Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property.

Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.