Team:LMU-Munich/Data/Suicideswitch

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Luminescence measurement of "Suicide switch-like" module

Luminescence measurement of 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)

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.