Synthetic fused sRNA for the simultaneous repression of multiple genes

Yeom, Jinho; Park, Jong Seong; Jeon, Yong Min; Song, Beom Seop; Yoo, Seung-Min

doi:10.1007/s00253-022-11867-5

Cited by 4 publications

(5 citation statements)

References 54 publications

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“…This is further highlighted by our data for OmrB-s8, which is less efficient to increase oxacillin susceptibility when compared to RybB-s8, MicA-s8, and MicF-s8, probably due to sequestration of the s8 seed region in a stem-loop structure formed with the OmrB scaffold. Consideration of structural features becomes especially important if multiple seed regions are fused upstream of a single scaffold as recently published by Yeom et al (2022) . Notably, the here promoted toolset would be capable of introducing multiple seed regions upstream of a single scaffold without the need for complex and cumbersome overlap extension PCR procedures.…”

Section: Discussionmentioning

confidence: 92%

“…Consideration of structural features becomes especially important if multiple seed regions are fused upstream of a single scaffold as recently published by Yeom et al (2022). 61 Notably, the here promoted toolset would be capable of introducing multiple seed regions upstream of a single scaffold without the need for complex and cumbersome overlap extension PCR procedures. With the respective planning, complex sRNAs can be constructed in a one-pot reaction in a highly parallelized manner.…”

Section: Discussionmentioning

confidence: 99%

“…Our work is based on a 16-nt seed region, which is the equivalent size of the natural RybB seed region. Other studies have used longer seed regions, which presumably enhance the translational repression based on their potentially lower binding energies but at the same time increasing the risk of introducing secondary structures. ,, Our presented toolset allows the quick alteration not just of the seed region itself but also its properties such as length or the concatenation of multiple seed regions for a multitargeting approach if needed by the user. Taking everything together, we provide a highly versatile and scalable resource for the generation of synthetic sRNAs with a “plug and play” character.…”

Section: Discussionmentioning

confidence: 99%

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An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria

et al. 2022

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Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a “plug and play” plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the β-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user’s needs.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria

et al. 2022

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“…Even though protein fusion strategies can achieve a substantial improvement of genetic manipulation efficiency, nucleotide-level fusion strategies can also achieve efficient genome modulation while leaving out the translation. The synthetic sRNA-mediated gene regulation system achieves the repression of target gene expression at the post-transcriptional level with the regulator Hfq protein ( Yeom et al, 2022 ), which involves the binding of sRNA and target mRNA. Moreover, the synthetic sRNA assembled with a specifically modified mRNA-binding module sequence can be expected to bind to any target mRNA via base-paring, resulting in specific repression and precise regulation of metabolic pathways.…”

Section: Recent Technologies and Future Trendsmentioning

confidence: 99%

“…However, the repeated use of the same scaffold sequences always leads to low regulatory efficiency, which can be attributed to unintentional HR. Therefore, the fusing of several mRNA binding modules that are specific for target genes to a single sRNA scaffold can achieve multiplex gene regulation, thus simplifying genetic procedures while imposing no additional burden on the cell ( Yeom et al, 2022 ).…”

Section: Recent Technologies and Future Trendsmentioning

confidence: 99%

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Wu,

Feng,

Sun

et al. 2023

Front. Bioeng. Biotechnol.

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Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

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Research progress of engineering microbial cell factories for pigment production

Gao

2023

Biotechnology Advances

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Synthetic fused sRNA for the simultaneous repression of multiple genes

Cited by 4 publications

References 54 publications

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Research progress of engineering microbial cell factories for pigment production

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