Team:TU-Munich/Project/Killswitch

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A novel mechanism preventing uncontrolled spread of transgenic plants

Background – Why generate a plant which kills itself under certain conditions?

Working on plants is uncomplicated since as photoautotrophic organisms they can provide their own energy. So creating a photosensitive plant might seem silly at first glance. "Crazy, stupid Germans!", you might think, but wait, there's more! Green biotechnology doesnt have an easy stand in Germany since the "German Angst" of uncontrolled spreading of transgenic plants. Therefore, we see it as our task and duty to meet the required high safety standards that minimize these risks for a maximum of biosafety. We created a plant that can only survive in a well defined environment. Plants do not neccessarily need the whole spectrum of light to supply themselves with energy, so reassigning part of the spectrum to other purposes is possible. Shielded from red light by filters, the moss survives without compromising vitality or growth. Unintended release of our protected environment leads to activation of a lethal process of no return and thus kills the moss.

Design of a red light triggered nuclease system for self-destruction

Choosing the effector that triggers cell-death quickly and safely

Programmed Cell-Death (PCD) is a endogenous mechanism of the cell itself for a regulated suicide. It is normally carried out e. g. upon tissue differentiation or due to a infection with a pathogen to prevent its propagiation.

Micococcal nuclease (also called: Thermonuclease, MNase) is a non-specific endo-exonuclease from Staphylococcus aureus which shows activy in digesting single-stranded DNA/RNA and also double-stranded DNA/RNA.

Nuclear Localization Signal/Sequence (NLS) is a short amino acid sequence of a nuclear targeted protein that is recognized by nuclear transporters, the so-called importins, and then is actively transported in the nucleus. NLS from SV40 large T antigen is mostly used to mark synthetic proteins for nuclear localization.

In plants, the complete regulation and mechanism of programmed cell-death (abbrev.: PCD) is still not fully understood, but it is known that as in all other eukaryotic organisms, fragmentation of the genomic DNA inevitably leads to PCD.

We chose the Micrococcal nuclease (BBa_K1159105) as effector to destroy the integrity of the genomic DNA. Since the genome is protected by the nuclear envelope, it was necessary to tag the nuclease with a Nuclear Localization Signal (NLS) (BBa_K1159109). Nucleases show their death-bringing activity already at low concentrations. Thus it was essential to put the kill-switch under tight spatiotemporal control and only induce activity under specific conditions.

Preventing the nuclease to develop its deadly potential under well-defined conditions

Transmembrane Domain (TMD) is the region of a membrane protein which traverses the plasma membrane.

TEV Protease is a site-specific cystein protease from the C4 peptidase family from Tabacco Etch Virus (TEV). Its typical recognition site is ENLYFQ(G/S).

Our solution is to anchor the nuclease in the cytoplasmatic membrane under basal conditions. In its membrane-associated form (BBa_K1159111), the nuclease cannot evolve its deadly potential as it is spatially separated from most genetic material. The reason is that the genomic DNA of eukaryotic organisms is subcellular located in the nucleus, sheltered by the nuclear envelope. By anchoring the nuclease in the cell membrane, the nuclease is spatially seperated from its site of action.

To make the nuclease releasable, we have design a long linker with a TEV recognition/cleavage site between the transmemmbrane domain (TMD) and NLS-tagged nuclease. In the presence of TEV protease activity, the nuclease is liberated from the membrane and translocates into the nucleus due to its NLS tag. There, it can fulfill its task as the executor of PCD by degrading the DNA with its endo/exonuclease activity.

The question is how to enable the lethal activity of the nuclease upon red light illumination? The first component consists a fusion protein composed of a signal peptide plus transmembrane region for membrane localization, a TEV cleavage site for cleaving off the nuclease upon red light exposition and nuclear localizaton signal plus the nuclease itself as the death bringing part.

Red light triggered reconstitution of split TEV Protease

Split TEV Protease is a split version of the TEV Protease which N- and C-terminal split parts shows no enzymatic activity. Bringing them to physical proximity leads to reconstitution of the cleavage activity.

Phytochrome B (PhyB) is a plant photoreceptor that exists in two interconvertible forms P_r and P_fr. In its P_fr (far red light sensititve) form upon red light illumination, PhyB interacts with Phytochrome Interacting Factors, e. g. PIF3 or PIF6.

Phytochrome Interacting Factor 3/6 (PIF3/PIF6) are both transcription factors with basic helix-loop-helix (bHLH) motifs that that bind to PhyB upon red light exposition

As mentioned, the nuclease is normally localized at the cytoplasmatic membrane and by cleavage through a TEV protease, the NLS-tagged nuclease is released from its cage and goes directly in the nucleus.

To liberate the nuclease after exposition to red light, we designed two fusion proteins; each of them contains either the N-terminal or the C-terminal splitted part of the TEV protease. The splitted TEV protease is not active as long as the catalytic residues are seperated. The first fusion protein has PhyB as its fusion partner which heterodimerizes with PIF3/PIF6, the fusion partner of the second fusion protein. Red light induces heterodimerization of PhyB with PIF3/PIF6 and thus reconstitutes the N- and C-terminal half of the TEV Protease resulting in a proteolytic activity for a specific TEV recognition site.

The reconstituted TEV Protease libarates the nuclease from its membrane anchor by cleaving it off from the transmembrane domain. The nuclear localization signal ensures nuclear translocation of the nuclease.

Reaching the nucleus, the nuclease can unfold its full deadly impact by digest the genomic DNA in fragments through its endo-/exonuclease activity. Genomic fragmentation automatically triggeres endogenous programmed cell-death.

Design of our construct

Results

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References:

[Edens et al., 1984]

[Edens et al., 1984] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.

@@ Line 36: / Line 36: @@
 '''TEV Protease''' is a site-specific cystein protease from the C4 peptidase family from Tabacco Etch Virus (TEV). Its typical recognition site is ENLYFQ(G/S).
 </div>
-Our solution is to anchor the nuclease in the cytoplasmatic membrane under basal conditions. In its membrane-associated form ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159111 BBa_K1159111]), the nuclease cannot evolve its deadly potential. The reason is that the genomic DNA of eukaryotic organisms is subcellular located in the nucleus, sheltered by the nuclear envelope. By anchoring the nuclease in the cell membrane, the nuclease is spatially seperated from its site of action.
+Our solution is to anchor the nuclease in the cytoplasmatic membrane under basal conditions. In its membrane-associated form ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159111 BBa_K1159111]), the nuclease cannot evolve its deadly potential as it is spatially separated from most genetic material. The reason is that the genomic DNA of eukaryotic organisms is subcellular located in the nucleus, sheltered by the nuclear envelope. By anchoring the nuclease in the cell membrane, the nuclease is spatially seperated from its site of action.
 To make the nuclease releasable, we have design a long linker with a TEV recognition/cleavage site between the '''transmemmbrane domain (TMD)''' and NLS-tagged nuclease. In the presence of '''TEV protease''' activity, the nuclease is liberated from the membrane and translocates into the nucleus due to its '''NLS''' tag. There, it can fulfill its task as the executor of '''PCD''' by degrading the DNA with its endo/exonuclease activity.