ISBN: 978-1-0716-3657-2