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]]. | + | 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> | + | <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 | + | <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 | + | <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 | + | <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'' | + | 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 ( | + | <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 | + | 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
The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".
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Inducible Bacillus Promoters
Luminescence measurements
The bacitracin-inducible promoter PliaI was evaluated in the reporter vector pSBBs3C-luxABCDE which contains the lux operon . 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).
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 PliaI shows a basal activity of about 10.000 Lumi/OD600. After induction with bacitracin the Lumi/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 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 PliaG, which should not be inducible by bacitracin. As expected there is no response to the induction with bacitracin and PliaG shows a constant maximum of about 10.000 Lumi/OD600 (Data not shown).
For better demonstration of the PliaI 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/OD600 values are shown for the time point t=3,5h.
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β-galactosidase assay
The inducible promoter PliaI was also evaluated with the reporter vector pSBBs1C-lacZ . (Fig.3).
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Promoter activity leads to the expression of the β-galactosidase. The β-galactosidase assay of the inducible Bacillus promoter PliaI was repeated three times. We induced with bacitracin (20μg/ml) when the cultures reached an OD600 of 0.4. PliaI 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.