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 derive from three independent experiments. The graphs show the mean value for three experiments 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 concentrations of bacitracin the growth curves decrease because the cells lyse. The promoter P_liaI shows a basal activity of about 10.000 Lumi/OD₆₀₀. After induction with bacitracin the Lumi/OD₆₀₀ increases depending of the concentration of bacitracin. 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. To see the induction of this inducible promoter in comparison to an uninducible promoter we also did induction experiments with P_liaG, which should not be inducible by bacitracin. As expected there is no response to the induction with bacitracin and P_liaG shows a constant maximum of about 10.000 Lumi/OD₆₀₀ (Data not shown).

For better demonstration of the P_liaI activity as a function of the bacitracin concentration, data from one timepoint at the experiment (Fig. 1) are shown depending on the bacitracin concentration (Fig. 2). Therefore Lumi/OD₆₀₀ values are shown for the time point t=3,5h.

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 derive from three independent experiments.

β-galactosidase assay

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 one experiment which was obtained in the same way from three independent experiments.

Promoter activity leads to the expression of the β-galactosidase. 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 does not show any activity without induction. After induction with bacitracin there is a strong promoter activity that the produced enzyme reaches an activity of about 500 Miller Units. Summing up, the promoter activity after induction is about 500 times higher than without induction.