Team:LMU-Munich/Data/gfp spore

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<p align="justify">For creating fusion proteins for the '''Sporo'''beads, the genes ''gfp'', ''cotZ'' and ''cgeA'' were amplified and brought into Freiburg Standard. The restriction site NgoMIV was inserted just after the start codon of the genes of the spore crust proteins. Since this restriction site adds six additional base pairs, the resulting genes are two amino acids longer ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823032 CotZ], CgeA). Since there is no way of knowing if this insertion has any effect on protein expression, we created an additional trancated version. The deletion of the six base pairs downstream of the restriction site resulted in fusion proteins that were two amino acids shorter than the constructs above (derivatives of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 CotZ-2aa] and CgeA-2aa).  
<p align="justify">For creating fusion proteins for the '''Sporo'''beads, the genes ''gfp'', ''cotZ'' and ''cgeA'' were amplified and brought into Freiburg Standard. The restriction site NgoMIV was inserted just after the start codon of the genes of the spore crust proteins. Since this restriction site adds six additional base pairs, the resulting genes are two amino acids longer ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823032 CotZ], CgeA). Since there is no way of knowing if this insertion has any effect on protein expression, we created an additional trancated version. The deletion of the six base pairs downstream of the restriction site resulted in fusion proteins that were two amino acids shorter than the constructs above (derivatives of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 CotZ-2aa] and CgeA-2aa).  
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<br>Afterwards, we first fused both versions of ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>. For the two ''cgeA'' variants, we only used the native promoter P<sub>''cgeA''</sub>, and P<sub>''cotYZ''</sub>, the stronger one of the two promoters of the ''cotVWXYZ'' (for more details see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters crust promotor evaluation]). In addtion [http://partsregistry.org/Part:BBa_K823039 ''gfp''] was ligated to the terminator [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 B0014]. After sequence confirmation, we fused the ''cgeA''/''cotZ''- and ''gfp''-constructs together, applying the [http://partsregistry.org/Help:Assembly_standard_25 Freiburg standard] to create in-frame fusion proteins. This way, we created C-terminal ''gfp'' fusion to both spore crust proteins flanked by the promoters and terminator above.  
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<br>Afterwards, we first fused both versions of ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>. For the two ''cgeA'' variants, we only used the native promoter P<sub>''cgeA''</sub>, and P<sub>''cotYZ''</sub>, the stronger one of the two promoters of the ''cotVWXYZ'' (for more details see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters crust promotor evaluation]). In addition [http://partsregistry.org/Part:BBa_K823039 ''gfp''] was ligated to the terminator [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 B0014]. After sequence confirmation, we fused the ''cgeA''/''cotZ''- and ''gfp''-constructs together, applying the [http://partsregistry.org/Help:Assembly_standard_25 Freiburg standard] to create in-frame fusion proteins. This way, we created C-terminal ''gfp'' fusion to both spore crust proteins flanked by the promoters and terminator above.  
<br>Since we want to display the fusion proteins on the surface of ''B. subtilis'' spores, we needed to clone our final constructs into an empty ''Bacillus'' vector, so that they would integrated into the chromosome of ''B. subtilis'' after transformation. We chose the empty vector pSB<sub>BS</sub>1C from our [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick'''B'''ox] for the ''cotZ'' constructs.  
<br>Since we want to display the fusion proteins on the surface of ''B. subtilis'' spores, we needed to clone our final constructs into an empty ''Bacillus'' vector, so that they would integrated into the chromosome of ''B. subtilis'' after transformation. We chose the empty vector pSB<sub>BS</sub>1C from our [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick'''B'''ox] for the ''cotZ'' constructs.  
<br>All resulting '''Sporo'''beads were investigated by fluorescence microscopy. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the distribution of fluorescence intensity across the spore surface. This correlates with the localization of our fusion proteins in the crust. For image analysis we measured the fluorescence intensity of an area of 750 pixel per spore by using ImageJ and evaluated the results with the statistical software '''R'''. The graph below shows a summery of our results obtained from all constructs.
<br>All resulting '''Sporo'''beads were investigated by fluorescence microscopy. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the distribution of fluorescence intensity across the spore surface. This correlates with the localization of our fusion proteins in the crust. For image analysis we measured the fluorescence intensity of an area of 750 pixel per spore by using ImageJ and evaluated the results with the statistical software '''R'''. The graph below shows a summery of our results obtained from all constructs.

Revision as of 01:17, 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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