Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is expressed from its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' and expressed from the promoter P<sub>''cotYZ''</sub>. Another promoter of this cluster, P<sub>''cotV''</sub>, is responsible for the transcription of the other three genes (Fig. 2). Those three promoters were [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters evaluated] with the ''lux'' reporter genes ([http://partsregistry.org/Part:BBa_K823025 pSB<sub>''Bs''</sub>3C-''lux''ABCDE]) to analyze their strength and the time point of their activation (see data link zu daten). Based on this data two of the three promoters could be used for expression of spore crust fusion proteins.</p>  
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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is expressed from its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' and expressed from the promoter P<sub>''cotYZ''</sub>. Another promoter of this cluster, P<sub>''cotV''</sub>, is responsible for the transcription of the other three genes (Fig. 2). Those three promoters were [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters evaluated] with the ''lux'' reporter genes ([http://partsregistry.org/Part:BBa_K823025 pSB<sub>''Bs''</sub>3C-''lux''ABCDE]) to analyze their strength and the time point of their activation (see data link zu daten). .</p>  
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<p align="justify">First, we used [http://partsregistry.org/Part:BBa_K823039 ''gfp''] as a proof of principle and fused it to ''cgeA'' and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 ''cotZ'']. This way we would determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation.</p>  
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<p align="justify">Based on this data, two of the three promoters could be used for expression of spore crust fusion proteins (Fig. 3). First, we used [http://partsregistry.org/Part:BBa_K823039 ''gfp''] as a proof of principle and fused it to ''cgeA'' and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 ''cotZ'']. This way, we can determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation.</p>  
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[[File:Spore crust proteins cycle.jpg|500px|center]]
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[[File:Spore crust proteins cycle.jpg|500px|center]] Fig. 3
   
   
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<p align="justify">We constructed the BioBrick for ''cotZ'', ''cgeA'' and ''gfp'' in [http://partsregistry.org/Help:Assembly_standard_25 Freiburg Standard]. ''cotZ''was then fused to its two native promoters, P<sub>''cotV''</sub> and to P<sub>''cotYZ''</sub>, and P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For ''cgeA'' we only used its native promoter P<sub>''cgeA''</sub> and the stronger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub> (for more details see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters crust promotor evaluation]. While [http://partsregistry.org/Part:BBa_K823039 ''gfp''] was ligated to the terminator B0014 (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 Registry]). When these constructs were finished and confirmed by sequencing, we fused them together by applying the Freiburg standard to create constructs, in which gfp is fused C-terminally to ''cotZ'' or ''cgeA'' flanked by one of the three promoters and the terminator. This way we created C-terminal fusion proteins.  
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<p align="justify">We constructed the BioBrick for ''cotZ'', ''cgeA'' and ''gfp'' in [http://partsregistry.org/Help:Assembly_standard_25 Freiburg Standard]. The ''cotZ'' gene was then fused to its two native promoters, P<sub>''cotV''</sub> and to P<sub>''cotYZ''</sub>, and P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For ''cgeA'' we only used its native promoter P<sub>''cgeA''</sub> and the stronger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub> (for more details see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters crust promotor evaluation]. While [http://partsregistry.org/Part:BBa_K823039 ''gfp''] was ligated to the terminator B0014 (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 Registry]). When these constructs were finished and confirmed by sequencing, we fused them together by applying the Freiburg standard to create constructs, in which ''gfp'' is fused C-terminally to ''cotZ'' or ''cgeA'' flanked by one of the three promoters and the terminator. This way we created C-terminal fusion proteins (Fig. 4). </p>
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<br>But as we did not know if C- or N-terminal fusion would influence the fusion protein expression, our second aim was to construct N-terminal fusion proteins as well. For this purpose we wanted to fuse the genes for the crust proteins ''cotZ'' and ''cgeA'' to the terminator and ''gfp'' to the three chosen promoters. Unfortunately, there occured a mutation in the XbaI site during construction of ''gfp'' in Freiburg Standard. Therefore we were not able to finish these constructs.
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<p align="justify">Finally we needed to clone our constructs into an empty ''Bacillus'' vector, so that they could get integrated into the genome of ''B. subtilis'' after transformation. Thus we picked the empty vector pSB<sub>BS</sub>1C from our [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors '''''Bacillus''B'''io'''B'''rick'''B'''ox],  for the ''cotZ'' constructs. This vector integrates into the ''amyE'' locus, which allows to easily check the integration via starch test. In oder to also express both crust protein constructs in one strain, the ''cgeA'' fusion proteins had to be cloned into one of our other empty vectors pSB<sub>BS</sub>4S. Unfortunately for unknown reasons, the cloning of the constructs with ''cgeA'' into this vector has so far not been successful.
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<p align="justify"><br>But as we did not know if C- or N-terminal fusion would influence the fusion protein expression, our second aim was to construct N-terminal fusion proteins as well. For this purpose we wanted to fuse the genes for the crust proteins ''cotZ'' and ''cgeA'' to the terminator and ''gfp'' to the three chosen promoters. Unfortunately, there occured a mutation in the XbaI site during construction of ''gfp'' in Freiburg Standard. Therefore we were not able to finish these constructs. Finally we needed to clone our constructs into an empty ''Bacillus'' vector, so that they could get integrated into the genome of ''B. subtilis'' after transformation. For that purpose, we picked the empty vector pSB<sub>BS</sub>1C from our [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors '''''Bacillus''B'''io'''B'''rick'''B'''ox],  for the ''cotZ'' constructs. This vector integrates into the ''amyE'' locus, which allows to easily check the integration by starch test. In order to also express both crust protein constructs in one strain, the ''cgeA'' fusion proteins had to be cloned into another empty vector, pSB<sub>BS</sub>4S. Unfortunately, for unknown reasons, the cloning of the constructs with ''cgeA'' into this vector has so far not been successful.
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<br>We were able to finish five constructs cloned into wildtype W168 and the Δ''cotZ'' mutant:
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<br>But we were able to finish five constructs and integrated them into wild type W168 and the Δ''cotZ'' mutant:
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!recipient strain W168
!recipient strain W168
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!recipient strain W168 Δ''cotZ'' (B 49)
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!recipient strain B 49 (W168 Δ''cotZ'')
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''-2aa-''gfp''-terminator
!pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''-2aa-''gfp''-terminator

Revision as of 00:13, 27 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".

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