Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

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===Cloning Results===
===Cloning Results===
{| "width=100%" style="text-align:center;" style="align:right"|
{| "width=100%" style="text-align:center;" style="align:right"|
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|<p align="justify">Main results of the various constructs we created</p>
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|<p align="justify">Main results of the various constructs that were created</p>
|[[File:LMU Firstspore.jpg|150px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
|[[File:LMU Firstspore.jpg|150px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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===Purification methods===
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<br>But we were able to finish five constructs and integrated them into wild type W168 and the Δ''cotZ'' mutant:
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{| "width=100%" style="text-align:center;" style="align:right"|
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</p>
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|<p align="justify">Description of the different purification methods of the spores</p>
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|[[File:Treatments.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/purification]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/purification]]
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!recipient strain W168
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!recipient strain B 49 (W168 Δ''cotZ'')
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''-2aa-''gfp''-terminator
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|B 53
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|B 70
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator
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|B 54
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|B 71
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!pSB<sub>''Bs''</sub>1C-P<sub>''cotV''</sub>-''cotZ''-2aa-terminator
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|B 55
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|B 72
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! pSB<sub>''Bs''</sub>1C-P<sub>''cotV''</sub>-''cotZ''-terminator
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|B 56
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|B 73
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!pSB<sub>''Bs''</sub>1C-P<sub>''cgeA''</sub>-''cotZ''-2aa-terminator
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|B 52
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|B 69
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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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|[[File:WT-B53 fluorescence.png|300px|center]]
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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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Revision as of 11:07, 24 October 2012

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