Team:LMU-Munich/Bacillus BioBricks

From 2012.igem.org

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<p align="justify">We also tested the [http://partsregistry.org/Promoters/Catalog/Anderson Anderson promoter collection] in ''Bacillus subtilis'' with pSB<sub>Bs</sub>3C-<i>luxABCDE</i>. The data will be online in our [https://2012.igem.org/Team:LMU-Munich/Data Data] section soon.</p>
<p align="justify">We also tested the [http://partsregistry.org/Promoters/Catalog/Anderson Anderson promoter collection] in ''Bacillus subtilis'' with pSB<sub>Bs</sub>3C-<i>luxABCDE</i>. The data will be online in our [https://2012.igem.org/Team:LMU-Munich/Data Data] section soon.</p>
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<p align="justify">Furthermore we plan to submit reporter genes optimized for ''Bacillus subtilis'', namely GFP, lacZ, luc and mKate. </p>
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<p align="justify">Furthermore we plan to submit reporter genes optimized for ''Bacillus subtilis'', namely ''GFP'', ''lacZ'', ''luc'' and ''mKate2''. </p>
Useful for the registry are also 5 tags in Freiburg Standard (cMyc, 10His, Flag, Strep, HA).
Useful for the registry are also 5 tags in Freiburg Standard (cMyc, 10His, Flag, Strep, HA).
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==''Bacillus'' Promoters==
==''Bacillus'' Promoters==
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<p align="justify">Another goal is to produce promoters of ''Bacillus subtilis'' in BioBrick mode and to evaluate them. These well-defined promoters will then be part of the [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks ''<b>Bacillus''B</b>io<b>B</b>rick<b>B</b>ox] which we will send to the registry, but they can also be useful in our project [https://2012.igem.org/Team:LMU-Munich/Project '''Bead'''zillus] to express our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins fusion crust proteins] on the outside of our spores. Therefore we will use different promoters which are the constitutive promoters from the [http://partsregistry.org/Promoters/Catalog/Anderson Anderson collection] from the Parts Registry, the constitutive promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis'' as well as the inducible promoter P<sub>''liaI''</sub> from ''B. subtilis''. For the characterization of the different promoters we will clone them upstream of reporter genes (lux operon, lacZ) to measure their activity in ''B. subtilis''. Therefore we use e.g. the two reporter vectors from ''B. subtilis'' which are also part of the [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks ''<b>Bacillus''B</b>io<b>B</b>rick<b>B</b>ox]. One of the reporter vectors pSB<sub>Bs</sub>3C-<i>luxABCDE</i> contains the ''lux'' operon as a reporter. This is why the activity of the promoter can be measured as luminescence with the plate reader (BioTek) which is a result of the expression of the luciferase. The second reporter vector used for the evaluation of the promoters is pSB<sub>Bs</sub>1C-''lacZ'' which contains the reporter gene ''lacZ''. The promoter activity leads to the production of the enzyme beta-galactosidase which cleaves the substrate ONPG. The product is detectable with the photometer and refers to the promoter activity.</p>
 
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=== Promoter Evaluation ===
 
<p align="justify">To get a set of promoters with different strength we characterized several promoters in ''B. subtilis''. They can be divided in three different groups: the constitutive promoters from the [http://partsregistry.org/Part:BBa_J23100 Anderson collection] from the Partsregistry, the constitutive promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis'', and the inducible promoters P<sub>''liaI''</sub> and P<sub>''xyl''</sub>-''XylR'' from ''B. subtilis''. For the characterization of the different promoters we used the two reporter vectors pSBBs4S-luxABCDE and pSBBs1C-lacZ as well as the reporters lacZ luc and mKate in BioBrick standard.</p>
<p align="justify">To get a set of promoters with different strength we characterized several promoters in ''B. subtilis''. They can be divided in three different groups: the constitutive promoters from the [http://partsregistry.org/Part:BBa_J23100 Anderson collection] from the Partsregistry, the constitutive promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis'', and the inducible promoters P<sub>''liaI''</sub> and P<sub>''xyl''</sub>-''XylR'' from ''B. subtilis''. For the characterization of the different promoters we used the two reporter vectors pSBBs4S-luxABCDE and pSBBs1C-lacZ as well as the reporters lacZ luc and mKate in BioBrick standard.</p>
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====Anderson promoters====
====Anderson promoters====
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<p align="justify">The first group of promoters evaluated are the promoters of the Anderson collection which we call ''Anderson promoters''. They have already been measured in ''Escherichia coli'' where they all showed a constitutive behavior with a different strength. In this project, eleven Anderson promoters were characterized in ''B. subtilis''. Therefore we used the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> from the BioBrickBox were they showed quiet a low activity in B. subtilis (see Data). To confirm this result some Anderson promoters were also evaluated in the reporter vector pSB<sub>Bs</sub>1C-''lacZ''(see Data).</p>
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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] which we call ''Anderson promoters''. They have already been measured in ''Escherichia coli'' where they all showed a constitutive behavior with a different strength. In this project, eleven Anderson promoters were characterized in ''B. subtilis''. Therefore we used the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> from the BioBrickBox were they showed quiet a low activity in B. subtilis (see Data). To confirm this result some Anderson promoters were also evaluated in the reporter vector pSB<sub>Bs</sub>1C-''lacZ''(see Data).</p>
====Constitutive promoters from ''B. subtilis''====
====Constitutive promoters from ''B. subtilis''====
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<p align="justify">The second group of promoters charaterized are constitutive promoters from ''B. subtilis''. We evaluated the promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub>. Therefore we used the reporter vectors pSB<sub>Bs</sub>3C-<i>luxABCDE</i> and pSB<sub>Bs</sub>1C-''lacZ'' as well as the reporter BioBricks lacZ, luc and mKate2.</p>
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<p align="justify">The second group of promoters charaterized are constitutive promoters from ''B. subtilis''. We evaluated the promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub>. Therefore we used the reporter vectors pSB<sub>Bs</sub>3C-<i>luxABCDE</i> and pSB<sub>Bs</sub>1C-''lacZ'' as well as the reporter BioBricks ''lacZ'', ''luc'' and ''mKate2''.</p>
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*'''P<sub>''liaG''</sub>'''
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<p align="justify">PliaG is a weak, constitutive promoter from the LiaRS system, were it is resposible for the production of the components for the resistence against cell wall antibiotics (Reference). This promoter showed a much higher activitx than the Anderson promoters which was still weak in comparison to the other evaluated ''Bacillus'' promoters.</p>
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K823000 BioBrick:BBa_K823000]
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P<sub>''liaG''</sub>
 

Revision as of 09:27, 12 September 2012

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