Team:LMU-Munich/TEV-System/Functional Principle

From 2010.igem.org

Revision as of 10:16, 16 August 2010 by Jara (Talk | contribs)


Contents

TEVdegron-System

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

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.

TEV Construct 1: Inserted in celline

To check the integration of tis 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 serration 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

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

TEV Construct 2: Contains gene of interest

The CMV-promoter is to read off construct 2. The task of TEV-protease is to cut TEV-recognition sites, espacially the one ahead of N-degron to signal protein degradation. p14 is interacting with SF3b155 and so increases the rate of TEV-proteases which "find" a TEV-recognition site. eGFP is our example of a gene of interest which can be verified by green flourescence.

Selection

So you have a cellline which has integrated construct 1 and transfect it with a plasmid containing construct 2. Then there are two possibilities:

a) the plasmid has not been incorporated

b) the plasmid has been incorporated

which leads to the following consequences.

Case a) the plasmid has not been incorporated

We induce the tet-on promoter and so 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 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 an will free the N-terminus of N-degron. This will cause the degradation of the whole protein complex - including bak. So only the gene of interest and the whole cell will "survive".

TEVdegron-System: TEV-degron-System


So you can see that cells that incorporated the plasmid will survive.