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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.


The combination of the novel theophylline-dependent riboswitch E1 and the P43 promoter in Bacillus subtilis. a Diagrams of the expression cassette driven by the native P43 promoter and the novel theophylline-dependent expression element (P43′-riboE1). The green fluorescent protein is represented by the green arrows, and the transcription start site is marked as TSS. The promoter fragment of the novel expression element, P43′, was produced by deletion of the entire sequence downstream of the native P43. The synthetic riboswitch E1 was placed immediately downstream of P43′, resulting in the novel expression element P43′-riboE1. The riboswitch was derived from the riboswitch E (previously reported by Topp et al.), in which a single A was inserted before the 5′ terminus of the original sequence. In the diagram, the inserted nucleotide is coloured red. The dash line denotes the difference between native P43 and the synthetic riboswitch E1 (riboE1). b The schematic diagram of mechanism of the novel genetic element. Theophylline is indicted in blue. c The growth curves of B. subtilis 168 harbouring pP43-gfp (BSG43) and pBSG11 BSG11). The recombinant B. subtilis strains were inoculated by pre-cultures with the initial OD600 of 0.05. The cultures were sampled periodically to measure the cell density until the cell density began to decrease at 27 h. d Expression levels of GFP (y-axis) against time (x-axis) during the culture period. The dashed line denotes the induction by 4 mM theophylline. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD
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Fig1: The combination of the novel theophylline-dependent riboswitch E1 and the P43 promoter in Bacillus subtilis. a Diagrams of the expression cassette driven by the native P43 promoter and the novel theophylline-dependent expression element (P43′-riboE1). The green fluorescent protein is represented by the green arrows, and the transcription start site is marked as TSS. The promoter fragment of the novel expression element, P43′, was produced by deletion of the entire sequence downstream of the native P43. The synthetic riboswitch E1 was placed immediately downstream of P43′, resulting in the novel expression element P43′-riboE1. The riboswitch was derived from the riboswitch E (previously reported by Topp et al.), in which a single A was inserted before the 5′ terminus of the original sequence. In the diagram, the inserted nucleotide is coloured red. The dash line denotes the difference between native P43 and the synthetic riboswitch E1 (riboE1). b The schematic diagram of mechanism of the novel genetic element. Theophylline is indicted in blue. c The growth curves of B. subtilis 168 harbouring pP43-gfp (BSG43) and pBSG11 BSG11). The recombinant B. subtilis strains were inoculated by pre-cultures with the initial OD600 of 0.05. The cultures were sampled periodically to measure the cell density until the cell density began to decrease at 27 h. d Expression levels of GFP (y-axis) against time (x-axis) during the culture period. The dashed line denotes the induction by 4 mM theophylline. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD

Mentions: Previously many diverse theophylline-dependent riboswitches were engineered and shown to function in various Gram-positive and Gram-negative bacteria at the translational level [12]. However, it was unclear if these riboswitches were compatible with most constitutive promoters. Usually, constitutive promoters are paired with theophylline riboswitches to achieve controllable and tuneable transcription [17, 18]. Here, a new riboswitch, theophylline riboswitch riboE1 containing a modified SD sequence AAAGGAGG was constructed by modifying the riboswitch E constructed by Topp et al. [12]. RiboE1 was genetically fused to a strong promoter P43, in which the native downstream SD sequence was deficient (P43′), yielding a novel dual expression element P43′-riboE1. The P43′ retains the full upstream sequence including the core region (−35 and −10), the transcriptional start site (TSS), and the 5′UTR between the TSS and the native SD sequence. The synthetic theophylline riboswitch is composed of an aptamer region, a synthetic SD sequence, and a spacer (Fig. 1a). Theoretically, the transcription of the riboE1 control element is triggered by P43 during the cell growth. However, the synthetic SD located downstream of the aptamer is sequestered via pairing with the bases in the stem of the riboswitch, resulting in translational block. The binding of theophylline to the aptamer domain initiates translation by altering the downstream base pairing, which releases the SD. Subsequently, GFP expression begins after the ribosome binding to the SD (Fig. 1b). We compared the cell growth and the expression pattern of GFP between the constitutive expression system driven by P43 and the inducible expression system driven by P43′-riboE1. The growth curve showed that the increase in cell density was similar for both the recombinant strain BSG11 (B. subtilis harbouring pBSG11, inducible expression of GFP) and the BSG43 (B. subtilis harbouring pP43-gfp, constitutive expression of GFP) strain during the culture period (Fig. 1c). This indicates that the recombinant strains BSG11 and BSG43 grow identically.Fig. 1


Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch
The combination of the novel theophylline-dependent riboswitch E1 and the P43 promoter in Bacillus subtilis. a Diagrams of the expression cassette driven by the native P43 promoter and the novel theophylline-dependent expression element (P43′-riboE1). The green fluorescent protein is represented by the green arrows, and the transcription start site is marked as TSS. The promoter fragment of the novel expression element, P43′, was produced by deletion of the entire sequence downstream of the native P43. The synthetic riboswitch E1 was placed immediately downstream of P43′, resulting in the novel expression element P43′-riboE1. The riboswitch was derived from the riboswitch E (previously reported by Topp et al.), in which a single A was inserted before the 5′ terminus of the original sequence. In the diagram, the inserted nucleotide is coloured red. The dash line denotes the difference between native P43 and the synthetic riboswitch E1 (riboE1). b The schematic diagram of mechanism of the novel genetic element. Theophylline is indicted in blue. c The growth curves of B. subtilis 168 harbouring pP43-gfp (BSG43) and pBSG11 BSG11). The recombinant B. subtilis strains were inoculated by pre-cultures with the initial OD600 of 0.05. The cultures were sampled periodically to measure the cell density until the cell density began to decrease at 27 h. d Expression levels of GFP (y-axis) against time (x-axis) during the culture period. The dashed line denotes the induction by 4 mM theophylline. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD
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Fig1: The combination of the novel theophylline-dependent riboswitch E1 and the P43 promoter in Bacillus subtilis. a Diagrams of the expression cassette driven by the native P43 promoter and the novel theophylline-dependent expression element (P43′-riboE1). The green fluorescent protein is represented by the green arrows, and the transcription start site is marked as TSS. The promoter fragment of the novel expression element, P43′, was produced by deletion of the entire sequence downstream of the native P43. The synthetic riboswitch E1 was placed immediately downstream of P43′, resulting in the novel expression element P43′-riboE1. The riboswitch was derived from the riboswitch E (previously reported by Topp et al.), in which a single A was inserted before the 5′ terminus of the original sequence. In the diagram, the inserted nucleotide is coloured red. The dash line denotes the difference between native P43 and the synthetic riboswitch E1 (riboE1). b The schematic diagram of mechanism of the novel genetic element. Theophylline is indicted in blue. c The growth curves of B. subtilis 168 harbouring pP43-gfp (BSG43) and pBSG11 BSG11). The recombinant B. subtilis strains were inoculated by pre-cultures with the initial OD600 of 0.05. The cultures were sampled periodically to measure the cell density until the cell density began to decrease at 27 h. d Expression levels of GFP (y-axis) against time (x-axis) during the culture period. The dashed line denotes the induction by 4 mM theophylline. The GFP fluorescence was measured in triplicates and the data were shown in mean ± SD
Mentions: Previously many diverse theophylline-dependent riboswitches were engineered and shown to function in various Gram-positive and Gram-negative bacteria at the translational level [12]. However, it was unclear if these riboswitches were compatible with most constitutive promoters. Usually, constitutive promoters are paired with theophylline riboswitches to achieve controllable and tuneable transcription [17, 18]. Here, a new riboswitch, theophylline riboswitch riboE1 containing a modified SD sequence AAAGGAGG was constructed by modifying the riboswitch E constructed by Topp et al. [12]. RiboE1 was genetically fused to a strong promoter P43, in which the native downstream SD sequence was deficient (P43′), yielding a novel dual expression element P43′-riboE1. The P43′ retains the full upstream sequence including the core region (−35 and −10), the transcriptional start site (TSS), and the 5′UTR between the TSS and the native SD sequence. The synthetic theophylline riboswitch is composed of an aptamer region, a synthetic SD sequence, and a spacer (Fig. 1a). Theoretically, the transcription of the riboE1 control element is triggered by P43 during the cell growth. However, the synthetic SD located downstream of the aptamer is sequestered via pairing with the bases in the stem of the riboswitch, resulting in translational block. The binding of theophylline to the aptamer domain initiates translation by altering the downstream base pairing, which releases the SD. Subsequently, GFP expression begins after the ribosome binding to the SD (Fig. 1b). We compared the cell growth and the expression pattern of GFP between the constitutive expression system driven by P43 and the inducible expression system driven by P43′-riboE1. The growth curve showed that the increase in cell density was similar for both the recombinant strain BSG11 (B. subtilis harbouring pBSG11, inducible expression of GFP) and the BSG43 (B. subtilis harbouring pP43-gfp, constitutive expression of GFP) strain during the culture period (Fig. 1c). This indicates that the recombinant strains BSG11 and BSG43 grow identically.Fig. 1

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.