Team:LMU-Munich/Cut'N'survive/Functional Principle

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== TEVdegron-System ==
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== Cut'N'Survive-System ==
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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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The Cut'N'Survive-System combines several proteins with specific properties to select eukaryotic cells incorporating the target plasmid by apoptosis. Two DNA constructs are needed and one of them (construct 1) should be integrated in the cell's genome.  
=== Construct 1 ===
=== Construct 1 ===
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Construct 1 contains the following parts in this order:
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Construct 1 contains the following parts in the following order:
Tetracyclin-inducible promoter, TEV-recognition site, N-degron, SF3b155, human bak and SV40 Polyadenylation site.
Tetracyclin-inducible promoter, TEV-recognition site, N-degron, SF3b155, human bak and SV40 Polyadenylation site.
[[Image:TEV11.jpg|400pxs|TEV Construct 1: Inserted in celline]]
[[Image:TEV11.jpg|400pxs|TEV Construct 1: Inserted in celline]]
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To check the integration of this construct into cellular DNA we do a double-transfection with a hygromycin resistence. 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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To select cells with this construct integrated into the cellular genome we perform a co-transfection with hygromycine resistence plasmid. The TEV-recognition site will be cut by the TEV-protease which is part of construct 2. Due to the digestion at the TEV-recognition site, the N-terminus of N-degron is free and thus provides a signal for the degeneration of the protein. SF3b155 is a protein interacting with p14* from construct 2. This interaction ascertains that the TEV-protease of construct 2 be brought to the recognition site. The human bak is an efficient apoptosis inducing membrane protein.
=== Construct 2 ===
=== Construct 2 ===
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[[Image:TEV12.jpg|400pxs|TEV Construct 2: Contains gene of interest]]
[[Image:TEV12.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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The CMV-promoter is for the expression of construct 2. The task of TEV-protease is to cut TEV-recognition sites, both the one upstream of N-degron to trigger protein degradation and the one upstream of the target gene (eGFP in this case) to ensure that the wanted product is not degraded. p14* is interacting with SF3b155 and so increases the rate of specific digestion at the TEV-recognition site by the TEV-proteases. eGFP is example of a gene of interest which can conveniently be verified by green fluorescence.
=== Selection ===
=== 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 cellline which is stably integrated with construct 1 and is transfected with construct 2 can contain the following two kinds of cells:
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a) the plasmid has not been incorporated
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a) cells not incorporated with construct 2
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b) the plasmid has been incorporated
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b) cells incorporated with construct 2
These lead to the following consequences.
These lead to the following consequences.
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==== Case a) the plasmid has not been incorporated ====
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==== Case a) cells not incorporated with construct 2====
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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.
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After induction of the tet-on promoter construct 1 will be translated into protein. The bak-protein will integrate into mitochondrial membrane and as a result induces apoptosis.
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==== Case b) the plasmid has been incorporated ====
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==== Case b) cells incorporated with construct 2 ====
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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.
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Construct 2 will be read off and after introduction of tetracycline the tet-on promoter will trigger the translation of construct 1 into protein. However in this case the TEV proteases will instantly separate eGFP from the rest of the protein as well as free the N-terminus of N-degron. This will cause the degradation of the whole protein complex - including bak. Therefore the cell will survive and only the protein product of the gene of interest will be left.
[[Image:TEV14.jpg|600pxs|TEVdegron-System: TEV-degron-System]]
[[Image:TEV14.jpg|600pxs|TEVdegron-System: TEV-degron-System]]

Revision as of 12:25, 27 August 2010


Contents

Cut'N'Survive-System

The Cut'N'Survive-System combines several proteins with specific properties to select eukaryotic cells incorporating the target plasmid by apoptosis. Two DNA constructs are needed and one of them (construct 1) should be integrated in the cell's genome.

Construct 1

Construct 1 contains the following parts in the following order: Tetracyclin-inducible promoter, TEV-recognition site, N-degron, SF3b155, human bak and SV40 Polyadenylation site.

TEV Construct 1: Inserted in celline

To select cells with this construct integrated into the cellular genome we perform a co-transfection with hygromycine resistence plasmid. The TEV-recognition site will be cut by the TEV-protease which is part of construct 2. Due to the digestion at the TEV-recognition site, the N-terminus of N-degron is free and thus provides a signal for the degeneration of the protein. SF3b155 is a protein interacting with p14* from construct 2. This interaction ascertains that the TEV-protease of construct 2 be brought to the recognition site. The human bak is an efficient apoptosis inducing membrane protein.

Construct 2

Construct 2 is composed of CMV-promoter, TEV-protease, p14, TEV-recognition site, eGFP, a double stop codon and SV40PA.

TEV Construct 2: Contains gene of interest

The CMV-promoter is for the expression of construct 2. The task of TEV-protease is to cut TEV-recognition sites, both the one upstream of N-degron to trigger protein degradation and the one upstream of the target gene (eGFP in this case) to ensure that the wanted product is not degraded. p14* is interacting with SF3b155 and so increases the rate of specific digestion at the TEV-recognition site by the TEV-proteases. eGFP is example of a gene of interest which can conveniently be verified by green fluorescence.

Selection

A cellline which is stably integrated with construct 1 and is transfected with construct 2 can contain the following two kinds of cells:


a) cells not incorporated with construct 2

b) cells incorporated with construct 2

These lead to the following consequences.

Case a) cells not incorporated with construct 2

After induction of the tet-on promoter construct 1 will be translated into protein. The bak-protein will integrate into mitochondrial membrane and as a result induces apoptosis.


Case b) cells incorporated with construct 2

Construct 2 will be read off and after introduction of tetracycline the tet-on promoter will trigger the translation of construct 1 into protein. However in this case the TEV proteases will instantly separate eGFP from the rest of the protein as well as free the N-terminus of N-degron. This will cause the degradation of the whole protein complex - including bak. Therefore the cell will survive and only the protein product of the gene of interest will be left.

TEVdegron-System: TEV-degron-System