Constitutive Bacillus Promoters
Fig. 3: Luminescence measurement of the constitutive Bacillus promoters PliaG and PlepA in the reporter vector pSBBs3C-luxABCDE. OD600 (up), LUMI (center) and LUMI per OD600
(down) depending on the time (h) are shown for two different clones (green/blue). Data come from three independent experiments. Curves were fitted over each other (t=0, OD600
=0,3) and smoothed by taking 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. Data derive from three undependant measurements (
Fig. 3). Curves were fitted over each other (t=0, OD
600=0,3) and smoothed by taking average of three neighboring values. OD
600 values shown are plate reader units and about one third of the usual OD values. All clones show a usual growth curves. The activity of the promoters raises during the pass from the transition to the 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 raise to their maximum. They show a similar behaviour in pertaining to the groth curve. P
liaG has an activity maximum of about 100.000 Lumi/OD
600 during the pass from logarithmic to the stationary phase. P
lepA shows an maximum of about 400.000 Lumi/OD
600. 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 PliaG (black) and Pveg (grey) in the reporter vector pSBBs1C-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 PliaG 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 Pveg and PliaG 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 PliaG with an maximum activity of about 12 Miller Units.
"
