Team:LMU-Munich/Data

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{{:Team:LMU-Munich/Templates/Page Header|File:Team-LMU_Photo2.jpg}}
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[[File:Data banner.resized WORDS.JPG|620px|link=]]
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==Data==
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[[Image:BacillusBioBrickBox.png|right|100px|link=https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks]]
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<br>
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<div class="box" style= "background-color:#e4f1d7">
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Here you will find all of our project data. For our big breakthroughs, or to follow specific projects, see the individual project pages:
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==='''''Bacillus''B'''io'''B'''rick'''B'''ox===
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====Evaluation of ''B. subtilis'' vectors====
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">Seven novel vectors were constructed and four already proven to work in ''B. subtilis''.</p>
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|[[File:LacZ plate.png|right|100px|link=Team:LMU-Munich/Data/Vectors]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Vectors]]
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction">
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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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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks">
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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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<a href="https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins">
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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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<a href="https://2012.igem.org/Team:LMU-Munich/Germination_Stop">
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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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==='''''Bacillus''B'''io'''B'''rick'''B'''ox===
 
</div>
</div>
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<div class="box">
<div class="box">
====Anderson Promoter Evaluation====
====Anderson Promoter Evaluation====
{| "width=100%" style="text-align:center;"|
{| "width=100%" style="text-align:center;"|
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|<p align="justify">Evaluation of the ''Anderson'' library in ''B. subtilis'' with pSB<sub>Bs</sub>3C-''lux'' (luminescence) and pSB<sub>Bs</sub>1C-''lacZ''</p>
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|<p align="justify">Eleven Anderson promoters were measured in ''B. subtilis'' and showed relatively weak activity.</p>
|[[File:LMU Anderson preview.jpg|200px|link=Team:LMU-Munich/Data/Anderson]]
|[[File:LMU Anderson preview.jpg|200px|link=Team:LMU-Munich/Data/Anderson]]
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====Constitutive ''Bacillus'' Promoters====
====Constitutive ''Bacillus'' Promoters====
{| "width=100%" style="text-align:center;"|
{| "width=100%" style="text-align:center;"|
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|<p align="justify">Evaluation of constitutive ''B. subtilis'' promoters with pSB<sub>Bs</sub>3C-''lux'' and pSB<sub>Bs</sub>1C-''lacZ''</p>
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|<p align="justify">Three novel ''B. subtilis'' promoter BioBricks (P<sub>''liaG''</sub>, P<sub>''veg''</sub>, P<sub>''lepA''</sub>) were constructed and functionality verified by luminescence and β-galactosidase assays.</p>
|[[File:LMU Constitutive preview.jpg|200px|link=Team:LMU-Munich/Data/Constitutive]]
|[[File:LMU Constitutive preview.jpg|200px|link=Team:LMU-Munich/Data/Constitutive]]
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====Inducible ''Bacillus'' Promoters====
====Inducible ''Bacillus'' Promoters====
{| "width=100%" style="text-align:center;"|
{| "width=100%" style="text-align:center;"|
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|<p align="justify">Evaluation of inducible ''B. subtilis'' promoters with pSB<sub>Bs</sub>3C-''lux'' and pSB<sub>Bs</sub>1C-''lacZ''</p>
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|<p align="justify">A novel antibiotic-inducible promoter, P<sub>''liaI''</sub>, was constructed and thoroughly evaluated.</p>
|[[File:Englisch Auswertung PliaI.png|200px|link=Team:LMU-Munich/Data/Inducible]]
|[[File:Englisch Auswertung PliaI.png|200px|link=Team:LMU-Munich/Data/Inducible]]
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[[File:SporeCoat.png|90px|right|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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<br>
<div class="box" style= "background-color:#e4f1d7">
<div class="box" style= "background-color:#e4f1d7">
=='''Sporo'''beads==
=='''Sporo'''beads==
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===Spore purification===
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===Crust Promoter Evaluation===
{| "width=100%" style="text-align:center;"|
{| "width=100%" style="text-align:center;"|
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|<p align="justify">We compared different ways of purifying ''Bacillus'' spores from vegetative cells</p>
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|<p align="justify">Three sporulation-dependent promoters (P<sub>''cotYZ''</sub>, P<sub>''cotV''</sub>, P<sub>''cgeA''</sub>) were constructed, two of which show the expected behavior.</p>
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|[[File:LMU Purification preview.tiff.png|200px|link=Team:LMU-Munich/Data/Sporepurification]]
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|[[File:Promotoren.png|200px|link=Team:LMU-Munich/Data/crustpromoters]]
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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Sporepurification]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/crustpromoters]]
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<div class="box">
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===GFP-'''Sporo'''bead Evaluation===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">They glow! GFP was successfully displayed on the spore crust as demonstrated by fluorescence microscopy.</p>
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|[[File:Sporobead Constructs Dada sheet preview.png|200px|link=Team:LMU-Munich/Data/gfp_spore]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/gfp_spore]]
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|}
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</div>
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<div class="box">
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==='''Sporo'''bead Purification===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">We compared different ways of purifying ''Bacillus'' spores from vegetative cells: Lysozyme is the best!</p>
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|[[File:Purification preview.png|200px|link=Team:LMU-Munich/Data/Sporepurification]]
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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Sporepurification]]
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|}
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</div>
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==='''Sporo'''beads===
 
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<p align="justify">To purify our spores from the vegetative cells we treated the samples, that were grown for 24 hours in DS-Medium, with three different methods. The data is summarized in the following table:</p>
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[[File:GerminationSTOP.png|90px|right|link=Team:LMU-Munich/Germination_Stop]]
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<br>
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<div class="box" style= "background-color:#e4f1d7">
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==='''Germination'''STOP===
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</div>
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{| class="colored" width="100%" align="center" style="text-align:center;"
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<div class="box">
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===Knockouts of germination genes ===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">We successfully prevented spore germination by multiple gene knockouts.</p>
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|[[File:LMU Knockout previewii.jpg|200px|link=Team:LMU-Munich/Data/Knockout]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Knockout]]
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<div class="box">
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==='''Suicide'''Switch ===
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!
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{| "width=100%" style="text-align:center;"|
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!all cells
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|<p align="justify">Death upon germination: We developed a novel safety strategy as a back-up to the gene knockouts.</p>
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!mature spores
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|[[File:PlatereaderSuicideSwitch1.jpg|200px|link=Team:LMU-Munich/Data/Suicideswitch]]
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!immature spores
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!untreated <br><font color="#EBFCE4" size="2"> wildtype </font>
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|7.29 ''x'' 10<sup>8</sup> /ml
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|1 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 13.71% </font>
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|0.04 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 0.55% </font>
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|-
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!untreated <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
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|6.79 ''x'' 10<sup>8</sup> /ml
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|1 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 14.72% </font>
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|0.13 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 1.9% </font>
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!French Press <br><font color="#EBFCE4" size="2">wildtype</font>
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|4.87 ''x'' 10<sup>8</sup> /ml
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|2.1 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 43% </font>
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|0.05 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 1% </font>
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|-
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!French Press <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
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|4.75 ''x'' 10<sup>8</sup> /ml
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|1.88 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 39.58% </font>
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|0.05 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 1% </font>
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!Sonification <br><font color="#EBFCE4" size="2">wildtype</font>
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|4.6 ''x'' 10<sup>8</sup> /ml
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|1.22 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 26.52% </font>
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|0.1 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 2% </font>
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!Sonification <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
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|6.72 ''x'' 10<sup>8</sup> /ml
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|1.53 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 22.77% </font>
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|0.23 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 3% </font>
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!Lysozyme <br><font color="#EBFCE4" size="2">wildtype</font>
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|2.48 ''x'' 10<sup>8</sup> /ml
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|1.58 ''x'' 10<sup>8</sup> /ml <br><font size="2"> 63.7% </font>
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|0 /ml
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!Lysozyme <font color="#EBFCE4" size="2">P<sub>''cotYZ''</sub>-''cotZre''-''gfp''-''terminator''</font>
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|1.05 ''x'' 10<sup>8</sup> /ml
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|1.05 ''x'' 10<sup>8</sup>/ml <br><font size="2"> 100% </font>
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|0 /ml
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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Suicideswitch]]
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</div>
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<p align="justify">Tab. 1 shows that after treatment with French Press and ultrasound the number of spores compared to the untreated samples was increased. We assume the reason for this was the impurity of these samples that derived from damaged vegetative cells. Thus, during counting it was not always possible to distinguish between mature spores and cell waste. However, a huge difference in number of vegetative cells and spores between untreated and lysozyme treated samples was noticeable under microscopy as it is visualized in the table above and the pictures below. Because the vegetative cells were lyzed and not damaged it was easy to recognize the mature spores.
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[[File:LMU-Munich-Invertersign.png|90px|right|link=Team:LMU-Munich/Inverter]]
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<div class="box" style= "background-color:#e4f1d7">
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===Inverter===
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<div class="box">
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===β-galactosidase assay of Inverter with ''lacZα'' ===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">It works! Invert the output of your promoter of choice.</p>
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|[[File:LMU Inverter graph.png|200px|link=Team:LMU-Munich/Data/Inverter]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Inverter]]
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|}
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</div>
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<div class="box" style= "background-color:#e4f1d7">
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==='''Trash'''can===
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Things that were a pain in the ''neck''!
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</div>
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<div class="box">
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===''cge''A and P<sub>''cge''A</sub>===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">
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Unexpected restriction sites combined with no activity at all - what a bummer!</p>
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|[[File:LMU cgea preview.jpg|200px|link=Team:LMU-Munich/Data/cgeA]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/cgeA]]
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|}
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</div>
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<div class="box">
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===MazF===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">
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The story of trying to clone a toxin from ''E. coli'' in ''E. coli'': They die, dammit! (...not a surprise, really...)
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</p>
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|[[File:MazFtrashcan.jpg|200px|link=Team:LMU-Munich/Data/MazF]]
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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/MazF]]
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|}
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</div>
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<div class="box">
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===Xylose promoters===
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">
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All constructs involving xylose-inducible promoters refused cloning or evaluation (sorry, Groningen...!).
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</p>
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|[[File:100px-D-Xylose Keilstrich.png|100px|link=Team:LMU-Munich/Data/Pxyl]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Pxyl]]
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<p></p>
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For our big breakthroughs, or to follow specific projects, see the individual project pages:
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====Project Navigation====
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{| width="100%" align="center" style="text-align:center;"
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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]
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|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]
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===Germination Stop===
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|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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We induced our germination-mutant strains to sporulate in Difco sporulation media. Then we measured the germination rate of mutant spores in a germination assay.
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|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]
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[[File:germination_assay_bahhumbug.jpg|Fig. 5: Comparison of colony growth on LB-agar (plus antibiotics) plates. Upper plates are plated with DSM cell/spore cultures that have been heated at 80°C for 1 hour to kill living cells (but does not affect spores). This prevents cells from forming colonies. Therefore, all colonies observed are from germinated spores. Lower plates contain cultures which have not been heated. Growth on these plates is from either cells or spores, and demonstrates that strains are able to grow. WT168 has no germination knockouts; strains B40-B43 have triple knockouts; strains B46 and B47 have quadruple knockouts; ''Spo0A'' has a knockout of the sporulation gene ''Spo0A'' (replaced with a tet cassette). WT168 is a positive control; ''Spo0A'' is a negative control. <br /> Strains: <br /> '''B40''' -- ''cwlD''::kan, ''sleB''::mls, ''cwlJ''::spec <br /> '''B41''' -- ''cwlD''::kan, ''sleB''::mls, ''gerD''::cat <br /> '''B42''' -- ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cat <br /> '''B43''' -- ''gerD''::cat, ''sleB''::mls, ''cwlJ''::spec <br /> '''B46''' -- ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cat, ''sleB''::mls <br /> '''B47''' -- ''gerD''::cat, ''sleb''::mls, ''cwlJ''::spec, ''cwlD''::kan|thumb|400px]]
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]
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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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The plate growth demonstrates the inability of our mutant spores to germinate. We can say that  fewer than 1 out of 3x10^7 spores of strains B40, B41, B43, B46, and B47 germinated.
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|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]
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|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]
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|}
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</div>
{{:Team:LMU-Munich/Templates/Page Footer}}
{{:Team:LMU-Munich/Templates/Page Footer}}

Latest revision as of 14:31, 22 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

Team-LMU Photo2.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/Data"