Team:LMU-Munich/Data/Suicideswitch

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Latest revision as of 13:41, 26 October 2012

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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Luminescence measurement of "Suicide switch-like" module

Luminescence measurement of the Bacillus subtilis strain W168 containing thrC::P_spoIVB-ecf41_{bli aa 1-204} (through transformation with pSB_Bs4S-P_spoIVB-ecf41_{bli aa 1-204}) and sacA::P_ydfG-luxABCDE (through transformation with pSB_Bs3C-luxABCDE-P_ydfG)

For this measurement, the Bacillus subtilis strain W168 B63 was used.
This puts the luxABCDE cassette in the place of the MazF later. Therefore, this output should reflect the expression of MazF in our Suicide switch.

σ^G, the last sigma factor in the forespore, activates P_spoIVB, which controls expression of ecf41_{bli aa 1-204} in our module. Ecf41_{bli aa 1-204} activates P_ydfG, and thereby the production of luciferase as a substitute of MazF.
This platereader measurement is the first and not reproduced to this point!
We get an expected activation of the module in late stationary phase and therefore during sporulation. It therefore seems to work.

But we also get a much too early peak of luminescence at about six hours during transition state, maybe due to unspecific (stress-induced?) activation. As the Ecf41_{bli aa 1-204} used is the truncated version which is constitutively "ON", this could lead to a high activation of the P_ydfG promoter.

This early peak, if it is activated in all cells and not a subset of the population, would probably be sufficient to kill the vegetative cells too early, before entering the sporulation cycle.

Though we will have to make this measurement reproducible, we are hoping that the other, weaker promoter P_sspK, which was not finished cloning, might be a better candidate. We hope that it will not have such an early peak and if so a much weaker one. But as the Ecf41_{bli aa 1-204} is so highly active it probably will still be sufficient to produce toxic levels of MazF.

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

@@ Line 2: / Line 2: @@
-===Platereader measurement of Suicide switch like module ===
+[[File:GerminationSTOP.png|100px|right|link=Team:LMU-Munich/Germination_Stop]]
-[[File:PlatereaderSuicideSwitch1.jpg|600px]]
-This module is the ''Bacillus subtilis'' strain W168 containing thrC::PspoIVB-ECF41 (through transformation with pSB<sub>Bs</sub>4S-PspoIVB-ECF41) and sacA::pydfG-luxABCDE (through transformation with pSB<sub>Bs</sub>3C-''luxABCDE''-PydfG). <br>
-This places the ''luxABCDE'' cassette in the place of the MazF later. So this output is the theoretical output of the MazF in our <b>Suicide</b> switch. <br>
+===Luminescence measurement of "<b>Suicide</b> switch-like" module ===
-Sigma G, the last sigma factor in the forespore, activates PspoIVB, producing ECF41. This then activates the PydfG promoter producing Lux ABCDE.<br>
-<b>This Platereader measurment is the first and not reproduced to this point!</b><br>
-In this platereader measurement we get a much too early peak of luminescence at about six hours, maybe due to unspecific small acitvation due to stress. As the ECF41 used is the short version which is constitutively on, this could lead to a high activation of the PydfG promoter. But there is also a significant activation at the point were sporulation should be in a late stage, though not finished.<br>
+{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
-The early peak, if it is activated in all cells and not a subset of cells, will probably be sufficient to kill the vegetative cells too early.<br>
+| style="width: 70%;background-color: #EBFCE4;" |
-Though we will have to make this measurement reproducible, we are hoping that the other, weaker promoter PsspK, which was not finished cloning, might be a better candidate.
+{|
+|[[File:PlatereaderSuicideSwitch1.jpg|600px|center]]
+|-
+| style="width: 70%;background-color: #EBFCE4;" |
+{| style="color:black;" cellpadding="0" width="100%" cellspacing="0" border="0" align="center" style="text-align:center;"
+|style="width: 70%;background-color: #EBFCE4;" |
+<font color="#000000"; size="2"><p align="justify"> Luminescence measurement of the ''Bacillus subtilis'' strain W168 containing ''thrC''::P<sub>''spoIVB''</sub>-''ecf41<sub>bli aa 1-204</sub>'' (through transformation with pSB<sub>Bs</sub>4S-P<sub>''spoIVB''</sub>-''ecf41<sub>bli aa 1-204</sub>'') and ''sacA''::P<sub>''ydfG''</sub>-''luxABCDE'' (through transformation with pSB<sub>Bs</sub>3C-''luxABCDE''-P<sub>''ydfG''</sub>) </p></font>
+|}
+|}
+|}
+<p align="justify">For this measurement, the ''Bacillus subtilis'' strain W168 [https://2012.igem.org/Team:LMU-Munich/Strains B63] was used. <br>
+This puts the ''luxABCDE'' cassette in the place of the MazF later. Therefore, this output should reflect the expression of MazF in our <b>Suicide</b> switch. </p>
+<p align="justify">σ<sup>G</sup>, the last sigma factor in the forespore, activates P<sub>''spoIVB''</sub>, which controls expression of ''ecf41<sub>bli aa 1-204</sub>'' in our module. Ecf41<sub>bli aa 1-204</sub> activates P<sub>''ydfG''</sub>, and thereby the production of luciferase as a substitute of MazF.<br>
+<b>This platereader measurement is the first and not reproduced to this point!</b><br>
+We get an expected activation of the module in late stationary phase and therefore during sporulation. It therefore seems to work.</p>
+<p align="justify">But we also get a much too early peak of luminescence at about six hours during transition state, maybe due to unspecific (stress-induced?) activation. As the Ecf41<sub>bli aa 1-204</sub> used is the truncated version which is constitutively "ON", this could lead to a high activation of the P<sub>''ydfG''</sub> promoter.</p>
+<p align="justify">This early peak, if it is activated in all cells and not a subset of the population, would probably be sufficient to kill the vegetative cells too early, before entering the sporulation cycle.</p>
+<p align="justify">Though we will have to make this measurement reproducible, we are hoping that the other, weaker promoter P<sub>''sspK''</sub>, which was not finished cloning, might be a better candidate. We hope that it will not have such an early peak and if so a much weaker one. But as the Ecf41<sub>bli aa 1-204</sub> is so highly active it probably will still be sufficient to produce toxic levels of MazF.</p>