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Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

View Article: PubMed Central - PubMed

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Comparison of the induced expression level with P43′-riboE1 and three strong promoters from B. subtilis. a SDS-PAGE analysis of GFP controlled by the constitutive promoters PsrfA, PaprE, P43 and by the theophylline-induced element P43′-riboE1. The BSG11 strain was activated by 8-mM theophylline for 24 h prior to sampling for SDS-PAGE. b Fluorescence intensity representing the relative expression level was driven by three constitutive promoters as well as by the P43′-riboE1 element after induction by 8-mM theophylline for total 31 and 24-h culture periods, respectively. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD
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Fig4: Comparison of the induced expression level with P43′-riboE1 and three strong promoters from B. subtilis. a SDS-PAGE analysis of GFP controlled by the constitutive promoters PsrfA, PaprE, P43 and by the theophylline-induced element P43′-riboE1. The BSG11 strain was activated by 8-mM theophylline for 24 h prior to sampling for SDS-PAGE. b Fluorescence intensity representing the relative expression level was driven by three constitutive promoters as well as by the P43′-riboE1 element after induction by 8-mM theophylline for total 31 and 24-h culture periods, respectively. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD

Mentions: To comprehensively compare the expression levels between commonly used strong constitutive promoters and P43′-riboE1, three wild-type promoters P43, PaprE and PsrfA, which have been characterized in previous studies [19, 20], were employed to express the GFP reporter. BSG43, BSG04, and BSG03 carrying the three strong promoters respectively, were cultured in LB for 31 h. The BSG11 harbouring pBSG11 was treated with 8 mM theophylline for 24 h. Then, the final expression level was determined by SDS-PAGE analysis. The GFP expression level controlled by P43′-riboE1 was far higher than expression by PaprE and P43 in the presence of 8-mM theophylline and was equivalent to expression by PsrfA. The basal level of P43′-riboE1 was relatively low, which was consistent with the fluorescence intensity shown in Fig. 2. Interestingly, the final expression level mediated by P43′-riboE1 in the presence of 8 mM theophylline was somewhat higher than expression by P43 (Fig. 4a). To quantitatively determine the differences in the expression levels controlled by the four types of gene expression elements, fluorescence intensity was measured. The fluorescence of GFP driven by wild-type promoters and the activated riboswitch element showed a trend similar to the SDS-PAGE analysis of GFP expression (Fig. 4b). This demonstrated that highly controllable and tuneable gene expression can be achieved by using P43′-riboE1 element.Fig. 4


Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch
Comparison of the induced expression level with P43′-riboE1 and three strong promoters from B. subtilis. a SDS-PAGE analysis of GFP controlled by the constitutive promoters PsrfA, PaprE, P43 and by the theophylline-induced element P43′-riboE1. The BSG11 strain was activated by 8-mM theophylline for 24 h prior to sampling for SDS-PAGE. b Fluorescence intensity representing the relative expression level was driven by three constitutive promoters as well as by the P43′-riboE1 element after induction by 8-mM theophylline for total 31 and 24-h culture periods, respectively. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5120567&req=5

Fig4: Comparison of the induced expression level with P43′-riboE1 and three strong promoters from B. subtilis. a SDS-PAGE analysis of GFP controlled by the constitutive promoters PsrfA, PaprE, P43 and by the theophylline-induced element P43′-riboE1. The BSG11 strain was activated by 8-mM theophylline for 24 h prior to sampling for SDS-PAGE. b Fluorescence intensity representing the relative expression level was driven by three constitutive promoters as well as by the P43′-riboE1 element after induction by 8-mM theophylline for total 31 and 24-h culture periods, respectively. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD
Mentions: To comprehensively compare the expression levels between commonly used strong constitutive promoters and P43′-riboE1, three wild-type promoters P43, PaprE and PsrfA, which have been characterized in previous studies [19, 20], were employed to express the GFP reporter. BSG43, BSG04, and BSG03 carrying the three strong promoters respectively, were cultured in LB for 31 h. The BSG11 harbouring pBSG11 was treated with 8 mM theophylline for 24 h. Then, the final expression level was determined by SDS-PAGE analysis. The GFP expression level controlled by P43′-riboE1 was far higher than expression by PaprE and P43 in the presence of 8-mM theophylline and was equivalent to expression by PsrfA. The basal level of P43′-riboE1 was relatively low, which was consistent with the fluorescence intensity shown in Fig. 2. Interestingly, the final expression level mediated by P43′-riboE1 in the presence of 8 mM theophylline was somewhat higher than expression by P43 (Fig. 4a). To quantitatively determine the differences in the expression levels controlled by the four types of gene expression elements, fluorescence intensity was measured. The fluorescence of GFP driven by wild-type promoters and the activated riboswitch element showed a trend similar to the SDS-PAGE analysis of GFP expression (Fig. 4b). This demonstrated that highly controllable and tuneable gene expression can be achieved by using P43′-riboE1 element.Fig. 4

View Article: PubMed Central - PubMed

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus