Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

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<p align="justify">Finally, we started with the most important experiment for our GFP-'''Sporo'''beads, the fluorescence microscopy. Therefore 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 mutant samples. The cells were fixed on agarose-pads and imaged in bright field and excited in blue wavelength. All '''Sporo'''beads showed green fluorescence on their surface. But B53-'''Sporo'''bead (containing the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator construct) illusidated the highest fluorescence intensity (see Figure 4). For further experiments, we chose this as it showed the brightest fluorescence.</p>
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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'''bead from strain B 53 (containing the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator construct)showed the highest fluorescence intensity (see Figure 5 and all data). Hence, this strain was chosen for further experiments.</p>
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<font color="#000000"; size="2"><p align="justify">Fig. 4: Fluorescence of wildtype spores and B53-'''Sporo'''beads (containing the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator construct)</p></font>
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<font color="#000000"; size="2"><p align="justify">Fig. 5: Fluorescence of wild type spores and B53-'''Sporo'''beads (containing the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator construct)</p></font>
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<p align="justify">Since there were still some vegetative cells left after 24 hours of growth in Difco Sporulation Medium, we wanted to purify the '''Sporo'''beads. We tried three different methods for this approach: treatment with French Press, sonification and lysozyme. By means of the microscopy results we were able to conclude that lysozyme treatment was the only successful method (see [https://2012.igem.org/Team:LMU-Munich/Data/Sporepurification data]). Additionally, it did not harm the crust fusion proteins as green fluorescence was still detectable afterwards.</p>
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<p align="justify">Since there were still some vegetative cells left after 24 hours of growth in Difco Sporulation Medium, we wanted to purify the '''Sporo'''beads. We tried three different methods for this approach: treatment with French Press, sonification and lysozyme. By means of the microscopy results we were able to conclude that lysozyme treatment was the only successful method (see [https://2012.igem.org/Team:LMU-Munich/Data/Sporepurification data]). Additionally, this treatment did not harm the crust fusion proteins as green fluorescence was still detectable afterwards (see data for details).</p>
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<p align="justify">Eventually, clean deletions of the native genes should reveal if there is any difference in fusion protein expression in our '''Sporo'''beads. Thus we deleted the native ''cotZ'' and ''cgeA'' using the cloning method described by [http://www.ncbi.nlm.nih.gov/pubmedterm=New%20Vector%20for%20Efficient%20Allelic%20Replacement%20in%20Naturally%20Nontransformable%2C%20Low-GC-Content%2C%20Gram-Positive%20Bacteria Arnaud ''et al''., 2004].</p>
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<p align="justify">Moreover, we wondered if clean deletions of the native spore crust genes would show any difference in fusion protein expression in our '''Sporo'''beads. Thus, we deleted the native ''cotZ'' and ''cgeA'' using the pMAD based gene deletion strategy described by [http://www.ncbi.nlm.nih.gov/pubmedterm=New%20Vector%20for%20Efficient%20Allelic%20Replacement%20in%20Naturally%20Nontransformable%2C%20Low-GC-Content%2C%20Gram-Positive%20Bacteria Arnaud ''et al''., 2004].</p>

Revision as of 00:21, 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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