Fig. 5: Orthogonal riboswitch-mediated gene regulation in E. coli.
a Schematic illustrating 5’ UTR orthogonal OFF riboswitch-mediated translational regulation via ribosome binding site (RBS) masking in the presence of the DHEF ligand. GOI denotes the gene of interest. The ribosome icon in a was created in BioRender. Rode (2025) https://BioRender.com/01y1hjp. In vitro coupled transcription-translation of synthetic riboswitches based on b Aptamer LP in the presence of FMN and DHEF (n = 2 replicates), and c Aptamer M15 in the presence of FMN and DHEF (n = 3 replicates), over a range of ligand concentrations (0 μM to 10 μM). The luciferase activity at 0 μM ligand concentration was defined as 100 %. d Relative mRFP1 expression for riboswitch constructs M15-RS A through E in the absence and presence of 2.5 mM DHEF (n = 3 biological replicates); the statistical significance was determined by two-way analysis of variance (ANOVA), with Sidak’s multiple comparisons test. e Dose-dependent suppression of mRFP1 expression under the control of the M15-RS-A riboswitch with varying DHEF concentrations (0-2.5 mM) (n = 3 biological replicates). f RT-qPCR analysis of relative mRFP mRNA levels in E. coli under untreated and DHEF-treated conditions (n = 3 biological replicates); the statistical significance was determined by the two-tailed t-test. g Representative Western blot showing mRFP1 detection in E. coli transformed with the M15-RS-A riboswitch under untreated and DHEF-treated conditions. GAPDH served as a loading control (left panel). The graph (right panel) shows Western blot quantification normalized to GAPDH (n = 3 biological replicates); the statistical significance was determined by the two-tailed t-test. For all the relevant panels, data are shown as mean ± standard deviation (SD). Source Data are provided as a Source Data file.
