title: Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.
abstract: BACKGROUND
  Synthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.
  
  
  RESULTS
  A novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43'-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43'-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.
  
  
  CONCLUSIONS
  The constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Laichuang
authors.initials: L
authors.lastname: Han
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Jintao
authors.initials: J
authors.lastname: Cheng
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Junling
authors.initials: J
authors.lastname: Guo
authors.affiliation: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhmzhou@jiangnan.edu.cn.|Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhmzhou@jiangnan.edu.cn.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic; D058928:Riboswitch / Q000235:genetics*; D013806:Theophylline / Q000378:metabolism*
chemical_list: D058928:Riboswitch; D013806:Theophylline
keywords: Bacillus subtilis; Controllable gene expression; RNA switch; Synthetic riboswitches; Theophylline