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Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli

Abstract

Gene knockout experiments are often essential in functional genomics and metabolic engineering studies. However, repeated multiple gene knockout experiments are laborious, time consuming and sometimes impossible to perform for those genes that are essential for cell function. Small regulatory RNAs (sRNAs) are short noncoding RNAs in prokaryotes that can finely control the expression of target genes in trans at the post-transcriptional level. Here we describe the protocol for synthetic sRNA-based gene expression control, including sRNA design principles. Customized synthetic sRNAs consist of a scaffold and a target-binding sequence, and they can be created by simply replacing an existing target-binding sequence with one that is complementary to the target mRNA to be repressed, while retaining the scaffold. Our plasmid-based synthetic sRNA system does not require chromosomal modifications, and it enables one to perform high-throughput studies on the effects of knockdowns on host cell physiology, and it further allows the simultaneous screening of target genes in different Escherichia coli strains for applications in metabolic engineering and synthetic biology. Once an sRNA scaffold-harboring plasmid is constructed, customized synthetic sRNAs can be made within 3–4 d; after this time, the synthetic sRNAs can be applied to the desired experiments.

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Figure 1: Mode of trans-acting and Hfq-dependent sRNA action.
Figure 2: Construction of customized synthetic sRNA plasmid library.
Figure 3: Schematic illustration of potential applications of tailor-made synthetic sRNAs.
Figure 4: Schematics of the template plasmid producing sRNAs, and of the dual regulatory expression system.
Figure 5: Design and construction of synthetic sRNAs (anti-lacZ as an example).

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Acknowledgements

This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) and the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Science, ICT and Future Planning through the National Research Foundation of Korea. This protocol paper describes the detailed strategies and step-by-step procedures for the work reported in our previous paper.

Author information

Authors and Affiliations

Authors

Contributions

D.N. and S.Y.L. conceived the project. S.M.Y. and D.N. performed all experiments and developed the experimental protocol. S.Y.L. supervised the project. All authors wrote the paper together and approved the final manuscript.

Corresponding author

Correspondence to Sang Yup Lee.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Scaffold selection for the construction of effective synthetic sRNAs. (a) Most sRNAs possess a consensus sequence that is able to recruit the Hfq protein. The left figure illustrates the process of analyzing sequence features of discovered sRNAs in E. coli and the selection of three candidate scaffolds from MicC, MicF and SgrS sRNAs. The respective scaffolds were fused with a sequence complementary to the TIR of DsRed2 mRNA in order to build anti-DsRed2 synthetic sRNAs with different scaffolds. The graph on the right shows that the three scaffolds are highly efficient (>90%) in repressing DsRed2 translation and that the MicC scaffold showed the highest repression efficiency. Consequently, the MicC scaffold was selected to be the synthetic sRNA scaffold. “C”, no synthetic sRNA; “-”, a scaffold without the DsRed2 targeting sequence; “+”, a scaffold with the DsRed2 targeting sequence. (b) Identification of effective mRNA binding regions and sRNA lengths are identified. The graph on the right shows repressed DsRed2 expression by synthetic sRNAs targeting different regions in the mRNA, as indicated in the left figure. “A” denotes the same anti-DsRed2 synthetic sRNA with a MicC scaffold used in (a). The TIR, denoted by a green bar, was estimated using a previously published algorithm27. (Reprinted from ref. 13; please see details there.) (PDF 316 kb)

Supplementary Figure 2

Testing the specificities of synthetic sRNA by using the web-based tools TargetRNA2 and RNAPredator. (a) TargetRNA2. Input information in the indicated boxes: select the target genome to search to E. coli (named ‘Replicon’), input the whole sequence of the synthetic sRNA (named ‘sRNA sequence’) – in this example anti-lacZ, enter ‘24’ in ‘Target parameters’, and then ‘Submit’. (b) Predicted target mRNAs for the anti-lacZ synthetic sRNA by TargetRNA2. (c) Predicted target mRNAs for the anti-lacZ synthetic sRNA by RNAPredator. (PDF 278 kb)

Supplementary Figure 3

Binding free energy–dependent repression capabilities of different anti-lacZ synthetic sRNA. (a) Primers for the anti-lacZ synthetic sRNAs with different binding free energies were designed. (b) The tunability of repression with respect to binding energy was investigated for lacZα (MITRegistry, BBa_I732006) and its corresponding synthetic sRNAs. The LacZ expression levels were measured according to the standard β-galactosidase assay in E. coli DH5α strain by α-complementation33. (Reprinted from ref. 13; please see details there.) (PDF 455 kb)

Supplementary Figure 4

Vector map of the pWAS plasmid. (a) This figure illustrates the brief structure of the pWAS plasmid and indicates promoters, coding sequences, synthetic sRNAs, etc. (b) The vector sequence is available in the Supplementary Data file, where the locations of relevant genetic components in the plasmid are listed. (PDF 136 kb)

Supplementary Discussion

Computational design of synthetic sRNAs and high-throughput library construction. (PDF 154 kb)

Supplementary Data

Sequence of the pWAS vector. (TXT 85 kb)

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Yoo, S., Na, D. & Lee, S. Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nat Protoc 8, 1694–1707 (2013). https://doi.org/10.1038/nprot.2013.105

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