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 brought into Freiburg Standard whereas we created two different versions of the crust proteins. The restriction site NgoMIV was inserted just after the startcodon of the gene of the crust protein. Since this restriction site adds six additional basepairs the resulting gene is two codons longer [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823032 CotZ]. It is not know if this insertion has any effect on protein expression that is why we created an additional version in which we deleted the following six basepairs, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 CotZ-2aa].
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[[File:SporeCoat.png|100px|right|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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<br>All the '''Sporo'''beads were investigated by fluorescence microscopy and analysed with ImageJ and the statistical software '''R'''. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the fluorescence intensity spread across the spore surface, which correlates with the distribution of our fusion proteins. For analysis we measured the fluorescence intensity of a area of 750px per spore by using ImageJ and evaluated it with the statistical software R. The following graph shows the results of microscopy and ImageJ analysis.
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[[File:LMU gfp spore data.png|620px]]
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<p align="justify">It appears the strain B70 with the construct P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-''terminator'' and the deleted native ''cotZ'' had the strongest fluorescence. Thus this would be the strain for future '''Sporo'''beads with special functions. In some 3D graphs the picked cell does not respresent the average fluorescence intensity depicted in the bar chart, because it was chosen randomly.
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== GFP-'''Sporo'''bead Evaluation ==
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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).
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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.
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<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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<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.
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|[[File:LMU gfp spore data.png|620px]]
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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.
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<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>
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[[File:Arrow purple Data.png|100px|link=Team:LMU-Munich/Data]]
 
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[[File:Arrow purple Data.png|center|100px|link=Team:LMU-Munich/Data]]
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Latest revision as of 13:36, 26 October 2012

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