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).  
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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 truncated 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).  
<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>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.  
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[[File:LMU gfp spore data.png|620px]]
 
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<p align="justify">Fig. 1 verifies the previously discribed diffuse background fluorescence of wild type spores (W168). The B49 strain even shows an increased fluorescence background. Nevertheless there is a signficant difference between gfp-'''Sporo'''beads and W168 and B 49,respectively. '''Sporo'''beads with present native ''cotZ'' revealed uniformal distribution around the edge, whereas the ones with the deletion of ''cotZ'' elucidated a clear "polar" distribution. Additionally there is a difference in fluorescence activity between the two versions of ''cotZ'' recognizable. It appears the strain B 70 with the construct [http://partsregistry.org/Part:BBa_K823049 P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-''terminator''] and the deleted native ''cotZ'' had the strongest fluorescence. But as it does not have a uniformal fusion protein distribution, B 53 would be the strain for future '''Sporo'''bead applications.  
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<font color="#000000"; size="2">Fig. 1: Data of the various''' Sporo'''bead constructs</font>
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<p align="justify">Fig. 1 verifies the previously described diffuse background fluorescence of wild type spores (W168). The B49 strain even shows an increased fluorescence background. Nevertheless there is a significant difference between gfp-'''Sporo'''beads and W168 and B 49, respectively. '''Sporo'''beads with present native ''cotZ'' revealed uniform distribution around the edge, whereas the ones with the deletion of ''cotZ'' elucidated a clear "polar" distribution. Additionally there is a difference in fluorescence activity between the two versions of ''cotZ'' recognizable. It appears the strain B 70 with the construct [http://partsregistry.org/Part:BBa_K823049 P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-''terminator''] and the deleted native ''cotZ'' had the strongest fluorescence. But as it does not have a uniform fusion protein distribution, B 53 would be the strain for future '''Sporo'''bead applications.  
<br>In some 3D graphs the picked cell does not represent the average fluorescence intensity depicted in the bar chart, because it was chosen randomly.</p>  
<br>In some 3D graphs the picked cell does not represent the average fluorescence intensity depicted in the bar chart, because it was chosen randomly.</p>  

Latest revision as of 13:36, 26 October 2012

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