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Team:LMU-Munich/Data/Suicideswitch
From 2012.igem.org
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| - | for the measurement 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>) was used. <br> | + | <p align="justify">for the measurement 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>) was used. <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. < | + | 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. </p> |
| - | σ G, 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> | + | <p align="justify">σ G, 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> | <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.< | + | We get an expected activation of the module in late stationary phase and therefore during Sporulation. It therefore seems to work.</p> |
| - | But we also get a much too early peak of luminescence at about six hours, maybe due to unspecific small activation due to stress. 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.<br> | + | <p align="justify">But we also get a much too early peak of luminescence at about six hours, maybe due to unspecific small activation due to stress. 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.<br> |
| - | 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.< | + | 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.</p> |
| - | 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. 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 align="justify">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. 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> |
Revision as of 18:01, 26 September 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".
[ more news ]
Luminescence measurement of Suicide switch like module
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for the measurement the Bacillus subtilis strain W168 containing thrC::PspoIVB-ecf41bli aa 1-204 (through transformation with pSBBs4S-PspoIVB-ecf41bli aa 1-204) and sacA::PydfG-luxABCDE (through transformation with pSBBs3C-luxABCDE-PydfG) was used.
This places the luxABCDE cassette in the place of the MazF later. So this output is the theoretical output of the MazF in our Suicide switch.
σ G, the last sigma factor in the forespore, activates PspoIVB, which controls expression of ecf41bli aa 1-204 in our module. ecf41bli aa 1-204 activates PydfG, 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, maybe due to unspecific small activation due to stress. As the ecf41bli aa 1-204 used is the truncated version which is constitutively on, this could lead to a high activation of the PydfG promoter.
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.
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. We hope that it will not have such an early peak and if so a much weaker one. But as the ecf41bli aa 1-204 is so highly active it probably will still be sufficient to produce toxic levels of MazF.
