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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.


Characterization of induction of GFP from pBSG11 harbouring P43′-riboE1 element after induction by 4-mM theophylline. GFP induction is denoted by green histograms at different time points. The solid circles within in the histograms represent the level of induced GFP expression. Similarly, the open circles within the histograms are the corresponding basal expression level. The activation ratio was obtained by dividing the induced expression level by the corresponding basal level
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Fig2: Characterization of induction of GFP from pBSG11 harbouring P43′-riboE1 element after induction by 4-mM theophylline. GFP induction is denoted by green histograms at different time points. The solid circles within in the histograms represent the level of induced GFP expression. Similarly, the open circles within the histograms are the corresponding basal expression level. The activation ratio was obtained by dividing the induced expression level by the corresponding basal level

Mentions: To characterize the activated level and efficiency driven by the P43′-riboE1 element, the BSG11 strain was cultivated in LB medium for 7 h prior to treatment with 4 mM theophylline. Figure 2 shows the theophylline-dependent induction of GFP fluorescence after culturing for 24 h. The histograms show that the activated expression levels of GFP and the induction ratios increased gradually during the induction period. After 24 h of induction, the expression level exceeded 350,000. The induction ratios constantly increased during the induction period, and were consistent with the expression levels. Expression due to leakage remained low during the induction period. Finally, the induction ratio peaked at 4.3 after 24 h of induction (Fig. 2).Fig. 2


Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch
Characterization of induction of GFP from pBSG11 harbouring P43′-riboE1 element after induction by 4-mM theophylline. GFP induction is denoted by green histograms at different time points. The solid circles within in the histograms represent the level of induced GFP expression. Similarly, the open circles within the histograms are the corresponding basal expression level. The activation ratio was obtained by dividing the induced expression level by the corresponding basal level
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Characterization of induction of GFP from pBSG11 harbouring P43′-riboE1 element after induction by 4-mM theophylline. GFP induction is denoted by green histograms at different time points. The solid circles within in the histograms represent the level of induced GFP expression. Similarly, the open circles within the histograms are the corresponding basal expression level. The activation ratio was obtained by dividing the induced expression level by the corresponding basal level
Mentions: To characterize the activated level and efficiency driven by the P43′-riboE1 element, the BSG11 strain was cultivated in LB medium for 7 h prior to treatment with 4 mM theophylline. Figure 2 shows the theophylline-dependent induction of GFP fluorescence after culturing for 24 h. The histograms show that the activated expression levels of GFP and the induction ratios increased gradually during the induction period. After 24 h of induction, the expression level exceeded 350,000. The induction ratios constantly increased during the induction period, and were consistent with the expression levels. Expression due to leakage remained low during the induction period. Finally, the induction ratio peaked at 4.3 after 24 h of induction (Fig. 2).Fig. 2

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.