Team:LMU-Munich/Spore Coat Proteins/result

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====GFP as a Proof of Principle====
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====Main Results====
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<p align="justify">Finally, we started with the most important experiment for our GFP-'''Sporo'''beads, the fluorescence microscopy. For a better understanding of our results, you can see in the table below the [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins/cloning five constructs] and strain numbers we used in the following experiments.
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<p align="justify">We were able to finish five constructs and integrated them into wild type W168 and the Δ''cotZ'' mutant:
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<p align="justify">Finally, we started with the most important experiment for our GFP-'''Sporo'''beads, the fluorescence microscopy. We developed a sporulation protocol (for details see [https://static.igem.org/mediawiki/2012/e/e9/LMU-Munich_2012_Protocol_for_enhancement_of_mature_spore_numbers.pdf Protocol for enhancement of mature spore numbers]) that increases the rates of mature spores in our samples. The cells were fixed on agarose pads and investigated by phase contrast and fluorescence microscopy. While spores of the wild type only showed the known background fluorescence, all '''Sporo'''beads showed bright green fluorescence at the edge (on the surface) of the spores. '''Sporo'''beads from strain B 53 (containing the P<sub>''cotYZ''</sub>-''cotZ''-2aa-''gfp''-terminator construct) showed the highest fluorescence intensity (see Fig. 5 and all [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]). Hence, this strain was chosen for further experiments.</p>
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<p align="justify">We developed a sporulation protocol (for details see [https://static.igem.org/mediawiki/2012/e/e9/LMU-Munich_2012_Protocol_for_enhancement_of_mature_spore_numbers.pdf Protocol for enhancement of mature spore numbers]) that increases the rates of mature spores in our samples. The cells were fixed on agarose pads and investigated by phase contrast and fluorescence microscopy. While spores of the wild type only showed the known background fluorescence, all '''Sporo'''beads showed bright green fluorescence at the edge (on the surface) of the spores. '''Sporo'''beads from strain B 53 (containing the P<sub>''cotYZ''</sub>-''cotZ''-2aa-''gfp''-terminator construct) showed the highest fluorescence intensity (see Fig. 5 and all [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]). Hence, this strain was chosen for further experiments.</p>
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<p align="justify">Because of the low but distinct fluorescence of wild type spores, we measured and compared the fluorescence intensity of 100 spores per construct (see [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]). We obtained significant differences between wild type spores and all of our '''Sporo'''beads (see [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]). The intensity bar charts in Fig. 6 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.
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This two short time lapse videos shows the formation of a spore and lollowing lysis of the mother cell.
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The following graph (Fig. 6) shows the results of microscopy and ImageJ analysis of the strongest construct integrated into wildtype W168 (B53) and the deletion strain B 49 (B70).</p>   
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<p align="justify">Because of the low but distinct fluorescence of wild type spores, we measured and compared the fluorescence intensity of 100 spores per construct (see [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]). We obtained significant differences between wild type spores and all of our '''Sporo'''beads (see [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]). The following graph (Fig. 6) shows the microscopy pictures and analysis of the strongest of the five constructs integrated into wildtype W168 (B53) and the deletion strain B 49 (B70).</p>   
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<font color="#000000"; size="2">Fig. 6: Result of fluorescence evaluation of the three strains W168, B53 and B70.</font>
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<font color="#000000"; size="2">Fig. 6: Result of fluorescence evaluation of the three strains W168, B53 and B70. The bar charts show the average fluorescence intensity over 100-200 spores. 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 3D graphs illustrate in one spore the distribution of fluorescence intensity across the spore surface.</font>
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<p align="justify">As shown in Fig. 6, the wild type spore has hardly any fluorescence, whereas both''' Sporo'''beads with the integrated construct  pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-terminator give a distinct fluorescence signal around the edge of the spore. Furthermore, it demonstrates that strain B 70 has the highest fluorescence intensity.</p>
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<p align="justify">As shown in Fig. 6, the wild type spore has hardly any fluorescence, whereas both''' Sporo'''beads with the integrated construct  pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-terminator give a distinct fluorescence signal around the edge of the spore. Furthermore, it demonstrates that strain B 70 has the highest fluorescence intensity. For more detailed information look at our [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore Data page]</p>
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<p align="justify">In summary we successfully developed functional sporobeads that are capable of displaying any protein of choice on the surface of modified ''B. subtilis'' endospores.</p>
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<p align="justify">In summary we successfully developed functional '''Sporo'''beads that are capable of displaying any protein of choice on the surface of modified ''B. subtilis'' endospores.</p>
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Latest revision as of 17:02, 26 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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