Team:LMU-Munich/Weekly Journal

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==Weekly Journal==
 
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|[[Team:LMU-Munich/Weekly Journal#July|July]]
|[[Team:LMU-Munich/Weekly Journal#July|July]]
|[[Team:LMU-Munich/Weekly Journal#October|October]]
|[[Team:LMU-Munich/Weekly Journal#October|October]]
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|[[Team:LMU-Munich/Weekly Journal# | ]]
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|[[Team:LMU-Munich/Weekly Journal# | ]]
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|[[Team:LMU-Munich/Weekly Journal#November|November]]
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===November===
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<div class="box">
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'''4-5 November 2012'''
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> Boston Jamboree!!</p>
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> OMG! We were finalist! We won "best wiki" together with Slovenia and best "new application"!</p>
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</div>
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<div class="box" style= "background-color:#e4f1d7">
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===October===
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</div>
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<div class="box">
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'''29 October - 3 November 2012'''
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> Flying across the Atlantic! On our way to the World Championship Jamboree in Boston!</p>
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</div>
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<div class="box">
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'''22-26 October 2012'''
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> Our dedicated presentation and website mini-team (Franzi, Korinna and Tamara) is working hard.</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html> Round two of time-lapse microscopy. We decided to try a new media combination and flow speed to see if we can prevent the cell lysis we experienced last week.</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html> We brain-stormed some ways to create a device for our '''Sporo'''beads. We asked our Gold Sponsor [https://2012.igem.org/Team:LMU-Munich/Sponsors Semadeni] if they would donate a few 0,20um filter spin columns to use for our envisioned sporo-device. We also batch-produced spores of our strain B53. Even with its modifications, it has definitely not lost its sporulation ability!</p>
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</div>
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<div class="box">
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'''15-19 October 2012'''
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> We got some great ideas from other teams at the European Jamboree about how to better present our project. We're working on a new version of our presentation for Boston.</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html> One of our collaborators, the [https://2012.igem.org/Team:Bielefeld-Germany Bielefeld iGEM team], graciously sent us two laccases to use on our '''Sporo'''beads.</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html> We wanted to demonstrate the formation of GFP-expressing spores in our cells, so we decided to try time-lapse microscopy. Our first round of time-lapse failed, as the cells spontaneously lysed after 10 hours. We were baffled by this reaction. Well, time to do some research.</p>
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</div>
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<div class="box">
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'''7-12 October 2012'''
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> On Sunday at the Jamboree, we got the great news that our team will take the '''Bead'''zillus project to Boston!</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> Back home to Munich, and a week of much-needed rest for most of the team before heading back to the lab.</p>
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</div>
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<div class="box">
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'''1-6 October 2012'''
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> Working on the final touches to our poster and presentation.</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> The whole team spent the entire Wednesday mixing, rolling, punching, baking and frosting between 600 and 800 '''Bead'''zillus cookies to bring to the European Jamboree poster session!</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> By planes and trains, we made our way to the European Jamboree in the lovely city of Amsterdam.</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> Friday and Saturday were the Jamboree: we gave our presentation, presented our poster, and distributed lots of '''Bead'''zillus cookies! We also met several potential collaboration partners, including [http://www.bsse.ethz.ch/bpl/people/panke Sven Panke] of [https://2012.igem.org/Team:ETH_Zurich ETH Zurich] who proposed the very interesting idea of using our '''Sporo'''beads for protein evolution.</p>
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</div>
<div class="box" style= "background-color:#e4f1d7">
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===September===
===September===
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'''24-28 September 2012'''
'''24-28 September 2012'''
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</div>
 
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html> Now, the [[Team:LMU-Munich/Bacillus_BioBricks/Sporovector|'''Sporo''' vector]] is cloned into [http://partsregistry.org/Part:BBa_K823022 pSB<sub>Bs</sub>4S] and we are trying to insert [http://partsregistry.org/Part:BBa_K823019 lacZ].</p>
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html> We all are busy setting up our website and finding the best ways to display our results!</p>
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
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Sitting on the computer every day and analyzing the sheer number of microscopy pictures... Check out our [https://2012.igem.org/Team:LMU-Munich/Data/gfp_spore data]</p>
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</div>
<div class="box">
<div class="box">
'''17-21 September 2012'''
'''17-21 September 2012'''
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
 +
Last microscopy experiments with the clean deletion mutants. They are glowing even brighter!</p> 
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
Finished cloning of [http://partsregistry.org/Part:BBa_K823029 '''''mKate2'''''] constructs. Now the evaluation of this reporter BioBrick with three different promoters, [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>], [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] and the Anderson promoter [http://partsregistry.org/Part:BBa_K823005 J23101], can start.</p>  
Finished cloning of [http://partsregistry.org/Part:BBa_K823029 '''''mKate2'''''] constructs. Now the evaluation of this reporter BioBrick with three different promoters, [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>], [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] and the Anderson promoter [http://partsregistry.org/Part:BBa_K823005 J23101], can start.</p>  
 +
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
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Finished cloning of [[Team:LMU-Munich/Bacillus_BioBricks/Sporovector|'''Sporo''' vector]] in pSB1C3.</p>
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
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Finished cloning of [[Team:LMU-Munich/Bacillus_BioBricks/Sporovector|'''Sporo''' vector]] in pSB1C3. Now, this device will be cloned into pSB<sub>Bs</sub>4S.</p>  
+
[https://2012.igem.org/Team:LMU-Munich/Data/Vectors#pSBBs0K-Pspac Evaluation] of [http://partsregistry.org/Part:BBa_K823026 pSB<sub>''Bs''</sub>0K-P<sub><i>spac</i></sub>] with [http://partsregistry.org/Part:BBa_K823019 ''lacZ''].</p>  
<p align="justify"><html><a>
<p align="justify"><html><a>
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<img src="https://static.igem.org/mediawiki/2012/0/0f/LMU-Munich-Invertersign.png" height=40"/></a></html>
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<img src="https://static.igem.org/mediawiki/2012/0/0f/LMU-Munich-Invertersign.png" height=30"/></a></html>
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Inverter measured quantitavely via ONPG assay</p>
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K823040 Inverter] measured quantitively via [https://2012.igem.org/File:LMU_Inverter_graph.png β-Galactosidase assay].</p>
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</div>
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<p align="justify"><html><a>
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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For our '''Suicide''' switch, the first plate reader measurement of ''Bacillus subtilis'' strain W168 containing ''thrC''::P<sub>''spoIVB''</sub>-''ecf41<sub>Bli aa  1-204</sub>'' (through transformation with pSBBs4S-P<sub>''spoIVB''</sub>-''ecf41<sub>Bli aa  1-204</sub>'') and ''sacA''::P<sub>''ydfG''</sub>-''luxABCDE'' (through transformation with pSBBs3C-''luxABCDE''-P<sub>''ydfG''</sub>) was performed.</p>
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<p align="justify"><html><a>
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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Knockouts: One last run of the germination assay on our triple and quadruple mutants. All results of these assays can be found compiled on our [https://2012.igem.org/Team:LMU-Munich/Data/Knockout data page].</p>
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</div>
<div class="box">
<div class="box">
'''10-14 September 2012'''
'''10-14 September 2012'''
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
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To clean up our '''Sporo'''beads from vegetative ''B. subtilis'' cells we tried three different methods: French Press, sonification and lysozymes. A significant difference was observed after the treatment with lysozyme! Furthermore the lysozyme did not damage our fusion protein as GFP fluorescence was still obtained in microscopy!</p>
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To clean up our '''Sporo'''beads from vegetative ''B. subtilis'' cells we tried three different methods: French Press, sonification and lysozymes. A great difference was observed after the [https://2012.igem.org/Team:LMU-Munich/Data/Sporepurification treatment with lysozyme]! Furthermore, the lysozyme did not damage our fusion protein, as GFP fluorescence was still obtained in microscopy!</p>
 +
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
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P<sub>''lepA''</sub> was fused into the finished vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823025 pSB<sub>''BS''</sub>3C-luxABCDE] to evaluate this BioBrick vector and compare in to the version where there is still one forbidden restriction site in it.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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PspoIVB-ECF41 in pSB<sub>BS</sub>4S, PydfG/PsspK/PspoIVB in pSB<sub>BS</sub>3C-''luxABCDE'' brought into ''B. subtilis''</p>
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For the '''Suicide''' switch, P<sub>''spoIVB''</sub>-''ecf41<sub>Bli aa 1-204</sub>'' in pSB<sub>BS</sub>4S, and P<sub>''ydfG''</sub>/P<sub>''sspK''</sub>/P<sub>''spoIVB''</sub> in pSB<sub>BS</sub>3C-''luxABCDE'' were brought into ''B. subtilis''.</p>
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<p align="justify"><html><a>
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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More work on the knockouts: We were really surprised at our great results so far, and repeated the germination assay on our triple and quadruple mutants once again.</p>
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<p align="justify"><html><a ">
<p align="justify"><html><a ">
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
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All our CotZ-GFP variants were examined with fluorescence microscopy! The brightest spores derived from our P<sub>''cotyz''</sub>-''cotZ''rep-''gfp''-terminator mutants!</p>
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All of our CotZ-GFP variants were examined with fluorescence microscopy! The brightest spores derived from our B 53 strain (containing P<sub>''cotyz''</sub>-''cotZ''rep-''gfp''-terminator).</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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Plates of our spores diluted at 10<sup>-2</sup>, 10<sup>-4</sup> and 10<sup>-6</sup> from the germination assay show NO GERMINATION for our triple and quadruple mutants, and plenty of germination for the WT168 positive control! We will try plating undiluted mutant spores to see if any germination occurs.</p>
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We worked more on the knockouts aspect of the '''Germination'''STOP module. We ran another germination assay for our triple and quadruple mutants. Plates of our spores diluted at 10<sup>-2</sup>, 10<sup>-4</sup> and 10<sup>-6</sup> show NO GERMINATION for our mutants, and plenty of germination for the WT168 positive control! We will try plating undiluted mutant spores to see if any germination occurs at higher concentrations.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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PsspK, PspoIVB and PydfG cloned into pSB1C3 and verified by sequencing <br> PspoIVB-ECF41 cloned into pSB<sub>BS</sub>4S and verified by sequencing <br> PydfG/PsspK/PspoIVB brought into pSB<sub>BS</sub>3C-''luxABCDE''</p>
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More work on the '''Suicide''' switch! [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823042 P<sub>''sspK''</sub>], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823048 P<sub>''spoIVB''</sub>] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823041 P<sub>''ydfG''</sub>] were cloned into pSB1C3 and verified by sequencing. <br> P<sub>''spoIVB''</sub>-''ecf41<sub>Bli aa1-204</sub>'' were cloned into pSB<sub>BS</sub>4S and verified by sequencing. <br> P<sub>''ydfG''</sub>/P<sub>''sspK''</sub>/P<sub>''spoIVB''</sub> were brought into pSB<sub>BS</sub>3C-''luxABCDE''.</p>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
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A collection of useful tags in Freiburg standard and with RBS included was cloned into pSB1C3. The tags are: 3xFlag, HA, cMyc, 10xHis and Streptavidin.</p>
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[http://partsregistry.org/Part:BBa_K823019 lacZ] was succesfully cloned into [http://partsregistry.org/Part:BBa_K823024 pSB<sub>''Bs''</sub>4S-P<sub><i>Xyl</i></sub>] and is functional (blue colonies with IPTG and X-Gal).</p>
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<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
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A collection of useful tags in Freiburg standard and with RBS included was cloned into pSB1C3. The tags are: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823034 3xFlag], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823035 HA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823036 cMyc], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823037 10xHis] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823038 Streptavidin].</p>
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<p align="justify"><html><a>
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<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
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Finally, we got our first glowing spores!! After 4 months of hard work we have the first proof that this module works.</p>
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Finally, we got our first glowing spores!! After 4 months of hard work, we have the first proof that this module works.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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Jara created quadruple mutants using two variations on past mutants: ''cwlD''::kan + ''cwlJ''::spec + ''gerD''::cm + ''sleB''::mls and ''gerD''::cm + ''sleB''::mls + ''cwlJ''::spec + ''cwlD''::kan.
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More on germination knockouts -- we created quadruple mutants using two variations on past mutants: ''cwlD''::kan + ''cwlJ''::spec + ''gerD''::cm + ''sleB''::mls and ''gerD''::cm + ''sleB''::mls + ''cwlJ''::spec + ''cwlD''::kan. See our [https://2012.igem.org/Team:LMU-Munich/Strains Strains Collection.]
Germination assay was performed on triple and quadruple mutants.</p>
Germination assay was performed on triple and quadruple mutants.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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MazF and ECF41 cloned into pSB1C3 and verified by sequencing</p>
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For the '''Suicide''' switch, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823044 MazF] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823043 Ecf41<sub>Bli aa 1-204</sub>] were cloned into pSB1C3 and verified by sequencing.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
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The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823019 lacZ] for ''B. subtilis'' was shown to be functional in <b>pSB<sub>Bs</sub>0K-P<sub><i>spac</i></sub> </b> in ''E. coli'' and ''B. subtilis''. (blue color on plates with IPTG and X-Gal)</p>
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The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823019 lacZ] for ''B. subtilis'' was shown to be functional in <b>pSB<sub>Bs</sub>0K-P<sub><i>spac</i></sub> </b> in ''E. coli'' and ''B. subtilis'' (blue color on plates with IPTG and X-Gal).</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
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The genes [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823028 luc+] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823029 mKate2], synthesized by [https://2012.igem.org/Team:LMU-Munich/Sponsors GeneArt] were successfully cloned into pSB1C3 and sequenced.</p>
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The genes [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823028 luc+] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823029 mKate2], synthesized by [https://2012.igem.org/Team:LMU-Munich/Sponsors GeneArt], were successfully cloned into pSB1C3 and sequenced.</p>
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<img src="https://static.igem.org/mediawiki/2012/0/0f/LMU-Munich-Invertersign.png" height=30"/></a></html>
-
Inverter with lacZa was finished and works qualitatively (Julia).</p>
+
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K823040 Inverter] with lacZα was finished and it works qualitatively.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
Good and bad news this week. First the good one: Another big step towards the GFP-Sporobeads is done! We have the CotZ constructs in pSB<sub>BS</sub>1C! Hopefully it will integrate easily! :D
+
Good and bad news this week. First the good news: Another big step towards the GFP-Sporobeads is done! We have the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823049 CotZ constructs] in pSB<sub>BS</sub>1C! Hopefully it will integrate easily! :D
-
Now the bad one: Finally we could start with the P<sub>cgeA</sub> evaluation! But contrary to our expectations this promoter did not show any activity :( </p>
+
Now the bad news: Finally we could start with the [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters P<sub>cgeA</sub> evaluation]! But contrary to our expectations, this promoter did not show any activity. </p>
-
 
+
-
 
+
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Jara and Jenny used last week's double mutants to create triple mutants as follows: ''cwlD''::kan + ''sleB''::mls + ''cwlJ''::spec ; ''cwlD''::kan + ''sleB''::mls + ''gerD''::cm ; ''cwlD''::kan + ''cwlJ''::spec + ''gerD''::cm ; ''gerD''::cm + ''sleB''::mls + ''cwlJ''::spec.</p>
+
For the knockouts, we used last week's double mutants to create triple mutants as follows: ''cwlD''::kan + ''sleB''::mls + ''cwlJ''::spec ; ''cwlD''::kan + ''sleB''::mls + ''gerD''::cm ; ''cwlD''::kan + ''cwlJ''::spec + ''gerD''::cm ; and ''gerD''::cm + ''sleB''::mls + ''cwlJ''::spec. See our [https://2012.igem.org/Team:LMU-Munich/Strains Strains Collection].</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
-
ß-glactosiase assay of the Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103] in <b>pSB<sub>Bs</sub>1C-<i>lacZ</i> </b> in ''B. subtilis''.
+
ß-glactosidase assay of the Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103] in <b>pSB<sub>Bs</sub>1C-<i>lacZ</i> </b> in ''B. subtilis'' was performed.
-
The xylose-inducible promoter with the according repressor (which has a constitutive promoter, RBS and terminator) P<sub><i>Xyl</i></sub> + <i>XylR</i> was cloned into pSB1C3 and sequenced.</p>
+
The xylose-inducible promoter with the according repressor (which has a constitutive promoter, RBS and terminator) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823015 ''xylR''-P<sub><i>Xyl</i></sub>] was cloned into pSB1C3 and sequenced.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
At last Bacillus was keen on integrating the pSB<sub>BS</sub>3C-luxABCDE-P<sub>''cgeA''</sub>!! And the clean deletions of CotZ and CgeA worked out, too!</p>
+
At last ''Bacillus'' was keen on integrating the pSB<sub>BS</sub>3C-luxABCDE-P<sub>''cgeA''</sub>!! And the clean deletions of CotZ and CgeA worked out, too!</p>
-
 
+
</div>
</div>
-
 
<div class="box">
<div class="box">
Line 203: Line 285:
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Jara and Jenny created four double-mutants using resistance-cassettes to knock out germination genes as follows: ''cwlD''::kan + ''sleB''::mls ; ''gerD''::cm + ''sleB''::mls ; ''gerD''::cm + ''cwlD''::kan ; ''cwlJ''::spec + ''cwlD''::kan. We also created the resistance cassette knockout ''cwlB''::kan.</p>
+
Germination knockouts: We created four double-mutants using resistance-cassettes to knock out germination genes as follows: ''cwlD''::kan + ''sleB''::mls ; ''gerD''::cm + ''sleB''::mls ; ''gerD''::cm + ''cwlD''::kan ; ''cwlJ''::spec + ''cwlD''::kan. See our [https://2012.igem.org/Team:LMU-Munich/Strains Strains Collection]. Of these double mutants, we tested three strains in our germination assay. We also created the resistance cassette knockout ''cwlB''::kan.</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
The final Promoter-CotZ-GFP-Terminator constructs in pSB1C3 are ready!! And Promoter-CgeAmut constructs are finally finished too. </p>
+
The final Promoter-CotZ-GFP-Terminator constructs in pSB1C3 are ready!! Our Promoter-CgeAmut constructs are finally finished too.</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html>
-
This week is our Human Practise week!
+
This week is our Human Practice week!
-
Form 23<sup>rd</sup> to 25<sup>th</sup> of July we enjoyed the [https://2012.igem.org/Team:LMU-Munich/Human_Practice/CAS_Conference CAS SynBio conference] and the visit of 10 iGEM teams!
+
From the 23<sup>rd</sup> to 25<sup>th</sup> of July we enjoyed the [https://2012.igem.org/Team:LMU-Munich/Human_Practice/CAS_Conference CAS SynBio conference] and the visit of 10 iGEM teams!
This weekend, high school students participated in our [https://2012.igem.org/Team:LMU-Munich/Human_Practice/Students_Practical_Course Students Practical Course] for three days and developed their ideas for useful '''Sporo'''beads!  
This weekend, high school students participated in our [https://2012.igem.org/Team:LMU-Munich/Human_Practice/Students_Practical_Course Students Practical Course] for three days and developed their ideas for useful '''Sporo'''beads!  
It was a great week! </p>   
It was a great week! </p>   
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>
-
Plate reader measurements of the ''Bacillus'' promoters [http://partsregistry.org/Part:BBa_K823000 P<sub>''liaG''</sub>], [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>] and [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] finished. Look at [https://2012.igem.org/Team:LMU-Munich/Data Data]!</p>
+
Plate reader measurements of the ''Bacillus'' promoters [http://partsregistry.org/Part:BBa_K823000 P<sub>''liaG''</sub>], [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>] and [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] are finished. Look at [https://2012.igem.org/Team:LMU-Munich/Data Data]!</p>
 +
</div>
-
 
-
</div>
 
<div class="box">
<div class="box">
'''16-20 July 2012'''
'''16-20 July 2012'''
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html>
-
Not many of us are working on our modules this week, as we are helping out for [https://2012.igem.org/Team:LMU-Munich/Human_Practice/CAS_Conference CAS SynBio conference] and organising the [https://2012.igem.org/Team:LMU-Munich/Human_Practice/Students_Practical_Course Students Practical Course].</p>
+
Not many of us are working on our modules this week, as we are helping out for the [https://2012.igem.org/Team:LMU-Munich/Human_Practice/CAS_Conference CAS SynBio conference] and organising the [https://2012.igem.org/Team:LMU-Munich/Human_Practice/Students_Practical_Course Students Practical Course].</p>
<p align="justify"><html><a>
<p align="justify"><html><a>
Line 264: Line 345:
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
-
The vectors [http://partsregistry.org/Part:BBa_K823024 <b>pSB<sub>Bs</sub>4S-P<sub><i>Xyl</i></sub> </b>], [http://partsregistry.org/Part:BBa_K823021 pSB<sub>''Bs''</sub>1C-''lacZ''] </b> and [http://partsregistry.org/Part:BBa_K823022 <b>pSB<sub>Bs</sub>4S </b>] were succesfully completed and tested by restriction digest as well as red colony color. </p>
+
The vectors [http://partsregistry.org/Part:BBa_K823024 <b>pSB<sub>Bs</sub>4S-P<sub><i>Xyl</i></sub> </b>], [http://partsregistry.org/Part:BBa_K823021 <b>pSB<sub>''Bs''</sub>1C-''lacZ''</b>]  and [http://partsregistry.org/Part:BBa_K823022 <b>pSB<sub>Bs</sub>4S </b>] were succesfully completed and tested by restriction digest as well as checked for red colony color. </p>
<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
We started with the clean deletions of CgeA and CotZ this week, seems like it will take for ever if you judge from the protocoll length... What took even longer is the pSB<sub>BS</sub>3C-luxABCDE-P<sub>''cgeA''</sub>, but it is finished now!</p>
+
We started with the clean deletions of CgeA and CotZ this week. It seems like it will take forever if you judge from the protocol length... What took even longer is the pSB<sub>BS</sub>3C-luxABCDE-P<sub>''cgeA''</sub>, but it is finished now!</p>
</div>  
</div>  
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Clean deletions of germination genes ''sleB'' and ''cwlB'' from PCR accomplished. DNA purified and frozen to be later transformed with ''Bacillus''.</p>
+
In the knockouts part of the '''Germination'''STOP module, clean deletions of germination genes ''sleB'' and ''cwlB'' from PCR were accomplished. DNA was purified and frozen to be later transformed with ''Bacillus''.</p>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
Our first plate reader experiments with P<sub>''cotyz''</sub> and P<sub>''cotv''</sub> are running! Like expected they both show activity in the late stationary phase, thus during sporulation!</p>
+
Our first plate reader experiments with [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters P<sub>''cotyz''</sub>] and [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters P<sub>''cotv''</sub>] are running! Like expected, they both show activity in the late stationary phase, thus during sporulation!</p>
</div>
</div>
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<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
pSB<sub>''BS''</sub>3C-luxABCDE-P<sub>''cotyz''</sub> and pSB<sub>''BS''</sub>3C-luxABCDE-P<sub>''cotv''</sub> integrated into the ''B. subtilis'' genome!  
pSB<sub>''BS''</sub>3C-luxABCDE-P<sub>''cotyz''</sub> and pSB<sub>''BS''</sub>3C-luxABCDE-P<sub>''cotv''</sub> integrated into the ''B. subtilis'' genome!  
-
Bad luck, we found an additional AgeI site in ''cgeA'', seems like this is the reason why we could not fuse ''gfp'' to it... Mutagenesis primers are designed and ordered!</p>
+
Bad luck, we found an additional AgeI site in ''cgeA''. It seems that this is the reason why we could not fuse ''gfp'' to it... Mutagenesis primers are designed and ordered!</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Clean deletions of germination genes ''cwlD'', ''cwlJ'', and ''gerD'' from PCR accomplished. DNA purified and frozen to be later transformed with ''Bacillus''.</p>
+
Knockouts: Clean deletions of germination genes ''cwlD'', ''cwlJ'', and ''gerD'' from PCR accomplished. DNA purified and frozen to be later transformed with ''Bacillus''. Edit: the clean deletions were never used for transformation, but remain ready for future use.</p>
</div>
</div>
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<div class="box">
<div class="box">
-
'''28-1 June 2012'''
+
'''28 May-1 June 2012'''
<p align="justify"><html><a>
<p align="justify"><html><a>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
pSB<sub>''BS''</sub>3C-''luxABCDE''-P<sub>''cotyz''</sub> and pSB<sub>''BS''</sub>3C-luxABCDE-P<sub>''cotv''</sub> are ready for transformation into Bacillus! </p>
+
pSB<sub>''BS''</sub>3C-''luxABCDE''-P<sub>''cotyz''</sub> and pSB<sub>''BS''</sub>3C-luxABCDE-P<sub>''cotv''</sub> are ready for transformation into ''Bacillus''! </p>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
-
Promoters [http://partsregistry.org/Part:BBa_K823000 P<sub>''liaG''</sub>], [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>], [http://partsregistry.org/Part:BBa_K823003 P<sub>''veg''</sub>] and [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] are now in the vector pSB1C3 as BioBrick standard for the registry. </p>
+
Promoters [http://partsregistry.org/Part:BBa_K823000 P<sub>''liaG''</sub>], [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>], [http://partsregistry.org/Part:BBa_K823003 P<sub>''veg''</sub>] and [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] are now in the vector pSB1C3 as per the BioBrick standard for the registry. </p>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
Combining the BioBricks and integrating them into different vectors works good! But still there are constructs missing, we'll keep on working! The fusion of the up and down fragment of CotZ finally works!</p>
+
Combining the BioBricks and integrating them into different vectors works well! But still there are constructs missing... We'll keep on working! The fusion of the up and down fragment of CotZ finally works!</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html>
-
The last weeks we tried to fuse the up and down fragment of our spore crust gene together for its clean deletion. For CgeA we have the first fused fragments! CotZ needs a little more attention :)</p>
+
The last weeks we tried to fuse the up and down fragment of our spore crust gene together for its clean deletion. For CgeA we have the first fused fragments! CotZ needs a little more attention. :)</p>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
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<div class="box">
<div class="box">
-
'''30 April-4 Mai 2012'''
+
'''30 April-4 May 2012'''
<p align="justify"><html><a>
<p align="justify"><html><a>
Line 403: Line 484:
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
-
The cloning of the reporter vector [http://partsregistry.org/Part:BBa_K823021 pSB<sub>''Bs''</sub>1C-''lacZ''] </b> was finished.</p>
+
The cloning of the reporter vector [http://partsregistry.org/Part:BBa_K823021 pSB<sub>''Bs''</sub>1C-''lacZ''] was finished.</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Jara created the single mutant ''cwlD''::kan.</p>
+
For the knockouts, we created the single mutant ''cwlD''::kan.</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Jara successfully created our first single knockouts of germination genes using a resistance cassettes: ''sleB''::mls, ''gerD''::cm and ''cwlJ''::spec.
+
In our germination genes knockout work, we successfully created our first single knockouts of germination genes using a resistance cassettes: ''sleB''::mls, ''gerD''::cm and ''cwlJ''::spec. See our [https://2012.igem.org/Team:LMU-Munich/Strains Strains Collection].
-
Jara tried knocking out ''cwlB'' using the kan resistance cassette. Mutants of ''cwlB''::kan grew very poorly.</p>
+
We tried knocking out ''cwlB'' using the kan resistance cassette. Mutants of ''cwlB''::kan grew very poorly.</p>
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
<p align="justify"><html><a><img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html>  
-
With <b>pSB<sub>Bs</sub>0K-P<sub><i>spac</i></sub> </b> our first vector for our <b>B</b>acillus <b>B</b>io<b>B</b>rick <b>B</b>ox was completed.</p>
+
With [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823026 <b>pSB<sub>Bs</sub>0K-P<sub><i>spac</i></sub> </b>] our first vector for our <b>''B''</b>''acillus'' <b>B</b>io<b>B</b>rick <b>B</b>ox was completed.</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
-
Jara tried knocking out ''cwlB'' using the tet resistance cassette. Mutants of ''cwlB''::tet grew very poorly.</p>
+
Knockouts: We tried knocking out ''cwlB'' using the tet resistance cassette. Mutants of ''cwlB''::tet grew very poorly.</p>
</div>
</div>
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<p align="justify"><html><a>
<p align="justify"><html><a>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html>
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We decided which genes to knock out for the germination stop. Based on the work of [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)], we decided to knock out genes ''cwlB'', ''gerD'', ''cwlJ'', and ''sleB''. From the research of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we also chose ''cwlD''.</p>
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For our germination gene knockouts, we decided which genes to knock out for the germination stop. Based on the work of [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)], we decided to knock out genes ''cwlB'', ''gerD'', ''cwlJ'', and ''sleB''. From the research of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we also chose ''cwlD''.</p>
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'''12-16 March 2012'''
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'''5-9 March 2012'''
'''5-9 March 2012'''
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<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html>
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We spent two full days (and nights) together planning our project in our "iGEM days." The idea of '''Bead'''zillus was born, and we got to know each other better as we divided the modules of the project, and learned each others' strengths and experience in different fields such as microbiology, ecology and biochemistry.</p>
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'''27 February - 2 March 2012'''
 
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'''20-24 February 2012'''
'''20-24 February 2012'''
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Team fully formed!
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<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=25"/></a></html>
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Team fully formed!</p>
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Latest revision as of 13:37, 14 November 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

Team-LMU Photo9.jpg

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".

IGEM HQ LMU prize.jpg

[ more news ]

Sporenfreunde

Retrieved from "http://2012.igem.org/Team:LMU-Munich/Weekly_Journal"