Team:LMU-Munich/Spore Coat Proteins

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=='''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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==Spore Crust Proteins==  
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===Scientific Background===
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{| "width=100%" style="text-align:center;" style="align:right"|
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|<p align="justify">Introduction to ''B. subtilis'' spores  and their use in our project</p>
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|[[File:Imamura, 2011 &amp; McKenney, 2010.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/Background]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/Background]]
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===Cloning Strategy===
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|<p align="justify">Cloning strategy to create different variants of our ''' Sporo'''beads</p>
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|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]
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<p align="justify">The aim of this project part is to create spores that display fusion proteins on their crust. There are several different proteins forming the spore coat layers of ''Bacillus subtilis'' spores. On the outermost layer, the so called spore crust, the CotZ and CgeA proteins are located ([http://www.ncbi.nlm.nih.gov/pubmed?term=imamura%20et%20al.%202011%20spore%20crust Imamura et al., 2011]). This is why we used them to create functional fusion proteins to be expressed on our '''Sporo'''beads.</p>  
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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">Main results of the various constructs that were created to find the best one!</p>
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|[[File:LMU Firstspore.jpg|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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===Laccases as functional enzymes===
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{|
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|<p align="justify">Creation of functional Laccase-''' Sporo'''beads</p>
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|[[File:Imamura, 2011 &amp; McKenney, 2010.png|Protein distribution in spore coat of ''Bacillus subtilis''|610px]]
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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/laccases]]
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<font color="#EBFCE4">Protein distribution in spore coat of ''Bacillus subtilis''</font>
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===Purification Methods===
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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is regulated by its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' regulated by the promoter P<sub>''cotYZ''</sub>. Another promoter of this cluster P<sub>''cotV''</sub> is responsible for the transcription of the other three genes. Those three promoters were evaluated with ''lux'' reporter genes to get an impression of their time of activation and their strength (see for more details [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick '''B'''ox]) so they could be used for expression of spore crust fusion proteins.</p>
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{| "width=100%" style="text-align:center;" style="align:right"|
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|<p align="justify">Description of the different purification methods of the spores</p>
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<p align="justify">The first step was to fuse ''gfp'' to ''cgeA'' and ''cotZ'' as a proof of principle. This way we would determine if it is possible to display proteins on the spore crust and if their expression has any effect on spore formation. Therefore we first fused ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For cgeA we only used its native promoter P<sub>''cgeA''</sub> and the stonger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub>. While ''gfp'' was ligated to the terminator B0014 (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 Registry]). When these constructs were finished and confirmed by sequencing, we fused them together applying the Freiburg standard to create in frame fusion proteins, flanked by one of the three promoters and the terminator.This way we created C-terminal fusion proteins.
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|[[File:Treatments.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/purification]]
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<br>But as we did not know if C- or N-terminal fusion would influence the fusion protein expression, our second aim was to construct N-terminal fusion proteins as well. For this purpose we wanted to fuse the genes for the crust proteins ''cotZ'' and ''cgeA'' to the terminator and ''gfp'' to the three chosen promoters. Unsuccessfully, there occured a mutation in the XbaI site during construction of ''gfp'' in Freiburg Standard which is why we were not able to finish these constructs.
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|[[File:Final construct-2.png|Scheme of variants of the final fusion constructs Promoter-Gene-GFP-Terminator|610px]]
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====Project Navigation====
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|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]
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<font color="#EBFCE4">Scheme of variants of the final fusion constructs Promoter-Crust Protein-GFP-Terminator</font>
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|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]
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<p align="justify">As we are working with B. subtilis spores, we needed to clone our final constructs into an empty Bacillus vector, so that they could get integrated into the genome of ''B. subtilis'' after transformation. Thus we picked the empty vector pSB<sub>BS</sub>1C from our '''''Bacillus''B'''io'''B'''rick'''B'''ox,  for the ''cotZ'' constructs. This vector integrates into the ''amyE'' locus in the ''B. subtilis'' genome and therefore we checked the integration of our construct via a starch test.  The clones with the right integrated constructs have then been chosen for further analysis. In oder to express both crust protein constructs in one strain the ''cgeA'' fusion proteins had to be cloned into one of the other empty vectors. Unfortunately for unknown reasons, the cloning of the constructs with ''cgeA'' into this vector have been unsuccessful so far.</p>
 
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<p align="justify">We were able to construct 5 ''B. subtilis'' strains with the following constructs integrated into the ''amyE'' locus:
 
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<br> P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''
 
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<br>P<sub>''cotYZ''</sub>-''cotZin''-''gfp''-''terminator''
 
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<br>P<sub>''cotV''</sub>-''cotZre''-''gfp''-''terminator''
 
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<br>P<sub>''cotV''</sub>-''cotZin''-''gfp''-''terminator''
 
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<br>P<sub>''cgeA''</sub>-''cotZre''-''gfp''-''terminator''
 
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<p align="justify">Finally we could start with the important experiment for our GFP-'''Sporo'''beads, fluorescence microscopy. Therefore we developed a sporulation protocol, that increases the rates of mature spores in our mutant samples (for details see link). The cells were fixed on agarose-pads and imaged in bright field and excited in blue wavelength.</p>
 
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<p align="justify">Because of the low but distinct fluorescence of wildtype sores, we measured and compared the fluorescence intensity of 100 spores per mutant. As you can see in the following graph, a significant difference was obtained. However we worked with the PcotYZ-CotZ-GFP-Terminator spores for further experiments as these showed the brightest fluorescence. In these experiments we had three different aims.
 
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<br>The first one was to show that the fusion proteins are really located on the outermost layer. Therefore we investiagted the fluoerscence of our spores after treatment with proteinase K and FRAP (fluorescence recovery after photobleaching). 
 
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<br>The second aim was to purify the '''Sporo'''beads from vegetative cells, which thereby should be deaden. We chose three different methods for this approach, the treatment with French Press, ultrasound (sonification) or lysozyme.</p>
 
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Latest revision as of 13:44, 14 November 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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