Team:LMU-Munich/Data/Inducible

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Inducible Bacillus Promoters

Luminescence measurements

The bacitracin-inducible promoter P_liaI was evaluated in the reporter vector pSB_Bs3C-luxABCDE which contains the lux operon . The promoter activity leads to gene expression and to the production of the protein luciferase. The luminescence produced by this protein can be measured with the plate reader Synergy2 (BioTek) (Fig. 1).

Fig. 1: Luminescence measurements of the inducible B. subtilis promoter PliaI after induction with different concentrations of bacitracin. OD600 (up), LUMI (middle) and LUMI per OD600 (down) of two clones (green/blue) depending on the time (h) after induction with bacitracin (0 μg/ml (left) to 100 μg/ml (right)). Data is derived from three independent experiments. The graphs show the mean value and the standard deviation. Curves were fitted over each other (t=0, OD600=0.3) and smoothed by taking the average of three neighboring values. t=0 is the time of induction.

All clones show a normal growth behaviour up to a bacitracin concentration of 10 μg/ml. At higher bacitracin concentrations, the growth curves decrease because of cell lyses. The promoter P_liaI shows a basal activity of about 10.000 Lumi/OD₆₀₀. After induction with bacitracin the Lumi/OD₆₀₀ increases in a concentration depending manner. The highest activity of about 1.5 Mio Lumi/OD₆₀₀ can be measured after induction with 10-30 μg/ml bacitracin. If the concentration is higher than 100 μg/ml the luminescence of both clones shows a different behaviour. In contrast, the constitutive promoter P_liaG shows a constant value of about 10,000 Lumi/_OD independent of the bacitracin concentration (data not shown).

To better illustrate the P_liaI activity as a function of the bacitracin concentration, data from one timepoint (t=3.5h) of the experiment (Fig. 1) is plotted against the bacitracin concentration (Fig. 2).

Fig. 2: Promoter activity of PliaI depending on the bacitracin concentration (0,1 μg/ml to 100 μg/ml). Values of the experiment (Fig. 1) are shown in a different way: Luminescence per OD600 of both clones (blue/green) from the time point t=3.5h. Data is derived from three independent experiments.

β-galactosidase assays

The inducible promoter P_liaI was also evaluated with the reporter vector pSB_Bs1C-lacZ . (Fig.3).

Fig. 3: β-galactosidase assay with (grey) and without (black) induction of bacitracin (20 μg/ml) of strains carrying the promoter PliaI fused to lacZ. The average of the β-galactosidase activity (Miller Units) and the standard deviation of two independent clones are shown depending on the time (minutes after induction). This graph shows representative data from three independent experiments.

The β-galactosidase assay of the inducible Bacillus promoter P_liaI was repeated three times. We induced with bacitracin (20μg/ml) when the cultures reached an OD₆₀₀ of 0.4. P_liaI only shows marginal basal activity without induction. After induction a promoter activity of up to 500 Miller Units was measured. In summary, the promoter activity is induced about 500 fold in the presence of bacitracin.