Team:LMU-Munich/Data/Constitutive

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Revision as of 12:57, 21 September 2012

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Constitutive Bacillus Promoters

Fig. 3: Luminescence measurement of the constitutive Bacillus promoters P_liaG and P_lepA in the reporter vector pSB_Bs3C-luxABCDE. OD₆₀₀ (up), LUMI (middle) and LUMI per OD₆₀₀ (down) depending on the time (h) are shown for two different clones (green/blue). Data derive from three independent experiments, the graphs show the mean fo the 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.

The constitutive promoters P_liaG and P_lepA were evaluated in the reporter vector pSB_Bs3C-luxABCDE which contains the lux operon. This is why the promoter activity leads to gene expression and to the production of the protein luciferase. The luminescence of this protein can be measured with the plate reader Synergy2 (Biotek) (Fig.1). All clones show a normal growth behaviour. The activity of the promoters raises during transition from log to stationary phase. The second clone of the promoters P_lepA and P_liaG did not show any luminescence activity. In the beginning of the growth curve the activity of both promoters increase to their maximum. This activity increase appears during the same growth phase. P_liaG has an activity maximum of about 100.000 Lumi/OD₆₀₀ during the transition from logarithmic to the stationary phase. P_lepA shows a maximum of about 400.000 Lumi/OD₆₀₀. Comparing these two constitutive promoters the activity of P_lepA is about four times higher than the activity of P_liaG. In the late stationary phase the activity completely disappears.

Fig. 4: β galactosidase assay and growth curve P_liaG (black) and P_veg (grey) in the reporter vector pSB_Bs1C-lacZ. β galactosidase activity (Miller Units)and growth curve are the average of two independant clones. Experiment shows representative data which was obtained in the same way from three independent experiments.

The β galactosidase assay of the constitutive Bacillus promoters Pveg and P_liaG was repeated three times. Data show one representative result. Two undependant clones of B. subtilis with the same construct were measured and their mean with standard deviation is show in the graph. In the beginning of the growth curve both promoters show a small activity. But then it raises to a maximum before it decreases to the begininng level after about seven hours (Data not shown). Summing up the course of activity of both promoters P_veg and P_liaG is very similar based on the growth curve. The highest beta galactosidase activity and therefore the highest activity of the promoter Pveg can with an maximum of 65 Miller units be found during the transfer from the logarithmic to the stationary phase. This is about five times higher than the acitivity of the promoter P_liaG with an maximum activity of about 12 Miller Units.