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

Wenjing Cui,Zhemin Zhou,Junling Guo,Zhongmei Liu,Laichuang Han,Li Zhou,Jintao Cheng

doi:10.1186/s12934-016-0599-z

Abstract

BackgroundSynthetic 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.ResultsA 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.ConclusionThe 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.

Highlights

Synthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms
We compared the cell growth and the expression pattern of green fluorescent protein (GFP) between the constitutive expression system driven by P43 and the inducible expression system driven by P43′-riboswitch E1 (riboE1)
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]

Summary

Introduction

Synthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. It has been broadly recognised that protein-based ligand-inducible tools that well functioned in E. coli do not work well in other bacteria, such as B. subtilis. To overcome the limitations of protein-based gene expression tools, RNA-based elements have increasingly been viewed as superior genetic tools for a range of applications in synthetic biology [4, 5]. One such RNA regulator is the riboswitch, which is an RNA-encoded genetic control element that regulates gene expression in a ligand-dependent fashion without the need for proteins [6]. Ligand-inducible expression systems are essential genetic tools for commonly used bacteria such as E. coli and B. subtilis. The glycine-riboswitch system only resulted in a sixfold induction, it is still a useful model for the development of more riboswitch-dependent gene expression systems and other biological devices

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Conclusion