title: Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property.
abstract: Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Jintao
authors.initials: J
authors.lastname: Cheng
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Shengnan
authors.initials: S
authors.lastname: Miao
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Junling
authors.initials: J
authors.lastname: Guo
authors.affiliation: School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China. zhmzhou@jiangnan.edu.cn.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, China. zhmzhou@jiangnan.edu.cn.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D018387:Codon, Initiator / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D058928:Riboswitch / Q000187:drug effects* / Q000235:genetics; D013806:Theophylline / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D018387:Codon, Initiator; D058928:Riboswitch; D013806:Theophylline
keywords: Bacillus subtilis; Gene expression; Synthetic riboswitches; Theophylline; Translation initiation