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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.


Determination of the effect of length of spacer on the induced level of GFP expression. a Schematic diagram displays the gene structure of riboE1, and riboE1-15 harbouring 15-bp spacer between SD sequence and start codon was produced by insertion of PstI restriction site immediately downstream of the SD sequence. The SD sequences within riboE1 and the modified riboE1-15 are in the azure box. b SDS-PAGE analysis was carried out to detect the expression level of GFP driven by P43′-riboE1 and P43′-riboE1-15 after induction with 8 mM theophylline for both 12 and 24 h. The strains harbouring recombinant plasmids treated with 4% DMSO are designated as 0 mM (controls). Protein extracts from Bacillus subtilis 168, which were collected at 19 and 31-h culture (equal to the induced groups with 12 and 24-h induction, respectively), are used as the negative controls. The GFP stands for the purified GFP protein, which is served as the specific marker to indicate the corresponding position of heterologous GFP. c Measurement of the relative fluorescent units of GFP corresponding to the SDS-PAGE in b
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Fig5: Determination of the effect of length of spacer on the induced level of GFP expression. a Schematic diagram displays the gene structure of riboE1, and riboE1-15 harbouring 15-bp spacer between SD sequence and start codon was produced by insertion of PstI restriction site immediately downstream of the SD sequence. The SD sequences within riboE1 and the modified riboE1-15 are in the azure box. b SDS-PAGE analysis was carried out to detect the expression level of GFP driven by P43′-riboE1 and P43′-riboE1-15 after induction with 8 mM theophylline for both 12 and 24 h. The strains harbouring recombinant plasmids treated with 4% DMSO are designated as 0 mM (controls). Protein extracts from Bacillus subtilis 168, which were collected at 19 and 31-h culture (equal to the induced groups with 12 and 24-h induction, respectively), are used as the negative controls. The GFP stands for the purified GFP protein, which is served as the specific marker to indicate the corresponding position of heterologous GFP. c Measurement of the relative fluorescent units of GFP corresponding to the SDS-PAGE in b

Mentions: To facilitate the cloning of the P43′-riboE1 element into other vectors, it was essential to modularize the element. One method is to insert restriction site either up- or down-stream of the element. Here, we inserted a PstI restriction site immediately downstream of the P43′-riboE1, to produce pBSG10 that contains P43′-riboE1-15 with a 15-bp spacer (Fig. 5a). To compare the effect of the longer spacer on GFP expression, BSG11 and BSG10 were treated with 8 mM theophylline for 12 and 24 h, and the production of GFP was analysed by SDS-PAGE. Obviously, the GFP protein expression was scarcely detected in BSG10 cultures treated with 8 mM theophylline or DMSO treatments in both 12- and 24-h cultures (Fig. 5b). In contrast, GFP expression in BSG11 for 12- and 24-h cultures was substantially higher than in BSG10. Noticeably, the basal expression level in BSG11 also was significantly higher than in BSG10 for both 12- and 24-h cultures (Fig. 5b). Additionally, the GFP fluorescence in BSG10 and BSG11 corresponding to the SDS-PAGE was determined to quantify the expression level. The data were consistent with that measured by SDS-PAGE, which authenticated the difference in heterologous expression level between the two different spacers (Fig. 5c). These results suggested that the failure of P43′-riboE1-15 to induce GFP expression after exposure to theophylline was due to increased spacer sequence length between the SD and the start codon, which probably affects ribosome binding in the translation initiation step [21].Fig. 5


Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch
Determination of the effect of length of spacer on the induced level of GFP expression. a Schematic diagram displays the gene structure of riboE1, and riboE1-15 harbouring 15-bp spacer between SD sequence and start codon was produced by insertion of PstI restriction site immediately downstream of the SD sequence. The SD sequences within riboE1 and the modified riboE1-15 are in the azure box. b SDS-PAGE analysis was carried out to detect the expression level of GFP driven by P43′-riboE1 and P43′-riboE1-15 after induction with 8 mM theophylline for both 12 and 24 h. The strains harbouring recombinant plasmids treated with 4% DMSO are designated as 0 mM (controls). Protein extracts from Bacillus subtilis 168, which were collected at 19 and 31-h culture (equal to the induced groups with 12 and 24-h induction, respectively), are used as the negative controls. The GFP stands for the purified GFP protein, which is served as the specific marker to indicate the corresponding position of heterologous GFP. c Measurement of the relative fluorescent units of GFP corresponding to the SDS-PAGE in b
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Fig5: Determination of the effect of length of spacer on the induced level of GFP expression. a Schematic diagram displays the gene structure of riboE1, and riboE1-15 harbouring 15-bp spacer between SD sequence and start codon was produced by insertion of PstI restriction site immediately downstream of the SD sequence. The SD sequences within riboE1 and the modified riboE1-15 are in the azure box. b SDS-PAGE analysis was carried out to detect the expression level of GFP driven by P43′-riboE1 and P43′-riboE1-15 after induction with 8 mM theophylline for both 12 and 24 h. The strains harbouring recombinant plasmids treated with 4% DMSO are designated as 0 mM (controls). Protein extracts from Bacillus subtilis 168, which were collected at 19 and 31-h culture (equal to the induced groups with 12 and 24-h induction, respectively), are used as the negative controls. The GFP stands for the purified GFP protein, which is served as the specific marker to indicate the corresponding position of heterologous GFP. c Measurement of the relative fluorescent units of GFP corresponding to the SDS-PAGE in b
Mentions: To facilitate the cloning of the P43′-riboE1 element into other vectors, it was essential to modularize the element. One method is to insert restriction site either up- or down-stream of the element. Here, we inserted a PstI restriction site immediately downstream of the P43′-riboE1, to produce pBSG10 that contains P43′-riboE1-15 with a 15-bp spacer (Fig. 5a). To compare the effect of the longer spacer on GFP expression, BSG11 and BSG10 were treated with 8 mM theophylline for 12 and 24 h, and the production of GFP was analysed by SDS-PAGE. Obviously, the GFP protein expression was scarcely detected in BSG10 cultures treated with 8 mM theophylline or DMSO treatments in both 12- and 24-h cultures (Fig. 5b). In contrast, GFP expression in BSG11 for 12- and 24-h cultures was substantially higher than in BSG10. Noticeably, the basal expression level in BSG11 also was significantly higher than in BSG10 for both 12- and 24-h cultures (Fig. 5b). Additionally, the GFP fluorescence in BSG10 and BSG11 corresponding to the SDS-PAGE was determined to quantify the expression level. The data were consistent with that measured by SDS-PAGE, which authenticated the difference in heterologous expression level between the two different spacers (Fig. 5c). These results suggested that the failure of P43′-riboE1-15 to induce GFP expression after exposure to theophylline was due to increased spacer sequence length between the SD and the start codon, which probably affects ribosome binding in the translation initiation step [21].Fig. 5

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.