Team:LMU-Munich/Data/Inducible

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==Inducible ''Bacillus'' Promoters==
 
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[[File:plate reader induzierbar promotor.png‎|thumb|right|400px|<p align="justify">'''Fig. 1: Luminescence measurements of the inducible ''B. subtilis'' promoter P<sub>''liaI''</sub> after induction with different concentrations of bacitracin.''' OD<sub>600</sub> (up), LUMI (middle) and LUMI per OD<sub>''600''</sub> (down) of the two clones (green/blue) depending on the time (h) after induction with  bacitracin (0 μg/ml(left) to 100μg/ml(right)). 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. t=0 is the time of induction.</p> ]]
 
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==Inducible ''Bacillus'' Promoters==
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===Luminescence measurements===
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The inducible promoter '''P<sub>''liaI''</sub>''' was evaluated in the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> which contains the ''lux'' operon [[File:Lux operon.png|100px]]. 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 up to a bacitracin concentration of 10 μg/ml. At higher concentrations of bacitracin the growth curves decrease because the cells die in presence of bacitracin. The promoter PliaI shows a basal activity level of about 10.000 Lumi/OD600 at the maximum. After induction with bacitracin the luminescence per 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 gets higher than 100 μg/ml the luminescence of both clones show a different behaviour. To see also the induction of this inducible promoter in comparison to an uninducible promoter we also did induction experiments with PliaG which should not be inducible. As expected there is no response in activity depending on the induction and PliaG shows a constant maximum of activity of about 10.000 Lumi/OD600 (Data not shown).</p>
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The bacitracin-inducible promoter '''P<sub>''liaI''</sub>''' was 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 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'' ([http://www.biotek.com/ BioTek]) (Fig. 1).</p>
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<font color="#000000"; size="2"><p align="justify">'''Fig. 1: Luminescence measurements of the inducible ''B. subtilis'' promoter P<sub>''liaI''</sub> after induction with different concentrations of bacitracin.''' OD<sub>600</sub> (up), LUMI (middle) and LUMI per OD<sub>600</sub> (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 is derived from three independent experiments. The graphs show the mean value 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. t=0 is the time of induction.</p></font>
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<p align="justify">All clones show a normal growth behaviour up to a bacitracin concentration of 10 μg/ml. At higher bacitracin concentrations, the growth curves decrease because of cell lyses. The promoter P<sub>''liaI''</sub> shows a basal activity of about 10.000 Lumi/OD<sub>600</sub>. After induction with bacitracin the Lumi/OD<sub>600</sub> increases in a concentration depending manner. The highest activity of about 1.5 Mio Lumi/OD<sub>600</sub> 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. In contrast, the constitutive promoter P<sub>''liaG''</sub> shows a constant value of about 10,000 Lumi/<sub>OD</sub> independent of the bacitracin concentration (data not shown).</p>
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<p align="justify">To better illustrate the P<sub>''liaI''</sub> activity as a function of the bacitracin concentration, data from one timepoint (t=3.5h) of the experiment (Fig. 1) is plotted against the bacitracin concentration (Fig. 2).</p>
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<font color="#000000"; size="2"><p align="justify">'''Fig. 2: Promoter activity of P<sub>''liaI''</sub> 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 OD<sub>600</sub> of both clones (blue/green) from the time point t=3.5h. Data is derived from three independent experiments.</p></font>
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[[File:Englisch Auswertung PliaI.png‎|thumb|right|400px|<p align="justify">'''Fig. 2: β-galactosidase assay with (grey) and without (black) induction of bacitracin (20μg/ml) of strains carrying the promoter P<sub>''liaI''</sub> 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). Experiment shows representative data which was obtained in the same way from three independent experiments.</p>]]
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===β-galactosidase assays===
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The inducible promoter P<sub>''liaI''</sub> was also evaluated with the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ'' [[File:LacZ.png|50px]]. (Fig. 3).</p>
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
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<font color="#000000"; size="2"><p align="justify">'''Fig. 3: β-galactosidase assay with (grey) and without (black) induction of bacitracin (20 μg/ml) of strains carrying the promoter P<sub>''liaI''</sub> 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 three independent experiments.</p></font>
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The inducible promoter P<sub>''liaI''</sub> was also evaluated with the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ'' which contains the ''lacZ'' reporter gene [[File:LacZ.png|50px]] ('''Fig.2'''). Promoter activity leads to the expression of the β-galactosidase which directly correlates to the promoter activity. The β-galactosidase assay of the inducible ''Bacillus'' promoter P<sub>''liaI''</sub> was repeated three times. Data show one representative result. We induced with bacitracin (20μg/ml) when OD600 reached 0,4. PliaI does not show any activity without induction. After induction with bacitracin (20μg/ml) there is a strong gene expression so that the produced enzyme reaches an activity of about 500 Miller Units. Summing up the activity of the enzyme and tht promoter activity after induction is about 500 times higher than without induction.
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The β-galactosidase assay of the inducible ''Bacillus'' promoter P<sub>''liaI''</sub> was repeated three times. We induced with bacitracin (20μg/ml) when the cultures reached an OD<sub>600</sub> of 0.4. P<sub>''liaI''</sub> only shows marginal basal activity without induction. After induction a promoter activity of up to 500 Miller Units was measured. In summary, the promoter activity is induced about 500 fold in the presence of bacitracin.
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