Team:LMU-Munich/Data/Anderson

From 2012.igem.org

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Eleven of the nineteen promoters of the [http://partsregistry.org/Part:BBa_J23100 '''Anderson collection'''] (J23100, J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118) were evaluated. For that purpose, we used the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE'' from the BioBrickBox containing the ''lux'' operon [[File:Lux operon.png|100px]] as a reporter for promoter activity. The promoter activity leads to the expression of the ''lux'' operon and to the production of the enzyme luciferase. The bioluminescence, which is produced by the luciferase, can be measured with the microtiter plate reader ''Synergy2'' (["http://www.biotek.com/"] BioTek) ('''Fig.1''').</p>
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Eleven of the nineteen promoters of the [http://partsregistry.org/Part:BBa_J23100 '''Anderson collection'''] (J23100, J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118) were evaluated. For that purpose, we used the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE'' from the BioBrickBox containing the ''lux'' operon [[File:Lux operon.png|100px]] as a reporter for promoter activity. The promoter activity leads to the expression of the ''lux'' operon and to the production of the enzyme luciferase. The bioluminescence, which is produced by the luciferase, can be measured with the microtiter plate reader ''Synergy2'' ([http://www.biotek.com/ BioTek]) ('''Fig.1''').</p>
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===β-galactosidase assay===
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===β-galactosidase assays===
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<p align="justify">To evaluate the activity not only with the ''lux'' reporter operon, four promoters of the Anderson collection were cloned into the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ'' [[File:LacZ.png|50px]] to do β-galactosidase assays and then to compare the results of the strength of these promoters in ''B. subtilis'' ('''Fig. 2'''). The results were compared to the results from the luminescence measurements.
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<p align="justify">To evaluate the activity not only with the ''lux'' reporter, four promoters of the Anderson collection were cloned into the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ'' [[File:LacZ.png|50px]] to perform β-galactosidase assays ('''Fig. 2'''). The results were then compared to the data of the luminescence measurements.
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Verification of the promoter activity by β-galactosidase assays revealed that the Anderson promoters do not seem to be as weak as measured by luminescence (Fig. 1). For direct comparison we should measure a constitutive promoter, e.g. P<sub>''liaG''</sub>, in the same experiment. But we think the luminescence measurements are more reliable because they were repeated three times with two independent clones.
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<p align="justify">Verification of the promoter activity by β-galactosidase assays revealed that the Anderson promoters do not seem to be as weak as measured by luminescence (Fig. 1). For a direct comparison, we need to include a constitutive promoter, e.g. P<sub>''liaG''</sub>, and repeat the same experiment. But we think the luminescence measurements are more reliable, because they were repeated three times with two independent clones.</p>
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Latest revision as of 01:52, 27 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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