Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

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=='''Sporo'''beads - What Protein Do ''You'' Want to Display?==  
=='''Sporo'''beads - What Protein Do ''You'' Want to Display?==  
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<p align="justify">'''Sporo'''beads are the first generation of ''Bacillus subtilis'' endospores displaying a protein of our choice on their outermost layer, the spore crust. In future '''Sporo'''beads could serve as a platform for protein display and thus be used for numerous versatile [https://2012.igem.org/Team:LMU-Munich/Application applications]. Our goal was to show that ''B. subtilis'' spores have the ability to do so. As a [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins/result proof of principle] we successfully fused GFP to the spore crust and obtained fluorescence with microscopy.</p> 
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===What are '''Sporo'''beads?===
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===Scientific Background===
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|<p align="justify">Introduction to ''B. subtilis'' spores  and their assignment for our project</p>
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|<p align="justify">Introduction to ''B. subtilis'' spores  and their use in our project</p>
|[[File:Imamura, 2011 &amp; McKenney, 2010.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/Background]]
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===Crust Promoter Evaluation===
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===Cloning Strategy===
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|<p align="justify">Three sporulation-dependent promoters (P<sub>''cotYZ''</sub>, P<sub>''cotV''</sub>, P<sub>''cgeA''</sub>) were constructed, two of which show the expected behavior.</p>
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|[[File:Promotoren.png|200px|link=Team:LMU-Munich/Data/crustpromoters]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/crustpromoters]]
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===Crust Protein Gene Organization===
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|<p align="justify">Location of the two crust protein genes in the genome</p>
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|<p align="justify">Cloning strategy to create different variants of our ''' Sporo'''beads</p>
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|[[File:Operons.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/crustproteins]]
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|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/crustproteins]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]
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===Cloning Strategy===
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===GFP as a Proof of Principle===
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{| "width=100%" style="text-align:center;" style="align:right"|
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|<p align="justify">Cloning strategy to create different variants of our ''' Sporo'''beads</p>
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|<p align="justify">Main results of the various constructs that were created to find the best one!</p>
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|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]
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|[[File:LMU Firstspore.jpg|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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===Cloning Results===
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===Laccases as functional enzymes===
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|<p align="justify">Main results of the various constructs that were created</p>
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|<p align="justify">Creation of functional Laccase-''' Sporo'''beads</p>
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|[[File:LMU Firstspore.jpg|150px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/laccases]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/laccases]]
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===Purification methods===
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===Purification Methods===
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|<p align="justify">Description of the different purification methods of the spores</p>
|<p align="justify">Description of the different purification methods of the spores</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 [https://2012.igem.org/Team:LMU-Munich/Data/Sporepurification data] for details).</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>
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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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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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<font color="#000000"; size="2">Fig. 6: Result of fluorescence evaluation of the three strains W168, B53 and B70.</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">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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====Project Navigation====
====Project Navigation====
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[[File:Spore crust proteins cycle.jpg|600px|right]]
 
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<font color="#000000"; size="2">Fig. 3: The '''Bead'''zillus cycle</font>
 
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Latest revision as of 13:44, 14 November 2012

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