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Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Authors :
Guobin Yin
Anqi Peng
Luyao Zhang
Yang Wang
Guocheng Du
Jian Chen
Zhen Kang
Source :
Synthetic and Systems Biotechnology, Vol 8, Iss 1, Pp 61-68 (2023)
Publication Year :
2023
Publisher :
KeAi Communications Co., Ltd., 2023.

Abstract

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B. subtilis are still not fully explored. Here, a bottom-up approach is proposed for designing artificial trans-acting sRNAs. By engineering the intrinsic sRNA SR6, a minimized core scaffold structure consisting of an 8 bp stem, a 4 nt loop, and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity (83%). Moreover, we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli (above 80%). A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences, which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence. By applying these auto-designed sRNAs, we rapidly modified the cell morphology and biofilm formation, and regulated metabolic flux toward acetoin biosynthesis. This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B. subtilis and E. coli but also enhanced our understanding of trans-acting sRNAs.

Details

Language :
English
ISSN :
2405805X
Volume :
8
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Synthetic and Systems Biotechnology
Publication Type :
Academic Journal
Accession number :
edsdoj.854596b518ca419bb0c494701ff66ff3
Document Type :
article
Full Text :
https://doi.org/10.1016/j.synbio.2022.11.003