FIG 3.

Characterizing activity of select double promoter system modifications by β-galactosidase assays. Assays were performed and shown as described for Fig. 2. (A) Pairwise combinations of the rhaI half-site series and RBS modifications from pKRJ17 and pKRJ18. (B) Combination of the rhaI₁-rhaI₁ half-sites and T7 gene 10 stem-loop; the inset is a representation of the new promoter construction. In panels A and B, the ratio between activity under the induced and uninduced condition was used to calculate the dynamic range for each variant. (C) l-Rhamnose dose-response of β-galactosidase activity with the parent (pKRJ12), double modification construct pKRJ38, and constituent single modification constructs (pKRJ23 and pKRJ24). (D to F) β-Galactosidase reporter activity of pKRJ12 and pKRJ38 in B. thailandensis E264 (D), B. multivorans ATCC 17616 (E), and B. vietnamiensis LMG 16232 (F). Values shown are means ± standard deviations from at least three biological replicates. Significance was assigned by one-way ANOVA followed by Dunnett’s post hoc test to pKRJ12. *, P < 0.05, and **, P < 0.005, inducing conditions; ‡, P < 0.05, and ‡‡, P < 0.005, noninducing conditions.