Team:LMU-Munich/Bacillus BioBricks/Promoters

From 2012.igem.org

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<p align="justify">To provide a set of promoters of different strength we characterized several promoters in ''Bacillus subtilis''. They can be divided in three different groups:  
<p align="justify">To provide a set of promoters of different strength we characterized several promoters in ''Bacillus subtilis''. They can be divided in three different groups:  
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*the constitutive promoters from the [http://partsregistry.org/Part:BBa_J23100 Anderson collection] from the Partsregistry
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*the constitutive promoters from the [http://partsregistry.org/Part:BBa_J23100 Anderson collection] from the [http://partsregistry.org/Main_Page Partsregistry]
*the constitutive promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis''
*the constitutive promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis''
*the inducible promoters P<sub>''liaI''</sub> and ''xylR''-P<sub>''xyl''</sub> from ''B. subtilis''</p>
*the inducible promoters P<sub>''liaI''</sub> and ''xylR''-P<sub>''xyl''</sub> from ''B. subtilis''</p>
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<p align="justify">For the characterization of the different promoters we used the ''lux'' operon [[File:Lux operon.png|100px]]. Here, promoter activity leads to expression of the luciferase and hence light production, which can be measured as luminescence.</p>
<p align="justify">For the characterization of the different promoters we used the ''lux'' operon [[File:Lux operon.png|100px]]. Here, promoter activity leads to expression of the luciferase and hence light production, which can be measured as luminescence.</p>
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<p align="justify">We also used the reporter gene ''lacZ'' [[File:LacZ.png|50px]]. Here, promoter activation results in expression of a β-galactosidase, whose activity can be measured by breakdown of the chromophoric substrate ONPG (β-galactosidase assays). We used the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ''. See this page for an overview and background information of all evaluated promoters and see the [https://2012.igem.org/Team:LMU-Munich/Data Data] page for more details.</p>
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<p align="justify">We also used the reporter gene ''lacZ'' [[File:LacZ.png|50px]]. Here, promoter activation results in expression of a β-galactosidase, whose activity can be measured by breakdown of the chromophoric substrate ONPG (β-galactosidase assays). We used the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ''. See below for an overview and background information of all evaluated promoters and see the [https://2012.igem.org/Team:LMU-Munich/Data Data] page for more details.</p>
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====Evaluation of Anderson promoters in ''B. subtilis''====
====Evaluation of Anderson promoters in ''B. subtilis''====
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<p align="justify">The first group of promoters evaluated are the promoters of the [http://partsregistry.org/Part:BBa_J23100 Anderson collection]  (''"Anderson promoters"''). They have already been extensively measured in ''Escherichia coli'' where they all showed a constitutive behavior with different strength. In this project, eleven Anderson promoters were characterized in ''B. subtilis'' with the ''lux'' operon as a reporter. In ''B. subtilis'' these promoters show quiet low activity (see [https://2012.igem.org/Team:LMU-Munich/Data/Anderson#Luminescence_measurements Data Anderson promoters] [[File:Lux operon.png|100px|link=https://2012.igem.org/Team:LMU-Munich/Data/Anderson#Luminescence_measurements]]).  
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<p align="justify">The first group of promoters evaluated are the promoters of the [http://partsregistry.org/Part:BBa_J23100 Anderson collection]  (''"Anderson promoters"''). They have already been extensively measured in ''Escherichia coli'' where they all showed a constitutive behavior with different strength. In this project, eleven Anderson promoters were characterized in ''B. subtilis'' with the ''lux'' operon as a reporter. In ''B. subtilis'' these promoters show quite low activity (see [https://2012.igem.org/Team:LMU-Munich/Data/Anderson#Luminescence_measurements Data Anderson promoters] [[File:Lux operon.png|100px|link=https://2012.igem.org/Team:LMU-Munich/Data/Anderson#Luminescence_measurements]]).  
To confirm these results some Anderson promoters were also evaluated with the reporter gene ''lacZ'' by doing β-galactosidase assays (see [https://2012.igem.org/Team:LMU-Munich/Data/Anderson Data Anderson promoters] [[File:LacZ.png|50px|link=https://2012.igem.org/Team:LMU-Munich/Data/Anderson#β-galactosidase_assays]]).</p>
To confirm these results some Anderson promoters were also evaluated with the reporter gene ''lacZ'' by doing β-galactosidase assays (see [https://2012.igem.org/Team:LMU-Munich/Data/Anderson Data Anderson promoters] [[File:LacZ.png|50px|link=https://2012.igem.org/Team:LMU-Munich/Data/Anderson#β-galactosidase_assays]]).</p>
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*'''P<sub>''liaI''</sub>'''    ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823001 BioBrick:BBa_K823001])
*'''P<sub>''liaI''</sub>'''    ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823001 BioBrick:BBa_K823001])
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<p align="justify">P''<sub>liaI</sub>'' is an inducible promoter from ''B. subtilis'', which responds to antibiotics that interfere with the integrity and biosynthesis of the cell wall [http://www.ncbi.nlm.nih.gov/pubmed/15273097 (Mascher ''et al.'', 2004)]. In the presence of a stimulus, the two-component system LiaRS is activated. The activated response regulator LiaR binds to the operator of the promoter and induces the transcription of the lia locus. When the promoter is turned on the two proteins LiaI and LiaH are expressed which play an important role in the stress response of ''B. subtilis''. The major strength of this promoter is its very low basal activity in the absence of an inducer and its high dynamic range [http://www.ncbi.nlm.nih.gov/pubmed/15273097 (Mascher ''et al.'', 2004)]. This promoter is evaluated with the reporter ''lux'' (see [https://2012.igem.org/Team:LMU-Munich/Data/Inducible#Luminescence_measurements Data inducible promoters] [[File:Lux operon.png|100px|link=https://2012.igem.org/Team:LMU-Munich/Data/Inducible#Luminescence_measurements]]) as well as ''lacZ'' (see [https://2012.igem.org/Team:LMU-Munich/Data/Inducible Data inducible promoters] [[File:LacZ.png|50px|link=https://2012.igem.org/Team:LMU-Munich/Data/Inducible]]). The induction was measured with different concentrations of bacitracin, demonstrating a concentration-dependent response over a large range of promoter activities.</p>
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<p align="justify">P''<sub>liaI</sub>'' is an inducible promoter from ''B. subtilis'', which responds to antibiotics that interfere with the integrity and biosynthesis of the cell wall [http://www.ncbi.nlm.nih.gov/pubmed/15273097 (Mascher ''et al.'', 2004)]. In the presence of a stimulus, the two-component system LiaRS is activated. The activated response regulator LiaR binds to the operator of the promoter and induces the transcription of the lia locus. When the promoter is turned on, the two proteins LiaI and LiaH (which play an important role in the stress response of ''B. subtilis'') are expressed. The major strength of this promoter is its very low basal activity in the absence of an inducer and its high dynamic range [http://www.ncbi.nlm.nih.gov/pubmed/15273097 (Mascher ''et al.'', 2004)]. This promoter is evaluated with the reporter ''lux'' (see [https://2012.igem.org/Team:LMU-Munich/Data/Inducible#Luminescence_measurements Data inducible promoters] [[File:Lux operon.png|100px|link=https://2012.igem.org/Team:LMU-Munich/Data/Inducible#Luminescence_measurements]]) as well as ''lacZ'' (see [https://2012.igem.org/Team:LMU-Munich/Data/Inducible Data inducible promoters] [[File:LacZ.png|50px|link=https://2012.igem.org/Team:LMU-Munich/Data/Inducible]]). The induction was measured with different concentrations of bacitracin, demonstrating a concentration-dependent response over a large range of promoter activities.</p>

Latest revision as of 12:53, 26 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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