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 does not 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.

How our red light triggered nuclease system for self-destruction works

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 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159105 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) ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159109 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 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159111 BBa_K1159111]), the nuclease cannot evolve its deadly potential as it is spatially seperated from its site of action: In eukaryotic organisms the genomic DNA is subcellular located in the nucleus, sheltered by the nuclear envelope.

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.

Key features of the system

During the design process of the kill-switch we developed a set of key features that we thought to be essential for the efficient activation of programmed cell death.

Signal transduction solely on protein level: By excluding transcriptional/translational transduction from the system we created a system that can assure PCD with tight temporal control. This design eliminates the risk of a negative feedback loop that would appear when transcriptional/translational efficiency is hampered due to lowered cell vitality. See: Modeling.

Strong lethal activity:

Cheap, non-toxic, automatic induction:

Design Notes

Releasable Membrane-associated Endo-exonuclease

For our membrane bound nuclease we have to choose a nuclease with following characteristics:

Our nuclease should digest any DNA wihtout any preferences for recognitions sites

Furthermore, that nuclease should not have disulfide bonds as they cannot be formed outside in the cytoplasmatic compartment.

Micrococcal nuclease ([http://parts.igem.org/Part:BBa_K1159105 BBa_K1159105]) (alias S7 Nuclease, MNase, Thermonuclease) from Staphylococcus aureus is choosed as our death bringing executor as it fulfills our requirements (see above). It shows endo-exonuclease activity for DNA and does not contain any disulfid bonds.

To anchor the nuclease in the cytoplasmatic membrane, we use the TMD ([http://parts.igem.org/Part:BBa_K1159315 BBa_K1159315]) and the N-terminal signal peptide ([http://parts.igem.org/Part:BBa_K1159303 BBa_K1159303]) from the chassis Physcomitrella patens itself.

To make the nuclease releasable from the membrane in the presence of TEV protease activity, we designed a long linker with a TEV recognition and cleavage site. We designed a long linker (GlyGlyGlyGlySer)x5 with TEV cleavage site at the C-terminal end of the linker ([http://parts.igem.org/Part:BBa_K1159310 BBa_K1159310]).

After beeing released from the membrane cage through the cleavage by TEV proteolysis, the nuclease has be guided to its site of action, the nucleus. For this purpose, we are using the NLS from SV40 ([http://parts.igem.org/Part:BBa_K801030 BBa_K801030]).

N-(SERK signal peptide)_(SERK transmembrane region)_(long flexible linker with TEV-site)_(SV40 NLS)_(micrococcal nuclease)-C

Red light triggered C-terminal Split-TEV Protease

For the first part of the light-induced reassembling of split TEV protease, we use the first 908 N-terminal amino acids ([http://parts.igem.org/Part:BBa_K801031 BBa_K801031]) for the reversible heterodimerization with PIF3/PIF6. It has been reported that the first 650 N-terminal amino acids indeed are sufficient for photoconversion of P_r (inactive form) to P_fr but are irreversible, so we take the first 908 N-terminal amino acids, which has been proven to be reversible to red light induced heterodimerization with PIF3/PIF6.

Red light triggered N-terminal Split-TEVV Protease

Results

Text

figure

References:

http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009 Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature, 461(7266):997–1001.
http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002 Mendelsohn, A. R. (2002). An enlightened genetic switch. Nat Biotechnol, 20(10):985–7.
http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002 Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. Nat Biotechnol, 20(10):1041–4.
http://www.ncbi.nlm.nih.gov/pubmed/17072307 Wehr MC et al., 2006 Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, Rossner MJ (2006). Monitoring regulated protein-protein interactions using split TEV. Nat Methods, 3(12):985-93.