FIG 6.

Regulated gene expression system in P. micra. (A) Genomic context (top) and corresponding sequence (bottom) for an inducible luciferase reporter strain (i-renG) in P. micra. The tuf gene Rho-independent terminator is indicated by a ball and stick icon. The sequences are shown for the promoter region of the luciferase reporter, with the original theophylline riboswitch (underlined) and Theo+ riboswitch (dashed lines, bottom) indicated. Promoter elements are shown in red font, while the ribosomal binding site is in blue font. (B) The mFold webserver (http://www.unafold.org/mfold/applications/rna-folding-form.php) was used to predict the secondary structures of the original (left image) and Theo+ (right image) theophylline riboswitches. (C) The original and Theo+ riboswitches were compared for their abilities to regulate luciferase reporter activity over a range of theophylline concentrations. Normalized luciferase activity (RLU) was calculated by dividing raw luciferase values by the respective OD₆₀₀ values. Data points represent the average values ± SD from triplicate independent determinations.