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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On this page, we will upload data to explain our project progress. More to come!!!
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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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Or see the results pages of our projects to follow our progress:
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==='''''Bacillus''B'''io'''B'''rick'''B'''ox===
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{|
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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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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Vectors]]
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|}
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</div>
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<div class="box">
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====Anderson Promoter Evaluation====
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">Eleven Anderson promoters were measured in ''B. subtilis'' and showed relatively weak activity.</p>
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|[[File:LMU Anderson preview.jpg|200px|link=Team:LMU-Munich/Data/Anderson]]
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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Anderson]]
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|}
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</div>
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<div class="box">
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====Constitutive ''Bacillus'' Promoters====
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{| "width=100%" style="text-align:center;"|
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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>
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|[[File:LMU Constitutive preview.jpg|200px|link=Team:LMU-Munich/Data/Constitutive]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Constitutive]]
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<div class="box">
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====Inducible ''Bacillus'' Promoters====
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{| "width=100%" style="text-align:center;"|
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|<p align="justify">A novel antibiotic-inducible promoter, P<sub>''liaI''</sub>, was constructed and thoroughly evaluated.</p>
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|[[File:Englisch Auswertung PliaI.png|200px|link=Team:LMU-Munich/Data/Inducible]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Inducible]]
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<html>
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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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<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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[[File:SporeCoat.png|90px|right|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks">
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<br>
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<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=70"/>
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<div class="box" style= "background-color:#e4f1d7">
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</a>
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=='''Sporo'''beads==
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<font size="1">
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</div>
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</font>
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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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|<html>
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<div class="box">
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<a href="https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins">
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===Crust Promoter Evaluation===
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<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=70"/>
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{| "width=100%" style="text-align:center;"|
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</a>
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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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<font size="1">
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|[[File:Promotoren.png|200px|link=Team:LMU-Munich/Data/crustpromoters]]
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</font>
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/crustpromoters]]
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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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|}
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</div>
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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/Germination_Stop">
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===GFP-'''Sporo'''bead Evaluation===
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=70"/>
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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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<font size="1">
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|[[File:Sporobead Constructs Dada sheet preview.png|200px|link=Team:LMU-Munich/Data/gfp_spore]]
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<br />
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/gfp_spore]]
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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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</div>
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===Bacillus BioBrick Box - Promoters===
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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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! 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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[[File:Auswertung Anderson promoters.png|thumb|right|400px|Fig. 1: luminescence measurement of Anderson promoters in the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE''. OD<sub>''600''</sub> (right), LUMI (center) and OD<sub>''600''</sub> per LUMI (left) depending on the time (h)are shown for two different clones (green/blue). Data derive from three independant experiments. Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smothed by taking average of three neighbouring values.]]
 
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Eleven (J23100,J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118) of the nineteen promoters of the Anderson collection were evaluated in the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE'' from the BioBrickBox containing the lux operon as a reporter for promoter activity. The gene expression which correlates to the promoter activity leads to the expression of the lux operon with the luciferase. The luminescence which is produced by the luciferase can be measured with the plate reader (BioTek).
 
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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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<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>
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<div class="box">
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==='''Suicide'''Switch ===
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{| "width=100%" style="text-align:center;"|
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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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|[[File:PlatereaderSuicideSwitch1.jpg|200px|link=Team:LMU-Munich/Data/Suicideswitch]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Suicideswitch]]
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|}
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</div>
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To measure the activity not only with the lux reporter operon, four promoters were cloned into the reporter vector pSBBs1C-lacZ to do beta-galactosidase assays and then to compare the results of the strength of these promoters in B. subtilis.  
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[[File:LMU-Munich-Invertersign.png|90px|right|link=Team:LMU-Munich/Inverter]]
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<br>
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<div class="box" style= "background-color:#e4f1d7">
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===Inverter===
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</div>
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[[File:Auswertung_plate_reader_andere_promotoren.png|thumb|right|400px|Fig. 2: luminescence measurement of the constitutive ''Bacillus'' promoters P<sub>''liaG''</sub> and P<sub>''lepA''</sub> in the reporter vector pSB<sub>''Bs''</sub>3C-''luxABCDE''. OD<sub>''600''</sub> (right), LUMI (center) and LUMI per OD<sub>''600''</sub>  (left) depending on the time (h) are shown for two different clones (green/blue). Data come from three independant experiments. Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smothed by taking average of three neighbouring values.]]
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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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The constitutive promoters P<sub>''liaG''</sub> and P<sub>''lepA''</sub> were evaluated in the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> which contains the ''lux'' operon.
 
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[[File:Englisch_Auswertung_PliaG_Pveg.png‎|thumb|right|400px|Fig. 3: β-galactosidase assay and growth curve P<sub>''liaG''</sub> (black) and P<sub>''veg''</sub> (grey) in the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ''. β-galactosidase activity (Miller Units)and growth curve are the average of two independant clones. Experiment shows representative data which was recieved in the same way from three independant experiments.]]
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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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[[File:plate reader induzierbar promotor.png‎|thumb|right|400px|Fig. 4: ]]
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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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! 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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[[File:Englisch Auswertung PliaI.png‎|thumb|right|400px|Fig. 4: ]]
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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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|}
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</div>
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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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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]
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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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|[[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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|[[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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===Germination Stop===
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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:germination_assay_plates_720_.jpg|frame|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''::mla, ''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]]
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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/Templates/Page Footer}}
{{:Team:LMU-Munich/Templates/Page Footer}}

Latest revision as of 14:31, 22 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".

IGEM HQ LMU prize.jpg

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