Team:LMU-Munich/Data/Inducible

From 2012.igem.org

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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''').</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'' (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 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></font>
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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 derive from three independent experiments. The graphs show the mean value for 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></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 concentrations of bacitracin the growth curves decrease because the cells die in presence of bacitracin. The promoter P<sub>''liaI''</sub> shows a basal activity level of about 10.000 Lumi/OD600 at the maximum. After induction with bacitracin the luminescence per OD<sub>600</sub> increases depending of the concentration of bacitracin. 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 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 P<sub>''liaG''</sub> which should not be inducible. As expected there is no response in activity depending on the induction and P<sub>''liaG''</sub> shows a constant maximum of activity of about 10.000 Lumi/OD<sub>600</sub> (Data not shown).</p>
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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 concentrations of bacitracin the growth curves decrease because the cells lyse. 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 depending of the concentration of bacitracin. 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. To see the induction of this inducible promoter in comparison to an uninducible promoter we also did induction experiments with P<sub>''liaG''</sub>, which should not be inducible by bacitracin. As expected there is no response to the induction with bacitracin and P<sub>''liaG''</sub> shows a constant maximum of about 10.000 Lumi/OD<sub>600</sub> (Data not shown).</p>
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<p align="justify">For better demonstration of the induction, data from the experiment (Fig. 1) are shown depending on the bacitracin concentration ('''Fig. 2'''). Therefore luminescence per OD<sub>600</sub> values are shown for the time point t=3,5h.</p>
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<p align="justify">For better demonstration of the P<sub>''liaI''</sub> activity as a function of the bacitracin concentration, data from one timepoint at the experiment (Fig. 1) are shown depending on the bacitracin concentration ('''Fig. 2'''). Therefore Lumi/OD<sub>600</sub> values are shown for the time point t=3,5h.</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 derive from three independent experiments.</p></font>
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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 derive 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.3''').</p>
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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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<font color="#000000"; size="2"><p align="justify">'''Fig. 3: 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). This graph shows representative data from one experiment which was obtained in the same way from three independent experiments.</p></font>
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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 one experiment which was obtained in the same way from three independent experiments.</p></font>
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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 OD<sub>600</sub> reached 0,4. P<sub>''liaI''</sub> 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 the promoter activity after induction is about 500 times higher than without induction.
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Promoter activity leads to the expression of the β-galactosidase. 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> does not show any activity without induction. After induction with bacitracin there is a strong promoter activity that the produced enzyme reaches an activity of about 500 Miller Units. Summing up, the promoter activity after induction is about 500 times higher than without induction.
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Revision as of 18:28, 25 September 2012

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