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).

Auswertung plate reader andere promotoren.png

Fig. 1: 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.

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 P_liaG (black) and P_veg (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.

Retrieved from "http://2012.igem.org/Team:LMU-Munich/Data/Constitutive"

@@ Line 1: / Line 1: @@
 {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}
-==Constitutive ''Bacillus'' Promoters==
-[[File:Auswertung_plate_reader_andere_promotoren.png|thumb|right|400px|'''<p align="justify">Fig. 3: Luminescence measurement of the constitutive ''Bacillus'' promoters P<sub>''liaG''</sub> and P<sub>''lepA''</sub> in the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE'''. OD<sub>600</sub> (up), LUMI (center) and LUMI per OD<sub>''600''</sub> (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, OD<sub>''600''</sub>=0,3) and smoothed by taking average of three neighboring values.</p>]]
+[[Image:BacillusBioBrickBox.png|100px|right|link=Team:LMU-Munich/Team:LMU-Munich/Bacillus_BioBricks]]
-<p align="justify">
-The '''constitutive promoters''' P<sub>''liaG''</sub> and P<sub>''lepA''</sub> were evaluated in the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> which contains the ''lux'' operon. Data derive from three undependant measurements ('''Fig. 3'''). Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smoothed by taking average of three neighboring values. OD<sub>600</sub> 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<sub>''lepA''</sub> and P<sub>''liaG''</sub> 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<sub>''liaG''</sub> has an activity maximum of about 100.000 Lumi/OD<sub>600</sub> during the pass from logarithmic to the stationary phase. P<sub>''lepA''</sub> shows an maximum of about 400.000 Lumi/OD<sub>600</sub>. Comparing these two constitutive promoters the activity of P<sub>''lepA''</sub> is about four times higher than the activity of P<sub>''liaG''</sub>. In the late stationary phase the activity completely disappears.</p>
-<p align="justify">[[File:Englisch_Auswertung_PliaG_Pveg.png‎|thumb|center|400px|'''Fig. 4: β galactosidase assay and growth curve P<sub>''liaG''</sub> (black) and P<sub>''veg''</sub> (grey) in the reporter vector pSB<sub>''Bs''</sub>1C-''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.]]</p>
+==Constitutive ''Bacillus'' Promoters==
+<br>
+===Luminescence measurements===
+<br>
+<p align="justify">
+The constitutive promoters '''P<sub>''liaG''</sub>''' and '''P<sub>''lepA''</sub>''' were evaluated in the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i>, which contains the ''lux'' operon [[File:Lux operon.png|100px]]. 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). </p>
+{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
+| style="width: 70%;background-color: #EBFCE4;" |
+{|
+|[[File:Auswertung_plate_reader_andere_promotoren.png|400px]]
+|-
+| style="width: 70%;background-color: #EBFCE4;" |
+{| style="color:black;" cellpadding="0" width="100%" cellspacing="0" border="0" align="center" style="text-align:center;"
+|style="width: 70%;background-color: #EBFCE4;" |
+<font color="#000000"; size="2"><p align="justify">'''Fig. 1: Luminescence measurement of the constitutive ''Bacillus'' promoters P<sub>''liaG''</sub> and P<sub>''lepA''</sub> in the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE'''''. OD<sub>600</sub> (up), LUMI (middle) and LUMI per OD<sub>600</sub> (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, OD<sub>600</sub>=0.3) and smoothed by taking the average of three neighboring values.</p></font>
+|}
+|}
+|}
+<p align="justify">All clones show a normal growth behaviour. The activity of both promoters increases during transition from log to stationary phase. P<sub>''liaG''</sub> has an activity maximum of about 100.000 Lumi/OD<sub>600</sub>. P<sub>''lepA''</sub> shows a maximum of about 400.000 Lumi/OD<sub>600</sub>. Comparing these two constitutive promoters, the activity of P<sub>''lepA''</sub> is about four times higher than the activity of P<sub>''liaG''</sub>. In the late stationary phase the activity completely disappears. The second clone of the promoters P<sub>''lepA''</sub> and P<sub>''liaG''</sub> did not show any luminescence activity. Therefore, additional clones need to be measured.</p>
-<p align="justify">The β galactosidase assay of the constitutive ''Bacillus'' promoters Pveg and P<sub>''liaG''</sub> 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<sub>''veg''</sub> and P<sub>''liaG''</sub> 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<sub>''liaG''</sub> with an maximum activity of about 12 Miller Units.
+===β-galactosidase assays===
+<br>
+<p align="justify"> The two constitutive promoters '''P<sub>''liaG''</sub>''' and '''P<sub>''veg''</sub>''' were evaluated with the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ'', which contains the ''lacZ'' reporter gene [[File:LacZ.png|50px]] (Fig. 2).</p>
+<br>
+{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
+| style="width: 70%;background-color: #EBFCE4;" |
+{|
+|[[File:Englisch_Auswertung_PliaG_Pveg.png‎|400px]]
+|-
+| style="width: 70%;background-color: #EBFCE4;" |
+{| style="color:black;" cellpadding="0" width="100%" cellspacing="0" border="0" align="center" style="text-align:center;"
+|style="width: 70%;background-color: #EBFCE4;" |
+<font color="#000000"; size="2"><p align="justify"> '''Fig. 2: β-galactosidase assay and growth curve of strains carrying the promoters P<sub>''liaG''</sub> (black) and P<sub>''veg''</sub> (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.</p></font>
+|}
+|}
+|}
+<br>
+<p align="justify">Promoter activity results in β-galactosidase expression. The β-galactosidase assays of P<sub>''veg''</sub> and P<sub>''liaG''</sub> 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<sub>''veg''</sub>= 65 Miller Units, P<sub>''liaG''</sub>= 12 Miller Units) can be found during the transition from logarithmic to stationary phase.
 </p>