http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Franzi.Duerr&year=&month=2012.igem.org - User contributions [en]2024-03-29T04:53:31ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:LMU-Munich/ApplicationTeam:LMU-Munich/Application2012-10-25T23:24:17Z<p>Franzi.Duerr: </p>
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What are the potential '''advantages''' of using ''B. subtilis'' spores as functional bioparticals?<br />
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<br>They are:<br />
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|[[File:BoxChecked.png|40px|left]]<br />
|Very stable even under severe environmental conditions<br />
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|[[File:BoxChecked.png|40px|left]]<br />
|Easy and cheap to produce in large quantities (check out the link to this commercial [http://www.alibaba.com/product-free/118791994/Bacillus_subtilis_spores_HU58_100_pure.html offer])<br />
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|[[File:BoxChecked.png|40px|left]]<br />
|Part of human nutrition in terms of [http://en.wikipedia.org/wiki/Bacillus_subtilis_R0179 food supplements] and [http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm generally regarded as safe]<br />
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|colspan="2"|Moreover, our '''Sporo'''beads are:<br />
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|[[File:BoxChecked.png|40px|left]]<br />
|[https://2012.igem.org/Team:LMU-Munich/Project_Safety Unable] to proliferate and therefore most likely not treated as genetically modified organisms by the law<br />
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<p align="justify">Our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] offer a wide variety of applications within the laboratory, and around the world. '''Sporo'''beads can be used for filtration and protein screening.</p><br />
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====Filtration====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the first possible application for our '''Sporo'''beads.</p><br />
|[[File:LMU Sporofilter.png|right|150px|link=Team:LMU-Munich/Application/Filtration]]<br />
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====Protein Screening====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:protein_libraries.jpg|right|150px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
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====Further Applications====<br />
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|<p align="justify">More information for further possible application with our diverse '''Sporo'''beads.</p><br />
|[[File:LMU Sporo diversity.png|right|150px|link=Team:LMU-Munich/Application/Further Applications]]<br />
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====Project Navigation====<br />
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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Why_BeadzillusTeam:LMU-Munich/Why Beadzillus2012-10-25T23:22:47Z<p>Franzi.Duerr: </p>
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==Why Beadzillus?==<br />
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Filters are widely used in everyday life and within the lab. Filters, such as ''Brita'' filters for removing calcium and other contaminants from drinking water and plumbing systems are abundant. In the lab, filters are used for DNA purification and protein purification.<br />
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
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{|<br />
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<font color="#000000"; size="2"><p align="justify"> Fig.: Filters for different applications. </p></font><br />
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All types of filters are filled with some type of matrix which determines the function of the filter. To maximize the surface in a minimal volume, beads are commonly used in all types of filters. These microbeads have a specific size and can display protein on their surface for functional use of the proteins. Then the features of the protein characterize the filter. The coupling of proteins to the beads is based on affinity binding. An example is nickle NTA-tags, in which a protein carrying a histidine tag binds to the nickel ion of the bead. Several companies offer such microbeads with proteins coupled to their surface using affinity tags.<br />
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Such beads are usually very expensive -- their production is laborious, as the protein has to be expressed, bound to the beads and then washed. Additionally, the binding of the protein is non-covalent. To solve this problem, we offer a synthetic biology solution in our project '''Bead'''zillus, in which we produced biological beads. We call these biological beads '''Sporo'''beads.<br />
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But what are our biological beads made of? Using clever natural engineering millions of years ago, evolution developed endospores of the soil bacteria ''Bacillus subtilis''. Endospores, which are highly resistant to environmental stressors and can survive harsh conditions, are a dormant life stage of ''B. subtilis''. To get to know more about the life cycle and the production of endospores, have a look at the life cycle of ''B. subtilis'' on the next page.<br />
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====Project Navigation====<br />
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|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Bacillus_IntroductionTeam:LMU-Munich/Bacillus Introduction2012-10-25T23:22:18Z<p>Franzi.Duerr: </p>
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==''Bacillus subtilis'' - a new chassis for iGEM==<br />
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====Introduction====<br />
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|<p align="justify">Introduction to ''B. subtilis'' and background information.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Data/introintro]]<br />
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There are two major differences between ''B. subtilis'' and ''E. coli'' that are of interest to us:<br />
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====Transformation of ''B. subtilis''====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Cloning strategy for the work of '' B. subtilis''</p><br />
|[[File:Integration.png|right|150px|link=Team:LMU-Munich/Data/integration]]<br />
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====Differentiation====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">The life cycle of B. subtilis and the different cell stages</p><br />
|[[File:Figures Bacillus Intro fig1.png|right|150px|link=Team:LMU-Munich/Data/differentiation]]<br />
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====Project Navigation====<br />
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|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricksTeam:LMU-Munich/Bacillus BioBricks2012-10-25T23:21:45Z<p>Franzi.Duerr: </p>
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=='''B<sup>4</sup>''' - 22 core parts for ''Bacillus subtilis''==<br />
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<p align="justify">A major goal of our iGEM project is to [https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction introduce ''B. subtilis''] as a new chassis for BioBrick-based Synthetic Biology. For that purpose, we created a toolbox of <i>Bacillus</i> BioBricks to contribute to the registry to make it accessible to many more future iGEM-Teams! This ''<b>Bacillus'' B</b>io<b>B</b>rick <b>B</b>ox ('''B<sup>4</sup>''') contains the following ''Bacillus'' specific parts:</p><br />
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| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]'''<br />
| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Promoters|Promoters]]'''<br />
| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Reporters|Reporters]]'''<br />
| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Affinity_Tags|Affinity tags]]<br />
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|[[File:LMU Backbone.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]<br />
|[[File:LMU PromoterIconBC.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Promoters]]<br />
|[[File:LMU Reporter.png|60px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Reporters]]<br />
|[[File:Proteinaffinitytagbutton.png|50px|link=Team:LMU-Munich/Bacillus_BioBricks#Affinity_Tags]]<br />
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pSB<sub>''Bs''</sub>1C<br><br />
pSB<sub>''Bs''</sub>4S<br><br />
pSB<sub>''Bs''</sub>2E<br><br />
pSB<sub>''Bs''</sub>1C-''lac''Z<br><br />
pSB<sub>''Bs''</sub>3C-''lux''ABCDE<br><br />
pSB<sub>''Bs''</sub>4S-P<sub>''xyl''</sub><br><br />
pSB<sub>''Bs''</sub>0K-P<sub>''spac''</sub><br><br />
'''Sporo'''vector</font><br />
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Anderson<br><br />
P<sub>''liaG''</sub><br><br />
P<sub>''veg''</sub><br><br />
P<sub>''lepA''</sub><br><br />
P<sub>''liaI''</sub><br><br />
''xylR''-P<sub>''xyl''</sub></font><br />
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''gfp''<br><br />
''mkate2''<br><br />
''lacZ''<br><br />
''luc+''<br></font><br />
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Flag<br>His<br>cMyc<br>Strep<br>HA</font><br />
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[[File:NEXT.png|right|80px|link=Team:LMU-Munich/Germination_Stop]] [[File:BACK.png|left|80px|link=Team:LMU-Munich/Data/differentiation_tour]]<br />
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<p align="justify">Since ''Bacillus subtilis'' is not an organism commonly used in iGEM, please check out our [https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction Introduction].</p><br />
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==''Bacillus'' Vectors [[File:LMU Backbone.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]==<br />
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|<p align="justify">We have generated a suit of BioBrick-compatible vectors: three empty insertional backbones with different antibiotic resistances and integration loci, two reporter and two expression vectors.</p> <br />
|[[File:LMU-Munich-PSBBs1C.png|200px|right|link=Team:LMU-Munich/Bacillus BioBricks/Vectors]]<br />
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==''Bacillus'' Promoters [[File:LMU PromoterIconBC.png|100px]]== <br />
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<p align="justify">To provide a set of promoters of different strength we characterized several promoters in ''Bacillus subtilis''. Both constitutive and inducible promoters are covered.</p><br />
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[[File:Promoters overview.png|200px|right|link=Team:LMU-Munich/Bacillus BioBricks/Promoters]]<br />
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==''Bacillus'' Reporters [[File:LMU Reporter.png|50px]]==<br />
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<p align="justify">We designed and codon-optimized a set of reporters that are commonly used in ''B. subtilis''.</p><br />
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[[File:LMU GFP.jpg|200px|right|link=Team:LMU-Munich/Bacillus BioBricks/Reporters]]<br />
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==Affinity Tags [[File:Proteinaffinitytagbutton.png|50px]]==<br />
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<p align="justify">We synthesized 5 affinity tags for protein purification. They all are designed in Freiburg standard with an optimized ribosome binding site upstream. We have not yet tested our tags.</p><br />
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====Project Navigation====<br />
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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Germination_StopTeam:LMU-Munich/Germination Stop2012-10-25T23:20:59Z<p>Franzi.Duerr: </p>
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== '''Germination'''STOP ==<br />
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<p align="justify">The goal of this module was to yield [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure throughout time). To achieve this, we sought to remove the germination capability of our spores, while keeping their necessary structural functions intact.</p> <br />
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[[File:GerminationSTOP_cycleIIii.jpg|620px|center]]<br />
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[[File:NEXT.png|right|80px|link=Team:LMU-Munich/safetytour]] [[File:BACK.png|left|80px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
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==Gene Knockouts==<br />
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|<p align="justify">We picked important germination genes and knocked them out. Subsequently we combined the single mutants up to quadruple mutants and prevented successfully germination.</p> <br />
|[[File:LMU Germination STop plate.png|200px|right|link=Team:LMU-Munich/Germination_Stop/Knockout]]<br />
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=='''Suicide'''switch==<br />
{| "width=100%" style="text-align:center;" style="align:right"|<br />
|<p align="justify">If germination knockout fail, we invented the suicide switch. In case spores still germinate, the production of a toxin leads to immediate cell death.</p> <br />
|[[File:LMU SuicideSwitch grafik.png|200px|right|link=Team:LMU-Munich/Germination_Stop/SuicideSwitch]]<br />
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====Project Navigation====<br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_ProteinsTeam:LMU-Munich/Spore Coat Proteins2012-10-25T23:19:57Z<p>Franzi.Duerr: </p>
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=='''Sporo'''beads - What Protein Do ''You'' Want to Display?== <br />
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===Scientific Background===<br />
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|<p align="justify">Introduction to ''B. subtilis'' spores and their assignment for our project</p><br />
|[[File:Imamura, 2011 &amp; McKenney, 2010.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/Background]]<br />
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===Cloning Strategy===<br />
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|<p align="justify">Cloning strategy to create different variants of our ''' Sporo'''beads</p><br />
|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]<br />
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===GFP as a Proof of Principle===<br />
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|<p align="justify">Main results of the various constructs that were created to find the best one!</p><br />
|[[File:LMU Firstspore.jpg|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]<br />
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|<p align="justify">Description of the different purification methods of the spores</p><br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_ProteinsTeam:LMU-Munich/Spore Coat Proteins2012-10-25T23:19:09Z<p>Franzi.Duerr: </p>
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=='''Sporo'''beads - What Protein Do ''You'' Want to Display?== <br />
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===Scientific Background===<br />
{| "width=100%" style="text-align:center;" style="align:right"|<br />
|<p align="justify">Introduction to ''B. subtilis'' spores and their assignment for our project</p><br />
|[[File:Imamura, 2011 &amp; McKenney, 2010.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/Background]]<br />
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===Cloning Strategy===<br />
{| "width=100%" style="text-align:center;" style="align:right"|<br />
|<p align="justify">Cloning strategy to create different variants of our ''' Sporo'''beads</p><br />
|[[File:Final construct.png|200px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]<br />
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===GFP as a Proof of Principle===<br />
{| "width=100%" style="text-align:center;" style="align:right"|<br />
|<p align="justify">Main results of the various constructs that were created to find the best one!</p><br />
|[[File:LMU Firstspore.jpg|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/result]]<br />
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===Purification Methods===<br />
{| "width=100%" style="text-align:center;" style="align:right"|<br />
|<p align="justify">Description of the different purification methods of the spores</p><br />
|[[File:Treatments.png|200px|right|link=Team:LMU-Munich/Spore_Coat_Proteins/purification]]<br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
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|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Data/differentiation_tourTeam:LMU-Munich/Data/differentiation tour2012-10-25T23:10:19Z<p>Franzi.Duerr: </p>
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===Overview on the life cycle of ''Bacillus subtilis''===<br />
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<p align="justify">''B. subtilis'' is able to differentiate into cells with different morphologies and functions (Fig. 1 and Fig. 3). The most characteristic form is the endospore, which is produced under nutrient starvation. In our project, we will exploit the production of endospores. Because they are extremely stable, they are perfect vehicles for the display of functional fusion proteins on their surface as illustrated by our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''bead] module.<br />
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<font color="#000000"; size="2"><p align="justify">Fig. 3: The vegetative cycle is very similiar to the one of ''E. coli.'' But if there is a stress like for example starvation, the cells enter sporulation, where they first undergo a polar cell division, followed by the formation of the endospore. If the enviromental conditions are suitable again, the spore will then germinate and reenter the vegetative cycle.</p></font><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Protein_Screening_tourTeam:LMU-Munich/Application/Protein Screening tour2012-10-25T23:06:26Z<p>Franzi.Duerr: </p>
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==Application for our Sporobeads: Protein Screening==<br />
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<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
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<font color="#000000"; size="2">Fig. 1: '''The simple protein-screening process in our Sporobeads'''. </font><br />
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<font color="#000000"; size="2">Fig. 2: '''The simple protein-screening process in our Sporobeads''' using affinity binding as a requisite. </font><br />
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==Application for our Sporobeads: Filtration==<br />
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<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
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<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
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<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/safetytourTeam:LMU-Munich/safetytour2012-10-25T23:02:54Z<p>Franzi.Duerr: </p>
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===Safety Aspect===<br />
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The '''Germination'''STOP includes the simple technical reason that the fusion proteins would lose their function if the spore were to become a vegetative cell. But also it means that our '''Sporo'''beads are no longer viable cells.<br />
In order to get a professional statement about the safety aspects of our project and how realistic it would be to release our spores in the environment, we invited the Commissioner for Bio-Safety in Bavaria to speak with us.<br />
In a two hour discussion, we explained the details of our projects and asked the Commissioner all of the "what ifs" on our minds<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins/tourendTeam:LMU-Munich/Spore Coat Proteins/tourend2012-10-25T23:00:28Z<p>Franzi.Duerr: </p>
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Further_Applications_tourTeam:LMU-Munich/Application/Further Applications tour2012-10-25T22:56:56Z<p>Franzi.Duerr: </p>
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==Further Applications==<br />
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<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
[[File:LMU Sporo diversity.png|left|200px]]<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Filtration_tourTeam:LMU-Munich/Application/Filtration tour2012-10-25T22:52:54Z<p>Franzi.Duerr: </p>
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==Possible Application: Filtration==<br />
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<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
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<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
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<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/SponsorenTeam:LMU-Munich/Sponsoren2012-10-25T22:49:02Z<p>Franzi.Duerr: </p>
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===Sponsors===<br />
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<p align="justify">Several public institutions generously supported our team and enabled us to take part at the iGEM competition 2012 by paying the registration fees for both the initial application and the jamborees and the travel expenses for Amsterdam and lab material.</p><br />
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<center><font size="6">Thank you!</font></center><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/SponsorenTeam:LMU-Munich/Sponsoren2012-10-25T22:48:40Z<p>Franzi.Duerr: </p>
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===Sponsors===<br />
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<p align="justify">Several public institutions generously supported our team and enabled us to take part at the iGEM competition 2012 by paying the registration fees for both the initial application and the jamborees and the travel expenses for Amsterdam and lab material.</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/File:Sponsoren_1.pngFile:Sponsoren 1.png2012-10-25T22:46:23Z<p>Franzi.Duerr: </p>
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<div></div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/SponsorenTeam:LMU-Munich/Sponsoren2012-10-25T22:43:27Z<p>Franzi.Duerr: </p>
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===Sponsors===<br />
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<p align="justify">Several public institutions generously supported our team and enabled us to take part at the iGEM competition 2012 by paying the registration fees for both the initial application and the jamborees and the travel expenses for Amsterdam and lab material.</p><br />
<center><font size="5">Thank you</font></center><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/SponsorenTeam:LMU-Munich/Sponsoren2012-10-25T22:43:16Z<p>Franzi.Duerr: </p>
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===Sponsors===<br />
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<p align="justify">Several public institutions generously supported our team and enabled us to take part at the iGEM competition 2012 by paying the registration fees for both the initial application and the jamborees and the travel expenses for Amsterdam and lab material.</p><br />
<center><font size="5">Thank you</font></center><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/safetytourTeam:LMU-Munich/safetytour2012-10-25T22:39:13Z<p>Franzi.Duerr: </p>
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===Safety Aspect===<br />
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The '''Germination'''STOP includes the simple technical reason that the fusion proteins would lose their function if the spore were to become a vegetative cell. But also it means that our '''Sporo'''beads are no longer viable cells.<br />
In order to get a professional statement about the safety aspects of our project and how realistic it would be to release our spores in the environment, we invited the Commissioner for Bio-Safety in Bavaria to speak with us.<br />
In a two hour discussion, we explained the details of our projects and asked the Commissioner all of the "what ifs" on our minds<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Further_Applications_tourTeam:LMU-Munich/Application/Further Applications tour2012-10-25T22:35:34Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}} [[File:Applications banner.resized WORDS.JPG|620..."</p>
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==Further Applications==<br />
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<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
[[File:LMU Sporo diversity.png|left|200px]]<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Protein_Screening_tourTeam:LMU-Munich/Application/Protein Screening tour2012-10-25T22:24:06Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}} [[File:Applications banner.resized WORDS.JPG|620..."</p>
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==Protein Screening==<br />
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<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
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<font color="#000000"; size="2">Fig. 1: '''The simple protein-screening process in our Sporobeads'''. </font><br />
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<font color="#000000"; size="2">Fig. 2: '''The simple protein-screening process in our Sporobeads''' using affinity binding as a requisite. </font><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Filtration_tourTeam:LMU-Munich/Application/Filtration tour2012-10-25T20:18:15Z<p>Franzi.Duerr: </p>
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==Possible Application: Filtration==<br />
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<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
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<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
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<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
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===GFP as a Proof of Principle===<br />
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<p align="justify">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. <br />
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> <br />
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<font color="#000000"; size="2">Fig. 6: Result of fluorescence evaluation of the three strains W168, B53 and B70.</font><br />
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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. For more detailed information look at our [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore Data page]</p><br />
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<p align="justify">In summary we successfully developed functional '''Sporo'''beads that are capable of displaying any protein of choice on the surface of modified ''B. subtilis'' endospores.</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Filtration_tourTeam:LMU-Munich/Application/Filtration tour2012-10-25T20:15:14Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}} [[File:Applications banner.resized WORDS.JPG|620..."</p>
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==Filtration==<br />
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<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
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<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
<br />
<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
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===GFP as a Proof of Principle===<br />
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<p align="justify">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. <br />
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> <br />
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{| style="color:black;" cellpadding="3" width="100%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
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<font color="#000000"; size="2">Fig. 6: Result of fluorescence evaluation of the three strains W168, B53 and B70.</font><br />
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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. For more detailed information look at our [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore Data page]</p><br />
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<p align="justify">In summary we successfully developed functional '''Sporo'''beads that are capable of displaying any protein of choice on the surface of modified ''B. subtilis'' endospores.</p><br />
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[[File:NEXT.png|right|80px|link=Team:LMU-Munich/Application]] [[File:BACK.png|left|80px|link=Team:LMU-Munich/Spore_Coat_ProteinsTour/Background]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_ProteinsTour/BackgroundTeam:LMU-Munich/Spore Coat ProteinsTour/Background2012-10-25T20:10:27Z<p>Franzi.Duerr: </p>
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===Scientific Background of our '''Sporo'''beads===<br />
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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 surface. There are several different proteins forming the spore coat layers of ''B. subtilis'' spores (Fig. 1). The outermost layer, the so-called spore crust, is composed of two proteins, CotZ and CgeA ([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> <br />
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
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{|<br />
|[[File:Imamura, 2011 &amp; McKenney, 2010.png|Protein distribution in spore coat of ''Bacillus subtilis''|500px|center]]<br />
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{| style="color:black;" cellpadding="0" width="70%" cellspacing="0" border="0" align="center" style="text-align:center;"<br />
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<font color="#000000"; size="2">Fig. 1: Composition of the ''Bacillus subtilis'' spore coat ([http://www.ncbi.nlm.nih.gov/pubmed?term=mckenney%202010%20spore%20crust McKenney ''et al''., 2010] & [http://www.ncbi.nlm.nih.gov/pubmed?term=imamura%20et%20al.%202011%20spore%20crust Imamura ''et al''., 2011])<br />
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=====Crust Protein Genes Organization=====<br />
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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is expressed from its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' and expressed from 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 (Fig. 2). Those three promoters were evaluated with the ''lux'' reporter genes ([http://partsregistry.org/Part:BBa_K823025 pSB<sub>''Bs''</sub>3C-''lux''ABCDE]) to analyze their strength and the time point of their activation (see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters data]).</p> <br />
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{| style="color:black;" cellpadding="3" width="100%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
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<font color="#000000"; size="2">Fig. 2: Gene clusters of ''cotZ'' and ''cgeA''</font><br />
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Based on this data, two of the three promoters could be used for expression of spore crust fusion proteins.<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins/BackgroundTeam:LMU-Munich/Spore Coat Proteins/Background2012-10-25T20:06:17Z<p>Franzi.Duerr: </p>
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===Scientific Background===<br />
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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 surface. There are several different proteins forming the spore coat layers of ''B. subtilis'' spores (Fig. 1). The outermost layer, the so-called spore crust, is composed of two proteins, CotZ and CgeA ([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> <br />
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|<br />
|[[File:Imamura, 2011 &amp; McKenney, 2010.png|Protein distribution in spore coat of ''Bacillus subtilis''|500px|center]]<br />
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| style="width: 70%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="0" width="70%" cellspacing="0" border="0" align="center" style="text-align:center;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 1: Composition of the ''Bacillus subtilis'' spore coat ([http://www.ncbi.nlm.nih.gov/pubmed?term=mckenney%202010%20spore%20crust McKenney ''et al''., 2010] & [http://www.ncbi.nlm.nih.gov/pubmed?term=imamura%20et%20al.%202011%20spore%20crust Imamura ''et al''., 2011])<br />
</font><br />
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=====Crust Protein Genes Organization=====<br />
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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is expressed from its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' and expressed from 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 (Fig. 2). Those three promoters were evaluated with the ''lux'' reporter genes ([http://partsregistry.org/Part:BBa_K823025 pSB<sub>''Bs''</sub>3C-''lux''ABCDE]) to analyze their strength and the time point of their activation (see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters data]).</p> <br />
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{| style="color:black;" cellpadding="3" width="100%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
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{|<br />
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{| style="color:black;" cellpadding="0" width="70%" cellspacing="0" border="0" align="center" style="text-align:center;"<br />
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<font color="#000000"; size="2">Fig. 2: Gene clusters of ''cotZ'' and ''cgeA''</font><br />
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Based on this data, two of the three promoters could be used for expression of spore crust fusion proteins.<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins/BackgroundTeam:LMU-Munich/Spore Coat Proteins/Background2012-10-25T20:05:38Z<p>Franzi.Duerr: </p>
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{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_eppis.resized.jpg|3}}<br />
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[[File:SporeCoat.png|100px|right|link=]]<br />
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===Scientific Background===<br />
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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 surface. There are several different proteins forming the spore coat layers of ''B. subtilis'' spores (Fig. 1). The outermost layer, the so-called spore crust, is composed of two proteins, CotZ and CgeA ([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> <br />
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|<br />
|[[File:Imamura, 2011 &amp; McKenney, 2010.png|Protein distribution in spore coat of ''Bacillus subtilis''|500px|center]]<br />
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| style="width: 70%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="0" width="70%" cellspacing="0" border="0" align="center" style="text-align:center;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 1: Composition of the ''Bacillus subtilis'' spore coat ([http://www.ncbi.nlm.nih.gov/pubmed?term=mckenney%202010%20spore%20crust McKenney ''et al''., 2010] & [http://www.ncbi.nlm.nih.gov/pubmed?term=imamura%20et%20al.%202011%20spore%20crust Imamura ''et al''., 2011])<br />
</font><br />
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=====Crust Protein Genes Organization=====<br />
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<p align="justify">The gene ''cgeA'' is located in the ''cgeABCDE'' cluster and is expressed from its own promoter P<sub>''cgeA''</sub>. The cluster ''cotVWXYZ'' contains the gene ''cotZ'' which is cotranscribed with ''cotY'' and expressed from 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 (Fig. 2). Those three promoters were evaluated with the ''lux'' reporter genes ([http://partsregistry.org/Part:BBa_K823025 pSB<sub>''Bs''</sub>3C-''lux''ABCDE]) to analyze their strength and the time point of their activation (see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters data]).</p> <br />
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{| style="color:black;" cellpadding="3" width="100%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
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{|<br />
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{| style="color:black;" cellpadding="0" width="70%" cellspacing="0" border="0" align="center" style="text-align:center;"<br />
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<font color="#000000"; size="2">Fig. 2: Gene clusters of ''cotZ'' and ''cgeA''</font><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Germination_StopTeam:LMU-Munich/Germination Stop2012-10-25T20:00:36Z<p>Franzi.Duerr: </p>
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{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_streaked_plate.resized.jpg|3}}<br />
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== '''Germination'''STOP ==<br />
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<p align="justify">The goal of this module was to yield [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure throughout time). To achieve this, we sought to remove the germination capability of our spores, while keeping their necessary structural functions intact.</p> <br />
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[[File:GerminationSTOP_cycleIIii.jpg|620px|center]]<br />
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[[File:NEXT.png|right|80px|link=Team:LMU-Munich/safetytour]] [[File:BACK.png|left|80px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
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==Gene Knockouts==<br />
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|<p align="justify">We picked important germination genes and knocked them out. Subsequently we combined the single mutants up to quadruple mutants and prevented successfully germination.</p> <br />
|[[File:LMU Germination STop plate.png|200px|right|link=Team:LMU-Munich/Germination_Stop/Knockout]]<br />
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=='''Suicide'''switch==<br />
{| "width=100%" style="text-align:center;" style="align:right"|<br />
|<p align="justify">If germination knockout fail, we invented the suicide switch. In case spores still germinate, the production of a toxin leads to immediate cell death.</p> <br />
|[[File:LMU SuicideSwitch grafik.png|200px|right|link=Team:LMU-Munich/Germination_Stop/SuicideSwitch]]<br />
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====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
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|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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=='''B<sup>4</sup>''' - 22 core parts for ''Bacillus subtilis''==<br />
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<p align="justify">A major goal of our iGEM project is to [https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction introduce ''B. subtilis''] as a new chassis for BioBrick-based Synthetic Biology. For that purpose, we created a toolbox of <i>Bacillus</i> BioBricks to contribute to the registry to make it accessible to many more future iGEM-Teams! This ''<b>Bacillus'' B</b>io<b>B</b>rick <b>B</b>ox ('''B<sup>4</sup>''') contains the following ''Bacillus'' specific parts:</p><br />
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| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]'''<br />
| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Promoters|Promoters]]'''<br />
| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Reporters|Reporters]]'''<br />
| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks#Affinity_Tags|Affinity tags]]<br />
|-<br />
|[[File:LMU Backbone.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]<br />
|[[File:LMU PromoterIconBC.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Promoters]]<br />
|[[File:LMU Reporter.png|60px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Reporters]]<br />
|[[File:Proteinaffinitytagbutton.png|50px|link=Team:LMU-Munich/Bacillus_BioBricks#Affinity_Tags]]<br />
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pSB<sub>''Bs''</sub>1C<br><br />
pSB<sub>''Bs''</sub>4S<br><br />
pSB<sub>''Bs''</sub>2E<br><br />
pSB<sub>''Bs''</sub>1C-''lac''Z<br><br />
pSB<sub>''Bs''</sub>3C-''lux''ABCDE<br><br />
pSB<sub>''Bs''</sub>4S-P<sub>''xyl''</sub><br><br />
pSB<sub>''Bs''</sub>0K-P<sub>''spac''</sub><br><br />
'''Sporo'''vector</font><br />
|valign="top"|<font size="2"><br />
Anderson<br><br />
P<sub>''liaG''</sub><br><br />
P<sub>''veg''</sub><br><br />
P<sub>''lepA''</sub><br><br />
P<sub>''liaI''</sub><br><br />
''xylR''-P<sub>''xyl''</sub></font><br />
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''gfp''<br><br />
''mkate2''<br><br />
''lacZ''<br><br />
''luc+''<br></font><br />
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Flag<br>His<br>cMyc<br>Strep<br>HA</font><br />
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<p align="justify">Since ''Bacillus subtilis'' is not an organism commonly used in iGEM, please check out our [https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction Introduction].</p><br />
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==''Bacillus'' Vectors [[File:LMU Backbone.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]==<br />
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|<p align="justify">We have generated a suit of BioBrick-compatible vectors: three empty insertional backbones with different antibiotic resistances and integration loci, two reporter and two expression vectors.</p> <br />
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==''Bacillus'' Promoters [[File:LMU PromoterIconBC.png|100px]]== <br />
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==''Bacillus'' Reporters [[File:LMU Reporter.png|50px]]==<br />
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==Affinity Tags [[File:Proteinaffinitytagbutton.png|50px]]==<br />
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====Project Navigation====<br />
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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
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|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Data/differentiation_tourTeam:LMU-Munich/Data/differentiation tour2012-10-25T19:42:43Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Bacillus in urban culture.jpg}} [[File:Bacillus introduction banner.re..."</p>
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===Differentiation===<br />
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<p align="justify">''B. subtilis'' is able to differentiate into cells with different morphologies and functions (Fig. 1 and Fig. 3). The most characteristic form is the endospore, which is produced under nutrient starvation. In our project, we will exploit the production of endospores. Because they are extremely stable, they are perfect vehicles for the display of functional fusion proteins on their surface as illustrated by our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''bead] module.<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Why_BeadzillusTeam:LMU-Munich/Why Beadzillus2012-10-25T19:38:51Z<p>Franzi.Duerr: </p>
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==Why Beadzillus?==<br />
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Filters are widely used in everyday life and within the lab. Filters, such as ''Brita'' filters for removing calcium and other contaminants from drinking water and plumbing systems are abundant. In the lab, filters are used for DNA purification and protein purification.<br />
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All types of filters are filled with some type of matrix which determines the function of the filter. To maximize the surface in a minimal volume, beads are commonly used in all types of filters. These microbeads have a specific size and can display protein on their surface for functional use of the proteins. Then the features of the protein characterize the filter. The coupling of proteins to the beads is based on affinity binding. An example is nickle NTA-tags, in which a protein carrying a histidine tag binds to the nickel ion of the bead. Several companies offer such microbeads with proteins coupled to their surface using affinity tags.<br />
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Such beads are usually very expensive -- their production is laborious, as the protein has to be expressed, bound to the beads and then washed. Additionally, the binding of the protein is non-covalent. To solve this problem, we offer a synthetic biology solution in our project '''Bead'''zillus, in which we produced biological beads. We call these biological beads '''Sporo'''beads.<br />
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But what are our biological beads made of? Using clever natural engineering millions of years ago, evolution developed endospores of the soil bacteria ''Bacillus subtilis''. Endospores, which are highly resistant to environmental stressors and can survive harsh conditions, are a dormant life stage of ''B. subtilis''. To get to know more about the life cycle and the production of endospores, have a look at the life cycle of ''B. subtilis'' on the next page.<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins/purificationTeam:LMU-Munich/Spore Coat Proteins/purification2012-10-25T19:25:55Z<p>Franzi.Duerr: </p>
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===Purification Methods===<br />
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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><br />
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<p align="justify">Next, we tested the fluorescence of Sporobeads after lysozyme treatement. This analysis revealed that lysozyme did not harm the gfp-fusion proteins, since fluorescence was not altered (see Fig. 2).</p><br />
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<font color="#000000"; size="2"><p align="justify">Fig. 2: Fluorescence of wild type spores and '''Sporo'''beads after treatment with lysozyme. '''Sporo'''beads still show undeminished fluorescence activity. </p></font><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Further_ApplicationsTeam:LMU-Munich/Application/Further Applications2012-10-25T19:25:39Z<p>Franzi.Duerr: </p>
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==Further Applications==<br />
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<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
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<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Further_ApplicationsTeam:LMU-Munich/Application/Further Applications2012-10-25T19:22:33Z<p>Franzi.Duerr: </p>
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==Further Applications==<br />
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<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
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<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Further_ApplicationsTeam:LMU-Munich/Application/Further Applications2012-10-25T19:22:00Z<p>Franzi.Duerr: </p>
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==Further Applications==<br />
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<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Protein_ScreeningTeam:LMU-Munich/Application/Protein Screening2012-10-25T19:20:56Z<p>Franzi.Duerr: </p>
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==Protein Screening==<br />
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<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/FiltrationTeam:LMU-Munich/Application/Filtration2012-10-25T19:20:12Z<p>Franzi.Duerr: </p>
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==Filtration==<br />
<br />
<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
<br />
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{| width="100%" cellpadding="20"<br />
|[[File:LMU Arrow purple BACK.png|right|80px|link=Team:LMU-Munich/Application]]<br />
|[[File:LMU Arrow purple NEXT.png|left|80px|link=Team:LMU-Munich/Protein_Screening]]<br />
|}<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/ApplicationTeam:LMU-Munich/Application2012-10-25T19:18:42Z<p>Franzi.Duerr: </p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
What are the potential '''advantages''' of using ''B. subtilis'' spores as functional bioparticals?<br />
<br> <br />
<br>They are:<br />
{|<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Very stable even under severe environmental conditions<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Easy and cheap to produce in large quantities (check out the link to this commercial [http://www.alibaba.com/product-free/118791994/Bacillus_subtilis_spores_HU58_100_pure.html offer])<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Part of human nutrition in terms of [http://en.wikipedia.org/wiki/Bacillus_subtilis_R0179 food supplements] and [http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm generally regarded as safe]<br />
|-<br />
|-<br />
|colspan="2"|Moreover, our '''Sporo'''beads are:<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|[https://2012.igem.org/Team:LMU-Munich/Project_Safety Unable] to proliferate and therefore most likely not treated as genetically modified organisms by the law<br />
|-<br />
|}<br />
<br />
<p align="justify">Our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] offer a wide variety of applications within the laboratory, and around the world. '''Sporo'''beads can be used for filtration and protein screening.</p><br />
<br />
<br />
<br />
<div class="box"><br />
====Filtration====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the first possible application for our '''Sporo'''beads.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Filtration]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Filtration]]<br />
|}<br />
</div><br />
<br />
<div class="box"><br />
====Protein Screening====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:protein_libraries.jpg|right|150px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|}<br />
</div><br />
<br />
<div class="box"><br />
====Further Applications====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">More information for further possible application with our diverse '''Sporo'''beads.</p><br />
|[[???|right|150px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="box"><br />
====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/ApplicationTeam:LMU-Munich/Application2012-10-25T19:16:22Z<p>Franzi.Duerr: </p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
What are the potential '''advantages''' of using ''B. subtilis'' spores as functional bioparticals?<br />
<br> <br />
<br>They are:<br />
{|<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Very stable even under severe environmental conditions<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Easy and cheap to produce in large quantities (check out the link to this commercial [http://www.alibaba.com/product-free/118791994/Bacillus_subtilis_spores_HU58_100_pure.html offer])<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Part of human nutrition in terms of [http://en.wikipedia.org/wiki/Bacillus_subtilis_R0179 food supplements] and [http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm generally regarded as safe]<br />
|-<br />
|-<br />
|colspan="2"|Moreover, our '''Sporo'''beads are:<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|[https://2012.igem.org/Team:LMU-Munich/Project_Safety Unable] to proliferate and therefore most likely not treated as genetically modified organisms by the law<br />
|-<br />
|}<br />
<br />
<p align="justify">Our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] offer a wide variety of applications within the laboratory, and around the world. '''Sporo'''beads can be used for filtration and protein screening.</p><br />
<br />
<br />
<br />
<div class="box"><br />
====Filtration====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the first possible application for our '''Sporo'''beads.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Filtration]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Filtration]]<br />
|}<br />
</div><br />
<br />
<div class="box"><br />
====Protein Screening====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|}<br />
</div><br />
<br />
<div class="box"><br />
====Further Applications====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="box"><br />
====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Further_ApplicationsTeam:LMU-Munich/Application/Further Applications2012-10-25T19:14:34Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}} [[File:Applications banner.resized WORDS.JPG|620..."</p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
==Further Applications==<br />
<br />
<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| width="100%" cellpadding="20"<br />
|[[File:LMU Arrow purple BACK.png|right|80px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:LMU Arrow purple NEXT.png|left|80px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/ApplicationTeam:LMU-Munich/Application2012-10-25T19:13:33Z<p>Franzi.Duerr: </p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
What are the potential '''advantages''' of using ''B. subtilis'' spores as functional bioparticals?<br />
<br> <br />
<br>They are:<br />
{|<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Very stable even under severe environmental conditions<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Easy and cheap to produce in large quantities (check out the link to this commercial [http://www.alibaba.com/product-free/118791994/Bacillus_subtilis_spores_HU58_100_pure.html offer])<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Part of human nutrition in terms of [http://en.wikipedia.org/wiki/Bacillus_subtilis_R0179 food supplements] and [http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm generally regarded as safe]<br />
|-<br />
|-<br />
|colspan="2"|Moreover, our '''Sporo'''beads are:<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|[https://2012.igem.org/Team:LMU-Munich/Project_Safety Unable] to proliferate and therefore most likely not treated as genetically modified organisms by the law<br />
|-<br />
|}<br />
<br />
<p align="justify">Our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] offer a wide variety of applications within the laboratory, and around the world. '''Sporo'''beads can be used for filtration and protein screening.</p><br />
<br />
<br />
<br />
<div class="box"><br />
====Filtration====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the first possible application for our '''Sporo'''beads.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Filtration]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Filtration]]<br />
|}<br />
</div><br />
<br />
==Filtration==<br />
<br />
<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
<br />
<div class="box"><br />
====Protein Screening====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|}<br />
</div><br />
<br />
==Protein Screening==<br />
<br />
<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 1: '''The simple protein-screening process in our Sporobeads'''. </font><br />
|}<br />
|}<br />
|}<br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries_affinities.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 2: '''The simple protein-screening process in our Sporobeads''' using affinity binding as a requisite. </font><br />
|}<br />
|}<br />
|}<br />
<br><br />
<br />
<br />
<div class="box"><br />
====Further Applications====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|}<br />
</div><br />
<br />
==Further Applications==<br />
<br />
<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| width="100%" cellpadding="20"<br />
|[[File:LMU Arrow purple BACK.png|right|80px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:LMU Arrow purple NEXT.png|left|80px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="box"><br />
====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/FiltrationTeam:LMU-Munich/Application/Filtration2012-10-25T19:13:29Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}} [[File:Applications banner.resized WORDS.JPG|620..."</p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
==Filtration==<br />
<br />
<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
<br />
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{| width="100%" cellpadding="20"<br />
|[[File:LMU Arrow purple BACK.png|right|80px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:LMU Arrow purple NEXT.png|left|80px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]<br />
|}<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Application/Protein_ScreeningTeam:LMU-Munich/Application/Protein Screening2012-10-25T19:13:24Z<p>Franzi.Duerr: Created page with "<!-- Include the next line at the beginning of every page --> {{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}} [[File:Applications banner.resized WORDS.JPG|620..."</p>
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[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
==Protein Screening==<br />
<br />
<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 1: '''The simple protein-screening process in our Sporobeads'''. </font><br />
|}<br />
|}<br />
|}<br />
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries_affinities.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 2: '''The simple protein-screening process in our Sporobeads''' using affinity binding as a requisite. </font><br />
|}<br />
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{| width="100%" cellpadding="20"<br />
|[[File:LMU Arrow purple BACK.png|right|80px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:LMU Arrow purple NEXT.png|left|80px|link=Team:LMU-Munich/Spore_Coat_Proteins/cloning]]<br />
|}<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/ApplicationTeam:LMU-Munich/Application2012-10-25T18:50:53Z<p>Franzi.Duerr: </p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
What are the potential '''advantages''' of using ''B. subtilis'' spores as functional bioparticals?<br />
<br> <br />
<br>They are:<br />
{|<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Very stable even under severe environmental conditions<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Easy and cheap to produce in large quantities (check out the link to this commercial [http://www.alibaba.com/product-free/118791994/Bacillus_subtilis_spores_HU58_100_pure.html offer])<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Part of human nutrition in terms of [http://en.wikipedia.org/wiki/Bacillus_subtilis_R0179 food supplements] and [http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm generally regarded as safe]<br />
|-<br />
|-<br />
|colspan="2"|Moreover, our '''Sporo'''beads are:<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|[https://2012.igem.org/Team:LMU-Munich/Project_Safety Unable] to proliferate and therefore most likely not treated as genetically modified organisms by the law<br />
|-<br />
|}<br />
<br />
<p align="justify">Our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] offer a wide variety of applications within the laboratory, and around the world. '''Sporo'''beads can be used for filtration and protein screening.</p><br />
<br />
<br />
<br />
<div class="box"><br />
====Filtration====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the first possible application for our '''Sporo'''beads.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Filtration]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Filtration]]<br />
|}<br />
</div><br />
<br />
==Filtration==<br />
<br />
<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
<br />
<div class="box"><br />
====Protein Screening====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|}<br />
</div><br />
<br />
==Protein Screening==<br />
<br />
<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 1: '''The simple protein-screening process in our Sporobeads'''. </font><br />
|}<br />
|}<br />
|}<br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries_affinities.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 2: '''The simple protein-screening process in our Sporobeads''' using affinity binding as a requisite. </font><br />
|}<br />
|}<br />
|}<br />
<br><br />
<br />
<br />
<div class="box"><br />
====Further Applications====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|}<br />
</div><br />
<br />
==Further Applications==<br />
<br />
<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="box"><br />
====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/ApplicationTeam:LMU-Munich/Application2012-10-25T18:44:46Z<p>Franzi.Duerr: </p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}<br />
[[File:Applications banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
What are the potential '''advantages''' of using ''B. subtilis'' spores as functional bioparticals?<br />
<br> <br />
<br>They are:<br />
{|<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Very stable even under severe environmental conditions<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Easy and cheap to produce in large quantities (check out the link to this commercial [http://www.alibaba.com/product-free/118791994/Bacillus_subtilis_spores_HU58_100_pure.html offer])<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|Part of human nutrition in terms of [http://en.wikipedia.org/wiki/Bacillus_subtilis_R0179 food supplements] and [http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm generally regarded as safe]<br />
|-<br />
|-<br />
|colspan="2"|Moreover, our '''Sporo'''beads are:<br />
|-<br />
|[[File:BoxChecked.png|40px|left]]<br />
|[https://2012.igem.org/Team:LMU-Munich/Project_Safety Unable] to proliferate and therefore most likely not treated as genetically modified organisms by the law<br />
|-<br />
|}<br />
<br />
<p align="justify">Our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] offer a wide variety of applications within the laboratory, and around the world. '''Sporo'''beads can be used for filtration and protein screening.</p><br />
<br />
<br />
<div class="box"><br />
====Filtration====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the first possible application for our '''Sporo'''beads.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Filtration]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Filtration]]<br />
|}<br />
</div><br />
<br />
==Filtration==<br />
<br />
<p align="justify">Our '''Sporo'''beads can express proteins on their outer coats to bind specific molecular targets. Such targets include heavy metals, toxins, and plastic.</p><br />
<p align="justify">One example of such a filtering protein could be a CPX-'''Sporo'''bead. [http://partsregistry.org/wiki/index.php/Part:BBa_I728500 CPX] is a peptide developed by the [https://2007.igem.org/MIT 2007 MIT iGEM team], which is extremely hydrophilic, and thus capable of binding microparticles of polystyrene from water. The excessive use of disposable plastic and the lack of universal recycling programs has led to the [http://www.ncbi.nlm.nih.gov/pubmed/22610295 pollution of the world's oceans]. In the ocean, large pieces of plastic litter are ground by sea currents and degraded by UV radiation into microscopic pieces, so called "plastic plankton," which is consumed by fish, filter feeders, and other marine organisms. Such plastic uptake can lead to poisoning, sterility and death.</p><br />
<p align="justify">CPX-'''Sporo'''beads in huge filter boxes could be put into place to mechanically filter microscopic plastic particles out of the water. Such specific filtration would be superior to blanket filtration systems, which also remove living phytoplankton important to ocean ecosystems. To prevent the beads from being released into the sea and to ensure the plastic be removed from the water, the '''Sporo'''beads could be attached to membranes in the filter boxes. Then the '''Sporo'''beads would need to not only display CPX but also a membrane binding protein on their surface.</p><br />
<br />
<div class="box"><br />
====Protein Screening====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Protein Screening]]<br />
|}<br />
</div><br />
<br />
==Protein Screening==<br />
<br />
<p align="justify">Designer protein molecules, which bind their desired targets specifically, have numerous uses. Designer proteins are frequently created by constructing and screening libraries of mutated protein variants. Proteins with desired properties are selected for in this method. Despite the success of the method, it is labor intensive and limited in throughput. Individual mutated proteins must be tracked throughout the entire screening process. Our '''Sporo'''beads would eliminate the need to track protein mutants, allowing researchers to screen huge quantities of mutated proteins, only identifying and sequencing these proteins after successful screening. Figures 1 and 2 offer schematics of the process of using '''Sporo'''beads for protein screening for tests and affinities.</p><br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 1: '''The simple protein-screening process in our Sporobeads'''. </font><br />
|}<br />
|}<br />
|}<br />
<br />
{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"<br />
| style="width: 70%;background-color: #EBFCE4;" |<br />
{|align:center<br />
|[[File:protein_libraries_affinities.jpg|620px|center]]<br />
|-<br />
| style="width: 80%;background-color: #EBFCE4;" |<br />
{| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;"<br />
|style="width: 70%;background-color: #EBFCE4;" |<br />
<font color="#000000"; size="2">Fig. 2: '''The simple protein-screening process in our Sporobeads''' using affinity binding as a requisite. </font><br />
|}<br />
|}<br />
|}<br />
<br><br />
<br />
<br />
<div class="box"><br />
====Further Applications====<br />
{| width="100%" style="text-align:center;"|<br />
|<p align="justify">Further information for the possible application of protein screening.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Application/Further Applications]]<br />
|}<br />
</div><br />
<br />
==Further Applications==<br />
<br />
<p align="justify">Ultimately, the ability to express virtually any protein of interest shows great potential for laboratory work. Our '''Sporo'''beads could additionally express TAL effectors, for the binding of sequence-specific DNA stretches. This could allow simple GMO detection of food crops. Besides simply expressing proteins capable of binding elements of interest, our '''Sporo'''beads could express proteins which have enzymatic activity. The 2011 University of Washington iGEM team developed an enzyme, Kumamolisin, which cleaves peptides. The specific substrate of this enzyme is the specific amino acid sequence which causes reactions in individuals with Celiac disease. Such a cleaving enzyme could be used to eliminate irritants from gluten-containing food products.</p><br />
<br />
<p align="justify">There are many possible applications for our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it is possible to display all kinds of different proteins on their surface. To easily create them, we designed a [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks/Sporovector '''Sporo'''vector] in which you just have to insert the gene encoding your fusion protein of choice. Since there are so many possible applications, we illustrate three examplary ideas for future '''Sporo'''beads in the following section:</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="box"><br />
====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Bacillus_IntroductionTeam:LMU-Munich/Bacillus Introduction2012-10-25T17:59:02Z<p>Franzi.Duerr: </p>
<hr />
<div><!-- Include the next line at the beginning of every page --><br />
{{:Team:LMU-Munich/Templates/Page Header|File:Bacillus in urban culture.jpg}}<br />
[[File:Bacillus introduction banner.resized WORDS.JPG|620px|link=]]<br />
<br />
<br />
[[File:Bacilluss_Intro.png|100px|right|link=]]<br />
<br />
<br />
<br />
==''Bacillus subtilis'' - a new chassis for iGEM==<br />
<br><br />
<div class="box"><br />
====Introduction====<br />
{| "width=100%" style="text-align:center;"|<br />
|<p align="justify">Introduction to ''B. subtilis'' and background information.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Data/introintro]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/introintro]]<br />
|}<br />
</div><br />
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There are two major differences between ''B. subtilis'' and ''E. coli'' that are of interest to us:<br />
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====Transformation of ''B. subtilis''====<br />
{| "width=100%" style="text-align:center;"|<br />
|<p align="justify">Cloning strategy for the work of '' B. subtilis''</p><br />
|[[File:Integration.png|right|150px|link=Team:LMU-Munich/Data/integration]]<br />
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/integration]]<br />
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====Differentiation====<br />
{| "width=100%" style="text-align:center;"|<br />
|<p align="justify">The life cycle of B. subtilis and the different cell stages</p><br />
|[[File:Figures Bacillus Intro fig1.png|right|150px|link=Team:LMU-Munich/Data/differentiation]]<br />
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/differentiation]]<br />
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====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerrhttp://2012.igem.org/Team:LMU-Munich/Bacillus_IntroductionTeam:LMU-Munich/Bacillus Introduction2012-10-25T17:58:11Z<p>Franzi.Duerr: </p>
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{{:Team:LMU-Munich/Templates/Page Header|File:Bacillus in urban culture.jpg}}<br />
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==''Bacillus subtilis'' - a new chassis for iGEM==<br />
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====Introduction====<br />
{| "width=100%" style="text-align:center;"|<br />
|<p align="justify">Introduction to ''B. subtilis'' and background information.</p><br />
|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Data/introintro]]<br />
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/introintro]]<br />
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</div><br />
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There are two major differences between ''B. subtilis'' and ''E. coli'' that are of interest to us:<br />
<br />
<div class="box"><br />
====Transformation of ''B. subtilis''====<br />
{| "width=100%" style="text-align:center;"|<br />
|<p align="justify">Cloning strategy with the work of '' B. subtilis''</p><br />
|[[File:Integration.png|right|150px|link=Team:LMU-Munich/Data/integration]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/integration]]<br />
|}<br />
</div><br />
<br />
<div class="box"><br />
====Differentiation====<br />
{| "width=100%" style="text-align:center;"|<br />
|<p align="justify">The life cycle of B. subtilis and the different cell stages</p><br />
|[[File:Figures Bacillus Intro fig1.png|right|150px|link=Team:LMU-Munich/Data/differentiation]]<br />
|-<br />
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/differentiation]]<br />
|}<br />
</div><br />
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====Project Navigation====<br />
{| width="100%" align="center" style="text-align:center;"<br />
|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]<br />
|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]<br />
|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]<br />
|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]<br />
|-<br />
|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]<br />
|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]<br />
|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]<br />
|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]<br />
|}<br />
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{{:Team:LMU-Munich/Templates/Page Footer}}</div>Franzi.Duerr