Team:LMU-Munich/Spore Coat Proteins

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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is regulated by its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' regulated by 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. Those three promoters were evaluated with ''lux'' reporter genes to get an impression of their time of activation and their strength (see for more details [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick '''B'''ox]) so they could be used for expression of spore crust fusion proteins.</p>  
<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is regulated by its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' regulated by 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. Those three promoters were evaluated with ''lux'' reporter genes to get an impression of their time of activation and their strength (see for more details [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick '''B'''ox]) so they could be used for expression of spore crust fusion proteins.</p>  
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<p align="justify">The first step was to fuse ''gfp'' to ''cgeA'' and ''cotZ'' as a proof of principle. 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. Therefore we first fused ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For cgeA we only used its native promoter P<sub>''cgeA''</sub> and the stonger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub>. While ''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 applying the Freiburg standard to create in frame fusion proteins, 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">The first step was to fuse ''gfp'' to ''cgeA'' and ''cotZ'' as a proof of principle. 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. All three genes were brought into Freiburg Standard whereas we created to different versions of the crust proteins. The restriction site NgoMIV is inserted just after the startcodon of the gene of the crust protein. Since this restriction site adds six additional bases the resulting gene is two codons longer. It is not know if this insertion has any effect on protein expression that's why created an additional version in which we deleted the following six bases. Therefore we first fused ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For cgeA we only used its native promoter P<sub>''cgeA''</sub> and the stonger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub>. While ''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 applying the Freiburg standard to create in frame fusion proteins, flanked by one of the three promoters and the terminator.This way we created C-terminal fusion proteins.  
<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. Unsuccessfully, there occured a mutation in the XbaI site during construction of ''gfp'' in Freiburg Standard which is why we were not able to finish these constructs.
<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. Unsuccessfully, there occured a mutation in the XbaI site during construction of ''gfp'' in Freiburg Standard which is why we were not able to finish these constructs.
</p>  
</p>  

Revision as of 12:52, 24 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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