Figure 2. Analysis of the reporter system’s regulatory properties after directed mutagenesis of the P_cat -10 element.

(A) Comparison of the P_cat and the S. Typhimurium (STY) σ⁷⁰ consensus promoter sequences. Differences are highlighted in red. (B) Western blot for determining the steady-state level of TetR expressed by P_cat or P_cat -10CATTTA (5 µg crude protein extract of each) with a polyclonal anti-TetR antibody. As controls, 5 µg crude protein extract of the strain P_tetA fluc (lacking TetR) and 30 ng of purified TetR were loaded onto the gel. (C) Dose-response curve to analyze the sensitivity of TetR induction by the plasmid-encoded Trx1-TIP2 fusion protein (pTrx1-TIP2) in strains expressing TetR either by P_cat or by P_cat -10CATTTA. These were incubated without and with 400 nM atc as control for maximum induction of TetR. Increasing IPTG concentrations were added for Trx1-TIP2 expression. The star marks the IPTG concentration at which the level of Trx1-TIP2 corresponds roughly to the endogenous Trx1 level. The bars illustrate the relative light units (RLU) which were normalized to a 1 ml culture with OD₅₉₅ = 1. The data are a representative set from at least three independent measurements and display the mean ± standard deviation.