Team:LMU-Munich/Data

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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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{|
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<html>
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction">
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<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=70"/>
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</a>
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<font size="1">
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</font>
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<br />
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction"><font size="1" face="verdana">Bacillus<BR>Intro</font></a>
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|<html>
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</div>
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks">
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<div class="box">
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<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=70"/>
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====Evaluation of ''B. subtilis'' vectors====
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</a>
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{| "width=100%" style="text-align:center;"|
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<font size="1">
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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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</font>
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|[[File:LacZ plate.png|right|100px|link=Team:LMU-Munich/Data/Vectors]]
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<br />
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|-
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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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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Vectors]]
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</html>
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|<html>
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<a href="https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins">
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<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=70"/>
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</a>
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<font size="1">
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</font>
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<br />
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<a href="https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins"><font size="1" face="verdana">SporeCoat<BR>FusionProteins</font></a>
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</html>
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|<html>
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<a href="https://2012.igem.org/Team:LMU-Munich/Germination_Stop">
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=70"/>
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</a>
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<font size="1">
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</font>
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<br />
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<a href="https://2012.igem.org/Team:LMU-Munich/Germination_Stop"><font size="1" face="verdana">Germination<BR>STOP</font></a>
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</html>
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|}
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<div class="box" style= "background-color:#e4f1d7">
 
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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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</div>
</div>
<div class="box">
<div class="box">
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====Constitutive ''Bacillus" Promoters====
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====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'' (luminescence) 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>
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|[[File:LMU Anderson preview.jpg|200px|link=Team:LMU-Munich/Data/Constitutive]]
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|[[File:LMU Constitutive preview.jpg|200px|link=Team:LMU-Munich/Data/Constitutive]]
|-
|-
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Constitutive]]
! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Constitutive]]
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|}
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</div>
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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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|-
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Inducible]]
|}
|}
</div>
</div>
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===Bacillus BioBrick Box - Promoters===
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[[File:SporeCoat.png|90px|right|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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<p align="justify">
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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 independent experiments. Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smoothed by taking average of three neighboring values.]]
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</p>
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<p align="justify">
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Eleven of the nineteen promoters of the [http://partsregistry.org/Part:BBa_J23100 '''Anderson collection'''] (J23100,J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118) 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). Data derive from three undependant measurements ('''Fig. 1'''). Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smoothed by taking average of three neighboring values. OD<sub>600</sub> values shown are plate reader units and about one third of the usual OD<sub>600</sub> values. All clones show a usual growth curves. The activity of the promoters raises during the pass from the transition to the stationary phase. This maximum (t=1h) reaches from 200Lumi/OD<sub>600</sub> (promoter J23115) to a maximum of 1500 Lumi/OD<sub>600</sub> for the strongest promoter (J23101). Afterwards the activity goes down to the beginning level (t=2h). The oscillation of luminescence (Lumi/ OD<sub>600</sub> in the beginning of the curves are due to the small OD<sub>600</sub> and do not mean a high promoter activity. The luminescence of one clone of the promoters J23107 and J23114 do not show activity where in future a second clone with promoter activity should be measured. In comparison to all the other evaluated ''Bacillus'' promoters these Anderson promoters showed a very low acitivity in ''B. subtilis''.</p>
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<p align="justify">To measure the activity not only with the ''lux'' reporter operon, four promoters of the Anderson collection were cloned into the reporter vector pSB<sub>''Bs''</sub>1C-''lacZ'' to do β galactosidase assays and then to compare the results of the strength of these promoters in ''B. subtilis''. ('''Fig. 2''')</p>
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<p align="justify">[[File:Auswertung_plate_reader_andere_promotoren.png|thumb|right|400px|'''Fig. 3: 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> (up), LUMI (center) and LUMI per OD<sub>''600''</sub> (down) depending on the time (h) are shown for two different clones (green/blue). Data come from three independent experiments. Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smoothed by taking average of three neighboring values.]]
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<br>
<br>
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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. Data derive from three undependant measurements ('''Fig. 3'''). Curves were fitted over each other (t=0, OD<sub>''600''</sub>=0,3) and smoothed by taking average of three neighboring values. OD<sub>600</sub> values shown are plate reader units and about one third of the usual OD values. All clones show a usual growth curves. The activity of the promoters raises during the pass from the transition to the stationary phase. The second clone of the promoters P<sub>''lepA''</sub> and P<sub>''liaG''</sub> did not show any luminescence activity. In the beginning of the growth curve the activity of both promoters raise to their maximum. They show a similar behaviour in pertaining to the groth curve. P<sub>''liaG''</sub> has an activity maximum of about 100.000 Lumi/OD<sub>600</sub> during the pass from logarithmic to the stationary phase. P<sub>''lepA''</sub> shows an maximum of about 400.000 Lumi/OD<sub>600</sub>. Comparing these two constitutive promoters the activity of P<sub>''lepA''</sub> is about four times higher than the activity of P<sub>''liaG''</sub>. In the late stationary phase the activity completely disappears.</p>
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<div class="box" style= "background-color:#e4f1d7">
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=='''Sporo'''beads==
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</div>
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<div class="box">
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===Crust Promoter Evaluation===
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{| "width=100%" style="text-align:center;"|
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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: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/crustpromoters]]
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|}
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</div>
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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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|-
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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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<p align="justify">[[File:Englisch_Auswertung_PliaG_Pveg.png‎|thumb|center|400px|'''Fig. 4: 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 obtained in the same way from three independent experiments.]]</p>
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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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<p align="justify">The β galactosidase assay of the constitutive ''Bacillus'' promoters Pveg and P<sub>''liaG''</sub> was repeated three times. Data show one representative result. Two undependant clones of ''B. subtilis'' with the same construct were measured and their mean with standard deviation is show in the graph. In the beginning of the growth curve both promoters show a small activity. But then it raises to a maximum before it decreases to the begininng level after about seven hours (Data not shown). Summing up the course of activity of both promoters P<sub>''veg''</sub> and P<sub>''liaG''</sub> is very similar based on the growth curve. The highest beta galactosidase activity and therefore the highest activity of the promoter Pveg can with an maximum of 65 Miller units be found during the transfer from the logarithmic to the stationary phase. This is about five times higher than the acitivity of the promoter P<sub>''liaG''</sub> with an maximum activity of about 12 Miller Units.
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[[File:GerminationSTOP.png|90px|right|link=Team:LMU-Munich/Germination_Stop]]
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</p>
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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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[[File:plate reader induzierbar promotor.png‎|thumb|center|400px|Fig. 5: ]]
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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:Englisch Auswertung PliaI.png‎|thumb|center|400px|Fig. 6: ]]
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|[[File:LMU Knockout previewii.jpg|200px|link=Team:LMU-Munich/Data/Knockout]]
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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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{| class="colored" width="100%" align="center" style="text-align:center;"
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|-
|-
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|style="width:35%;"|
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Knockout]]
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|style="width:20%;"|
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|}
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|style="width:20%;"|
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</div>
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|style="width:20%;"|
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<div class="box">
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|-
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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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|-
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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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|-
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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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|-
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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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|-
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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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|-
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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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|-
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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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<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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<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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|-
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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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|-
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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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|-
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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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<div class="box">
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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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|[[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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|-
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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_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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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".

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