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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|<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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===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 module 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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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
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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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|[[File:Imamura, 2011 &amp; McKenney, 2010.png|Protein distribution in spore coat of ''Bacillus subtilis''|500px|center]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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<font color="#000000"; size="2">Protein distribution in spore coat of ''Bacillus subtilis''</font>
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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 [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters evaluated] with ''lux'' reporter genes to get an impression of their time of activation and their strength (see for more details [http://partsregistry.org/Part:BBa_K823025 pSB<sub>''Bs''</sub>3C-''lux''ABCDE]) so they could be used for expression of spore crust fusion proteins.</p>  
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===Laccases as functional enzymes===
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{| "width=100%" style="text-align:center;" style="align:right"|
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[[File:Example.jpg operons]]
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|<p align="justify">Creation of functional Laccase-''' Sporo'''beads</p>
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|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/laccases]]
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<p align="justify">The first step was to fuse [http://partsregistry.org/Part:BBa_K823039 ''gfp''] to ''cgeA'' and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 ''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.
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All three genes were brought into Freiburg Standard whereas we created to different versions of the crust proteins. The restriction site NgoMIV was inserted just after the startcodon of the gene of the crust protein. Since this restriction site adds six additional basepairs the resulting gene is two codons longer [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823032 CotZ]. It is not know if this insertion has any effect on protein expression that is why we created an additional version in which we deleted the following six basepairs, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 CotZ-2aa].
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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 [http://partsregistry.org/Part:BBa_K823039 ''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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<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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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Spore_Coat_Proteins/laccases]]
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<font color="#000000">Scheme of variants of the final fusion constructs Promoter-Crust Protein-GFP-Terminator</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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===Purification Methods===
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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">Description of the different purification methods of the spores</p>
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<br>The second aim was to purify the '''Sporo'''beads from vegetative cells, which thereby should be deadened. We chose three different methods for this approach, the treatment with French Press, ultrasound (sonification) or lysozyme. By means of the microscopy results we were able to conclude that lysozyme treatment was the only successful method. Additionally, it did not harm the crust fusion proteins as green fluorescence was detectable afterwards. This is why we use this treatment for purifying spores since.
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|[[File:Treatments.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/purification]]
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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. We obtained significant differences in fluorescence intensity between wildtype spores and all our Sporobeads. The following graph shows a significant difference between wildtype and all our '''Sporo'''beads.  We only worked with the P<sub>''cotYZ''</sub>-''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 investigated the fluorescence of our spores.
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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. We deleted ''cotZ'' and ''cgeA'' using the cloning method described by [http://www.ncbi.nlm.nih.gov/pubmed?term=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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Latest revision as of 13:44, 14 November 2012

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