Figure 1.

Schematic overview of transcriptional and translational layers of control for the four systems developed in this study. Different promoters and riboswitches were combined to control the toxin’s (red) gene expression. (A) PfdeA-RS_B12 combination. The naringenin-inducible promoter PfdeA in light blue is positively regulated by the FdeR activator (grey) in presence of naringenin and is combined with the negatively regulated vitamin B₁₂ riboswitch (RS_B12, green). Only in absence of vitamin B₁₂, the ribosome binding site (RBS, orange) is accessible for ribosome binding. (B) Ptac-RS_B12 combination. The IPTG-inducible tac promoter (Ptac) in light purple is repressed by binding of the LacI repressor (brown) to the lac operator (lacO, purple) in absence of IPTG. Presence of IPTG allows transcription initiation. This is combined with the B₁₂ riboswitch. (C) Ptac-RS_theo combination. The lacI/lacO operator system is combined with the positively regulated synthetic theophylline riboswitch (RS_theo, yellow). Presence of theophylline (blue) alters the riboswitch conformation, allowing access to the RBS and therefore translation. (D) P_BAD-RS_theo combination. The arabinose-inducible promoter P_BAD (light green) is positively regulated through the binding of the AraC activator (magenta) to the I1 and I2 sites when bound to arabinose (turquoise). Combination with the RS_theo allows tight regulation of the toxic gene.