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Team:LMU-Munich/TEV-System/Functional Principle
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| - | + | == TEVdegron-System == | |
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| - | The TEV-protease is | + | The TEVdegron-System uses and combines several proteins with different properties to select the incorporation of a plasmid by apoptosis. We need two DNA constructs and one of them (construct 1) should be integrated in the cell's DNA. |
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| - | [[Image: | + | === Construct 1 === |
| - | [[Image: | + | |
| + | Construct 1 contains the following parts in this order: | ||
| + | CMV-promoter for the hygromycin resistance, hygromycin resistance, tetracyclin-inducible promoter, TEV-recognition site, N-degron, SF3b155, human bak. | ||
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| + | [[Image:TEV1.jpg|400pxs|TEV Construct 1: Inserted in celline]] | ||
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| + | To check the integration of this construct into cellular DNA we need the hygromycin resistance which can be read-off thanks to the CMV-promoter. The TEV-recognition site will be cut by the TEV-protease which is part of construct 2. Due to the scission of TEV-recognition site the N-terminus of N-degron is free which is a signal for the degeneration of this protein. SF3b155 is a protein interacting with p14 from construct 2. This interaction should make sure that the TEV-protease of construct 2 will really "find" the recognition site. The human bak is a apoptosis triggering membrane protein. | ||
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| + | === Construct 2 === | ||
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| + | Construct 2 is composed of CMV-promoter, TEV-protease, p14, TEV-recognition site, eGFP and a double stop codon. | ||
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| + | [[Image:TEV2.jpg|400pxs|TEV Construct 2: Contains gene of interest]] | ||
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| + | The CMV-promoter is to read off construct 2. The task of TEV-protease is to cut TEV-recognition sites, especially the one ahead of N-degron to signal protein degradation. p14 is interacting with SF3b155 and so increases the rate of finding the TEV-recognition site by the TEV-proteases. eGFP is our example of a gene of interest which can be verified by green flourescence. | ||
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| + | === Selection === | ||
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| + | A cellline which has integrated construct 1 and is transfected with construct 2 leads to two outgoings: | ||
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| + | a) the plasmid has not been incorporated | ||
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| + | b) the plasmid has been incorporated | ||
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| + | These lead to the following consequences. | ||
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| + | ==== Case a) the plasmid has not been incorporated ==== | ||
| + | After induction of the tet-on promoter construct 1 will be translated into protein. The bak-part will integrate into mitochondrial membrane and as a result will induce apoptosis. | ||
| + | |||
| + | |||
| + | ==== Case b) the plasmid has been incorporated ==== | ||
| + | Construct 2 will be read off and already existing when tet-on promoter will be induced to create construct 1 as protein. Therefore TEV proteases will instantly seperate eGFP from the remain of the protein and will free the N-terminus of N-degron. This will cause the degradation of the whole protein complex - including bak. So the cell will survive and only the gene of interest will be left. | ||
| + | |||
| + | [[Image:TEV4.jpg|400pxs|TEVdegron-System: TEV-degron-System]] | ||
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| + | |||
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{{:Team:LMU-Munich/Templates/Page Footer}} | {{:Team:LMU-Munich/Templates/Page Footer}} | ||
Latest revision as of 10:29, 16 August 2010
The TEVdegron-System uses and combines several proteins with different properties to select the incorporation of a plasmid by apoptosis. We need two DNA constructs and one of them (construct 1) should be integrated in the cell's DNA.
Construct 1 contains the following parts in this order:
CMV-promoter for the hygromycin resistance, hygromycin resistance, tetracyclin-inducible promoter, TEV-recognition site, N-degron, SF3b155, human bak.
To check the integration of this construct into cellular DNA we need the hygromycin resistance which can be read-off thanks to the CMV-promoter. The TEV-recognition site will be cut by the TEV-protease which is part of construct 2. Due to the scission of TEV-recognition site the N-terminus of N-degron is free which is a signal for the degeneration of this protein. SF3b155 is a protein interacting with p14 from construct 2. This interaction should make sure that the TEV-protease of construct 2 will really "find" the recognition site. The human bak is a apoptosis triggering membrane protein.
Construct 2 is composed of CMV-promoter, TEV-protease, p14, TEV-recognition site, eGFP and a double stop codon.
The CMV-promoter is to read off construct 2. The task of TEV-protease is to cut TEV-recognition sites, especially the one ahead of N-degron to signal protein degradation. p14 is interacting with SF3b155 and so increases the rate of finding the TEV-recognition site by the TEV-proteases. eGFP is our example of a gene of interest which can be verified by green flourescence.
A cellline which has integrated construct 1 and is transfected with construct 2 leads to two outgoings:
b) the plasmid has been incorporated
These lead to the following consequences.
After induction of the tet-on promoter construct 1 will be translated into protein. The bak-part will integrate into mitochondrial membrane and as a result will induce apoptosis.
Construct 2 will be read off and already existing when tet-on promoter will be induced to create construct 1 as protein. Therefore TEV proteases will instantly seperate eGFP from the remain of the protein and will free the N-terminus of N-degron. This will cause the degradation of the whole protein complex - including bak. So the cell will survive and only the gene of interest will be left.
Contents
TEVdegron-System
Construct 1
Construct 2
Selection
a) the plasmid has not been incorporated
Case a) the plasmid has not been incorporated
Case b) the plasmid has been incorporated
