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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=='''Sporo'''beads - what protein do you want them to display?==  
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===Scientific Background===
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<p align="justify">'''Sporo'''beads are ''Bacillus subtilis spores modulated in a way that they can be used as a platform for individual protein display. The aim of this module is to create these 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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|<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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|[[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/Background]]
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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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===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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<font color="#000000"; size="2">Gene clusters of ''cotZ'' and ''cgeA''</font>
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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.</p>
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===GFP as a Proof of Principle===
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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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<p align="justify">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> (for more details see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters crust promotor evaluation]. 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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|[[File:LMU Firstspore.jpg|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]
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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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<font color="#000000"; size="2">Section of the genome of ''B. subtilis'' with the various integrated constructs.</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. Therefore we checked the integration of our construct via a starch test.  The clones with the right integrated device 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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===Laccases as functional enzymes===
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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. 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 between wildtype spores and all our '''Sporo'''beads [link data]. We only worked with the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-''terminator'' spores for further experiments as these showed the brightest fluorescence.</p>
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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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<font color="#000000"; size="2">Fluorescence of wildtype spore and B 53 '''Sporo'''bead</font>
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<br>Since there were still some vegetative cells left after 24 hours of growth in DS-Medium, we wanted to purify the '''Sporo'''beads from them, 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 [link to data]. Additionally, it did not harm the crust fusion proteins as green fluorescence was detectable afterwards [link zu data]. This is why we use this treatment for purifying spores since.</p>
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===Purification Methods===
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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">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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<font color="#000000"; size="2">Section of the genome of ''B. subtilis'' with the various integrated constructs and the deletion of ''cotZ''.</font>
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<p align="justify">The '''Sporo'''beads-Δ''cotZ'' were investigated by fluorescence microscopy and analysed like the other '''Sporo'''beads. The intensity bar charts should thereby show the fluorescence difference between wildtype (W168), B53- and B70-'''Sporo'''beads. To demonstrate the distribution of the fusion proteins we created 3D graphs, which show the fluorescence intensity spread across the spore surface. For analysis we measured the fluorescence intensity of a area of 750px per spore by using ImageJ and evaluated it with the statistical software '''R'''. The following graph shows the results of microscopy and ImageJ analysis.</p>
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<font color="#000000"; size="2">Result of fluorescence evaluation of the three strains: W168, B53 and B70.</font>
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==Applications==
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<p align="justify">There are many possible applications for our '''Sporo'''beads as it is possible to modify their outermost protein layer. This way they could be used as beads with special functions. To easily create any kind of '''Sporo'''bead we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector], where you just have to insert your protein of choice. Since there are so many possible applications we picked three examplary ideas for future '''Sporo'''beads: </p>
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<br>'''Kumamolisin'''-'''Sporo'''beads: the solution for carefree enjoyment of everyday meals
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World wide one out of [http://www.enriquecastro.net/index.php/term/,9da4ab975b545ba0ae53646c58a5a265aa5d535892a89b979fa4b1a49297a261a260555c5a.xhtml 3350] people cannot eat that contains wheat products and other foods with traces of gluten. Kumamolisin is an [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 enzyme] that cleaves peptides and was produced by the iGEM-Team from the University of [https://2011.igem.org/Team:Washington Washington]] last year. The substrate includes a specific sequence of amino acids which causes celiac disease in sensitive people when they consume food containing gluten. Our beads could carry Kumamolisin and offer a protected passage through the stomach, so that the enzyme can work properly where it is needed in the intestines. The [https://2012.igem.org/Team:LMU-Munich/Germination_Stop <b>Germination</b>STOP] we put in place in our spores would ensure a correct dosage. This project is a pharmaceutical application and therefore would have to fulfill the laws for pharmaceuticals. This includes several verification steps of non-toxicity and efficacy.
 
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'''CPX'''-'''Sporo'''beads: the relieve of the marine life
 
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The excessive use of disposable plastic and the lack of universal recycling programs has led to the pollution of the world's oceans. In the ocean, large pieces of Polystyrene litter are ground by sea currents into very small pieces, so called plastik plankton, that are consumed by fish, filter feeders, and other organisms living in the oceans. Such plastic uptake can lead to poisoning, sterility and death. The [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX-peptide] generated by MIT (2007) can bind to Polystyrene. CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles, like polystyrene plankton, out of the water. To prevent the beads from being released into the sea and the plastic to be kept the '''Sporo'''beads could be attached to membranes in the boxes. The spores would then not only express CPX but also a membrane binding protein on their surface.
 
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'''TALE'''-'''Sporo'''beads: easy and cheap detection of GMOs
 
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Since 1990 green biotechnology releases many transgenic plants into the environment by selling genetically modified seeds. Thus organic farmers need to prove today, that their products meet the requirements for organic crops. Usually they pay laboratories to    The new tool for molecular biology, TAL effector, combined with our Sporobeads could be an easy and cheap
 
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Latest revision as of 13:44, 14 November 2012

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