Team:LMU-Munich/Spore Coat Proteins

From 2012.igem.org

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<p align="justify">Finally we needed to clone our 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 [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors '''''Bacillus''B'''io'''B'''rick'''B'''ox],  for the ''cotZ'' constructs. This vector integrates into the ''amyE'' locus, which allows to easily check the integration via starch test. In oder to also express both crust protein constructs in one strain, the ''cgeA'' fusion proteins had to be cloned into one of our other empty vectors pSB<sub>BS</sub>4S. Unfortunately for unknown reasons, the cloning of the constructs with ''cgeA'' into this vector has so far not been successful.</p>  
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<p align="justify">Finally we needed to clone our 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 [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors '''''Bacillus''B'''io'''B'''rick'''B'''ox],  for the ''cotZ'' constructs. This vector integrates into the ''amyE'' locus, which allows to easily check the integration via starch test. In oder to also express both crust protein constructs in one strain, the ''cgeA'' fusion proteins had to be cloned into one of our other empty vectors pSB<sub>BS</sub>4S. Unfortunately for unknown reasons, the cloning of the constructs with ''cgeA'' into this vector has so far not been successful.
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<br>We were able to finish five constructs cloned into wildtype W168 and the Δ''cotZ'' mutant:
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</p>  
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{| class="colored" width="100%" align="center" style="text-align:center;"
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|-
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|style="width:50%;"|
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|style="width:25%;"|
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|style="width:25%;"|
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|-
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!
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!recipient strain W168
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!recipient strain Δ''cotZ''
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|-
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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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|-
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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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|-
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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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|-
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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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|-
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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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|-
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|}
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<p align="justify">Finally, we started with the most important experiment for our GFP-'''Sporo'''beads, the fluorescence microscopy. Therefore 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 mutant samples. The cells were fixed on agarose-pads and imaged in bright field and excited in blue wavelength. All '''Sporo'''beads showed green fluorescence on their surface. But B53-'''Sporo'''bead (containing the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator construct) illusidated the highest fluorescence intensity (see Figure 4). For further experiments, we chose this as it showed the brightest fluorescence.</p>
<p align="justify">Finally, we started with the most important experiment for our GFP-'''Sporo'''beads, the fluorescence microscopy. Therefore 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 mutant samples. The cells were fixed on agarose-pads and imaged in bright field and excited in blue wavelength. All '''Sporo'''beads showed green fluorescence on their surface. But B53-'''Sporo'''bead (containing the P<sub>''cotYZ''</sub>-''cotZ''-''gfp''-terminator construct) illusidated the highest fluorescence intensity (see Figure 4). For further experiments, we chose this as it showed the brightest fluorescence.</p>
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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 attest that this is the case. The new tools of molecular biology, TAL effectors, combined with our Sporobeads could be an easy and cheap solution for organic agriculture. Farmers could in future use our kit with TALE-''lacZ''-'''Sporo'''beads to detect genetically modified crops themselves. As spores are stable and safe vehicles, they could be send by mail without any considerations. The kit would be suitable for use outside of laboraty. The protocol for this could work as follows:
 
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<br>The DNA extracted from plants with solutions provided by the kit, is immobilized and fixed on a nitrocellulose membrane. This membrane is then washed, incubated with '''Sporo'''beads in solution and washed again. With addition of the substrate X-Gal, the ''lacZ'' of bound '''Sporo'''beads will catalyze the reaction so that a blue staining appears. If no such DNA is present, the spores will not bind and no blue color will appear.</p>
 
   
   

Revision as of 22:17, 26 September 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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