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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

Validation of the theophylline-responsive gene expression system with gus reporter gene. a SDS-PAGE analysis showed the expression level of GUS in the absence and presence of theophylline. The induced expression level of GUS in the recombinant strain, BSGgus, was determined by treatment with 8 mM theophylline for 20 h culture, and the culture treated with 4% DMSO for the same time after induction was designated as controls (0 mM). The solid triangle pointed the overproduced GUS. Protein extracts from B. subtilis 168 sampled simultaneously with the induced recombinant strains was used to be the negative control. GUS, denoted the purified GUS protein, was used to be a specific marker to indicate the bands of GUS on SDS-PAGE. b Enzymatic assay for GUS in BSGgus after induction by 8 mM theophylline for 16 and 20 h. The induced activities of GUS and the levels for leakage are shown by solid and open circles, respectively. The histograms represent the corresponding induction ratios. The enzymatic activity assay was performed in triplicates and the data are presented in mean ± SD
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Fig6: Validation of the theophylline-responsive gene expression system with gus reporter gene. a SDS-PAGE analysis showed the expression level of GUS in the absence and presence of theophylline. The induced expression level of GUS in the recombinant strain, BSGgus, was determined by treatment with 8 mM theophylline for 20 h culture, and the culture treated with 4% DMSO for the same time after induction was designated as controls (0 mM). The solid triangle pointed the overproduced GUS. Protein extracts from B. subtilis 168 sampled simultaneously with the induced recombinant strains was used to be the negative control. GUS, denoted the purified GUS protein, was used to be a specific marker to indicate the bands of GUS on SDS-PAGE. b Enzymatic assay for GUS in BSGgus after induction by 8 mM theophylline for 16 and 20 h. The induced activities of GUS and the levels for leakage are shown by solid and open circles, respectively. The histograms represent the corresponding induction ratios. The enzymatic activity assay was performed in triplicates and the data are presented in mean ± SD

Mentions: To test the usefulness and the compatibility of the gene system, the reporter gene gus was used to verify the expression level driven by P43′-riboE1 element. The SDS-PAGE analysis showed some level of gus expression in both the presence and absence of theophylline. The gus was successfully activated at very high level after treatment with 8 mM theophylline for 16 and 20 h. In contrast, the control groups treated with 4% DMSO for the same time showed relatively low GUS expression levels (Fig. 6a).Fig. 6


Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch
Validation of the theophylline-responsive gene expression system with gus reporter gene. a SDS-PAGE analysis showed the expression level of GUS in the absence and presence of theophylline. The induced expression level of GUS in the recombinant strain, BSGgus, was determined by treatment with 8 mM theophylline for 20 h culture, and the culture treated with 4% DMSO for the same time after induction was designated as controls (0 mM). The solid triangle pointed the overproduced GUS. Protein extracts from B. subtilis 168 sampled simultaneously with the induced recombinant strains was used to be the negative control. GUS, denoted the purified GUS protein, was used to be a specific marker to indicate the bands of GUS on SDS-PAGE. b Enzymatic assay for GUS in BSGgus after induction by 8 mM theophylline for 16 and 20 h. The induced activities of GUS and the levels for leakage are shown by solid and open circles, respectively. The histograms represent the corresponding induction ratios. The enzymatic activity assay was performed in triplicates and the data are presented in mean ± SD
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Validation of the theophylline-responsive gene expression system with gus reporter gene. a SDS-PAGE analysis showed the expression level of GUS in the absence and presence of theophylline. The induced expression level of GUS in the recombinant strain, BSGgus, was determined by treatment with 8 mM theophylline for 20 h culture, and the culture treated with 4% DMSO for the same time after induction was designated as controls (0 mM). The solid triangle pointed the overproduced GUS. Protein extracts from B. subtilis 168 sampled simultaneously with the induced recombinant strains was used to be the negative control. GUS, denoted the purified GUS protein, was used to be a specific marker to indicate the bands of GUS on SDS-PAGE. b Enzymatic assay for GUS in BSGgus after induction by 8 mM theophylline for 16 and 20 h. The induced activities of GUS and the levels for leakage are shown by solid and open circles, respectively. The histograms represent the corresponding induction ratios. The enzymatic activity assay was performed in triplicates and the data are presented in mean ± SD
Mentions: To test the usefulness and the compatibility of the gene system, the reporter gene gus was used to verify the expression level driven by P43′-riboE1 element. The SDS-PAGE analysis showed some level of gus expression in both the presence and absence of theophylline. The gus was successfully activated at very high level after treatment with 8 mM theophylline for 16 and 20 h. In contrast, the control groups treated with 4% DMSO for the same time showed relatively low GUS expression levels (Fig. 6a).Fig. 6

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