Team:LMU-Munich/Cut'N'survive

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Cut'N'survive System

In this system, the apoptosis triggering protein Bak is controlled by the tet-on system for inducible apoptosis. Bak can be degraded when the target gene is successfully expressed, therefore ensuring the survival of the wished cells. Bak is combined with a certain interacting protein p17*, TEV-protease recognition site and N-Degron. This construct should be stably integrated into the cell line genome. The target plasmid consists of eGFP as target gene, another TEV-protease recognition site, the interaction partner of p17* and TEV-protease. When the target plasmid is expressed, the interaction proteins bring the TEV-protease together with its recognition site; TEV cuts Bak, together with N-Degron, free from the rest of the protein complex, where N-Degron with its free N-terminal acts as degradation signal of protease in the cells. Thus this system enables selection of cells expressing the target gene while the cells not transfected undergo induced apoptosis.

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 ==Cut'N'survive System==
-The Cut'N'survive System conists of two multi-protein-complexes. The DNA of the first one that you can see below shall integrate into the genom of a HeLa-cell line. The DNA of the second one will be added by a plsmid transfection. At the site where you can see the eGFP, other scientists shuold be able to integrate their gene of interest.
+In this system, the apoptosis triggering protein Bak is controlled by the tet-on system for inducible apoptosis. Bak can be degraded when the target gene is successfully expressed, therefore ensuring the survival of the wished cells. Bak is combined with a certain interacting protein p17*, TEV-protease recognition site and N-Degron. This construct should be stably integrated into the cell line genome. The target plasmid consists of eGFP as target gene, another TEV-protease recognition site, the interaction partner of p17* and TEV-protease. When the target plasmid is expressed, the interaction proteins bring the TEV-protease together with its recognition site; TEV cuts Bak, together with N-Degron, free from the rest of the protein complex, where N-Degron with its free N-terminal acts as degradation signal of protease in the cells. Thus this system enables selection of cells expressing the target gene while the cells not transfected undergo induced apoptosis.
-...to be continued
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