Team:LMU-Munich/Data/Inducible

From 2012.igem.org

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===Luminescence measurements===
===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''').</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 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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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 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">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 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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===β-galactosidase assay===
===β-galactosidase assay===
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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'''). 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.
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'''). 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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Revision as of 17:54, 25 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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