Team:LMU-Munich/Data/Constitutive

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Constitutive Bacillus Promoters

Luminescence measurements

The constitutive promoters P_liaG and P_lepA were evaluated in the reporter vector pSB_Bs3C-luxABCDE, which contains the lux operon . The promoter activity results in gene expression and production of the protein luciferase. The luminescence produced by this protein can be measured with the plate reader Synergy2 ([http://www.biotek.com/ BioTek]) (Fig. 1).

Fig. 1: Luminescence measurement of the constitutive Bacillus promoters PliaG and PlepA in the reporter vector pSBBs3C-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.

All clones show a normal growth behaviour. The activity of both promoters increases during transition from log to stationary phase. P_liaG has an activity maximum of about 100.000 Lumi/OD₆₀₀. 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. The second clone of the promoters P_lepA and P_liaG did not show any luminescence activity. Therefore, additional clones need to be measured.

β-galactosidase assays

The two constitutive promoters P_liaG and P_veg were evaluated with the reporter vector pSB_Bs1C-lacZ, which contains the lacZ reporter gene (Fig. 2).

Fig. 2: β-galactosidase assay and growth curve of strains carrying the promoters PliaG (black) and Pveg (grey) fused to lacZ. β-galactosidase activity (Miller Units) and the growth curve values are the average of two independent clones with their standard deviation. Experiment shows representative data from three independent experiments.

Promoter activity results in β-galactosidase expression. The β-galactosidase assays of P_veg and P_liaG were repeated three times. The graph shows data of one representative experiment. In the beginning of the growth curve, both promoters show only low activity before it increases to a maximum value at three hours. It then decreases steadily until reaching initial level after about seven hours (data not shown). Summing up, the time course of activity is very similar for both promoters. The highest β-galactosidase activity (P_veg= 65 Miller Units, P_liaG= 12 Miller Units) can be found during the transition from logarithmic to stationary phase.