Team:LMU-Munich/Data/Inducible
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+ | [[Image:BacillusBioBrickBox.png|100px|right|link=Team:LMU-Munich/Team:LMU-Munich/Bacillus_BioBricks]] | ||
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+ | ==Inducible ''Bacillus'' Promoters== | ||
+ | <br> | ||
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+ | ===Luminescence measurements=== | ||
<p align="justify"> | <p align="justify"> | ||
- | 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'' ([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:plate reader induzierbar promotor.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 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> | ||
<br> | <br> | ||
<br> | <br> | ||
+ | <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> | ||
+ | {| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;" | ||
+ | | style="width: 70%;background-color: #EBFCE4;" | | ||
+ | {| | ||
+ | |[[File:Titration PliaI.png|400px]] | ||
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+ | | 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: 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> | ||
+ | |} | ||
+ | |} | ||
+ | |} | ||
<br> | <br> | ||
<br> | <br> | ||
- | [[File:Englisch Auswertung PliaI.png| | + | ===β-galactosidase assays=== |
+ | <br> | ||
+ | <p align="justify"> | ||
+ | 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> | ||
+ | <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 PliaI.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. 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> | ||
+ | |} | ||
+ | |} | ||
+ | |} | ||
<p align="justify"> | <p align="justify"> | ||
- | + | 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. | |
</p> | </p> | ||
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Latest revision as of 13:27, 26 October 2012
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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 ([http://www.biotek.com/ BioTek]) (Fig. 1).
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 PliaI shows a basal activity of about 10.000 Lumi/OD600. After induction with bacitracin the Lumi/OD600 increases in a concentration depending manner. 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. In contrast, the constitutive promoter PliaG shows a constant value of about 10,000 Lumi/OD independent of the bacitracin concentration (data not shown).
To better illustrate the PliaI 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).
|
β-galactosidase assays
The inducible promoter PliaI was also evaluated with the reporter vector pSBBs1C-lacZ . (Fig. 3).
|
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 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.