Team:LMU-Munich/Jump-or-die/Functional Principle

Jump-or-die-System

The jump-or-die-system selects the itegration of a gene of interest by apoptosis. Therefore three constructs ae needed.

Construct 1

Construct 1 contains a tet-on promoter, bacterial attachment site (attB), human bak (bak) and SV40 polyadenylation site (SV40PA). It has to be integrated to the genom of the cellline. The selection if this construct is integrated will be made by double transfection with hygromycin resistance and tet-on-inducible apoptosis.

Construct 2

Construct 2 will be inserted in a vector and consist of the following parts: phage attachment site (attP), eGFP (our gene of interest), doubled stop codon and SV40PA.

Construct 3

This Biobrick is containing CMV-promoter, PhiC31o (mouse-codon optimized integrase) and SV40PA. It will be transfected to the cellline and read-off so that PhiC31o will be existing.

Transfection possibilities

A cellline which has integrated construct 1 into genom will be transfected with construct 2 and 3 at the same time. Construct 3 will be read-off instantly, in contrast to construct 1 which has to be induced by tetracycline and because of the lack of a promoter, construct 2 won't be translated.

This transfection has several outgoings:

a) none of the plasmids entered the cell

b) only construct 2 entered the cell

c) only construct 3 entered the cell

d) construct 2 and 3 entered the cell

a) None of the plasmids entered the cell

If none of the plamids will be tranfsected and the tet-on.promoter will be induced, bak will be translated, insert to mitochandrial membrane and therefore induce apoptosis.

b) Only construct 2 entered the cell

Induction of tet-on promoter will cause translation of bak and so induce apoptosis. The gene of interest will not be read off, because there is no promoter in construct 2.

c) only construct 3 entered the cell

PhiC31o will emerge because of CMV promoter. But after tet-on induction bak will also be read-off and therefore cause cell death.

d) construct 2 and 3 entered the cell

PhiC31o will be read off because of CMV promoter. Now the integrase will combine attB and attP site and integrate construct 2 into genom. Up next, the gene of interest is behind tet-on promoter and will be read off after induction. The doubled stop codon will efficiently prevent the tranlation of bak. This cell will express the gene of interest and survive.