Team:LMU-Munich/Germination Stop

From 2012.igem.org

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[[File:GerminationSTOP.png|100px|right]]
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'''Germination'''STOP
 
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The goal of this project is to remove the germination capability, while keeping the necessary structural functions of the spores intact.
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[[File:GerminationSTOP.png|100px|right|link=]]
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There are two approaches to achive this:
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== '''Germination'''STOP ==
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<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>
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* Knock out as much genes as possible that are involved in germination
 
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* [[Team:LMU-Munich/Germination_Stop#Suicideswitch|'''Suicide'''switch]]: Toxin production if first fails
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[[File:GerminationSTOP_cycleIIii.jpg|620px|center]]
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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]]
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Based on the work of [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] and [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we decided to knock out genes ''cwlJ'', ''sleB'', ''cwlB'', ''gerD'', and ''cwlD''. The genes ''cwlJ'' and ''sleB'' code for lytic enzymes which are active in the process of germination. In the work of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], when ''cwlJ'' and ''sleB'' were knocked out together, germination frequency was reduced by 5 orders of magnitude. [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] reports a similar reduction in germination when ''gerD'' and ''cwlB'' are knocked out together, and a reduction in germination ability when the gene ''cwlD'' is knocked out. When all five of these genes are knocked out, we hope to yield a ''B. subtilis'' strain which produces spores completely incapable of germination.
 
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Two methods are being employed to knock out germination: resistance cassette (RC) knockouts and clean deletions. Single RC knockouts were created, and have then been combined to create multiple knockouts.
 
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This will be checked with germination assays.
 
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Further, we will investigate the possibility of using a redundant toxin system to immediately kill off any spores which somehow germinate.
 
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=='''Suicide'''switch==
 
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<div class="box">
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'''Project Navigation'''
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==Gene Knockouts==
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{| "width=100%" style="text-align:center;" style="align:right"|
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{|
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|<p align="justify">We picked important germination genes and knocked them out. Subsequently we combined the single mutants into quadruple mutants and successfully prevented germination.</p>  
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|<font color="#FFFFFF">+--+--+--+--+--+--+--</font>
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|[[File:LMU Germination STop plate.png|200px|right|link=Team:LMU-Munich/Germination_Stop/Knockout]]
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|<font color="#FFFFFF">+--+--+--+--+--</font>
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|<font color="#FFFFFF">+--+--+--+--+--+--</font>
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|<font color="#FFFFFF">+--+--+--+--+--+--</font>
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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Germination_Stop/Knockout]]
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<html>
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|}
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction">
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</div>
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<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=70"/>
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</a>
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<font size="1">
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</font>
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<br />
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction"><font size="1" face="verdana">Bacillus<BR>Intro</font></a>
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</html>
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|<html>
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<div class="box">
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks">
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=='''Suicide'''switch==
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<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=70"/>
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{| "width=100%" style="text-align:center;" style="align:right"|
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</a>
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|<p align="justify">In case of germination gene knockout failure, we invented the suicide switch. If spores still germinate, the production of a toxin leads to immediate cell death.</p>
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<font size="1">
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|[[File:LMU SuicideSwitch grafik.png|200px|right|link=Team:LMU-Munich/Germination_Stop/SuicideSwitch]]
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</font>
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|-
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<br />
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Germination_Stop/SuicideSwitch]]
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks"><font size="1" face="verdana">Bacillus<BR>BioBrickBOX</font></a>
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|}
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</html>
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</div>
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<br>
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|<html>
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<br>
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<a href="https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins">
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<br>
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<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=70"/>
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<br>
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</a>
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<div class="box">
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<font size="1">
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====Project Navigation====
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</font>
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{| width="100%" align="center" style="text-align:center;"
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<br />
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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]
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<a href="https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins"><font size="1" face="verdana">SporeCoat<BR>FusionProteins</font></a>
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|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]
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</html>
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|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]
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|<html>
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|-
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<a href="https://2012.igem.org/Team:LMU-Munich/Germination_Stop">
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=70"/>
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|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]
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</a>
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|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]
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<font size="1">
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|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]
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</font>
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<br />
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<a href="https://2012.igem.org/Team:LMU-Munich/Germination_Stop"><font size="1" face="verdana">Germination<BR>STOP</font></a>
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</html>
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|}
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</div>
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{{:Team:LMU-Munich/Templates/Page Footer}}
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Latest revision as of 16:42, 26 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".

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