Team:TU Darmstadt/Lab/KILLswitch
KILL(switch)
ABSTRACT
Synthetic suicide systems have been safeguards in synthetic biology for as long as the field exists. There are different kinds of designs, often based on a regulatory mechanism and a toxin such as a host-killing protein or a metabolic inhibitor. However, these mechanisms often don't tackle the problem of DNA surviving the death of the host cell.
Here we show a possible design for a simple synthetic killswitch based on an endonuclease called colicin E2 and a corresponding suppressing immunity protein, Im2. Our immunity protein is regulated by amber suppression - the usage of an amber stop codon coding for the non-natural amino acid O-methyl-L-tyrosine. The aim of the system is to make survival of a transgenic organism and its genetic information dependent on the addition of O-methyl-L-tyrosine. In case of a ceasing OMT concentration, the inhibition of colicin E2 drops due to a lack of translated immunity protein. The free DNase cuts all DNA within the cell and its surroundings, killing the host and preventing the escape of transgenic DNA
Killswitch
There are a lot of fears in the general public's mind when it comes to genetics and genetically modified organisms (GMOs). These represent a major burden, that synthetic biology has to overcome before it will gain widespread acceptance for more applications in daily life. Since the advent of synthetic biology, scientists have been hard at work to implement safeguards in their genetically modified organisms. They were making use of lots of different designs such as controlled essential gene regulations1, engineered auxotrophies2 and inducible toxin switches3 . One problem that arises with most of these systems is that they don't tackle the problem of bioorthogonal DNA escaping into the environment. The synthetic genetic systems remain, even if their host has died by the means of a killswitch. That DNA can not only be used for industrial espionage, but bacteria in the environment could get transformed with these synthetic plasmids and thus propagate the pathways introduced into the lab-grown bacteria throughout ecosystems. The general aim of a killswitch is to kill its host, most often bearer of plasmids or other forms of non-wild-type DNA. They are generally designed with some sort of regulating mechanism followed by some form of host killing toxin. The toxin can be any sort of host killing mechanism, be that inhibiting proteins, toxin creating enzymes or other things. The toxin we chose for the design of our killswitch is colicin E2, a nicking endonuclease4. Colicins occur in some wild type coliform bacteria and are used to kill rivaling strains of Escherichia coli. An essential part of the design is the way one regulates the release of the toxin. Colicin E2 has a natural counterpart, the immunity protein Im2, which suppresses colicin by tightly binding to it. It can be used as a suppression mechanism for our toxin to protect the bearer of the colicin from its martial effects. We chose to use the so-called amber suppression5 system as our regulating mechanism for the expression of the immunity protein. By introducing a bioorthogonal pair of a tRNA (for more information on that, press this link) and synthase into our cells, we can insert a non-natural amino acid into our immunity protein. You can find more on the rational design of the insertion on our modeling page and on the specifics on our suppression system in our text Incorporation of OMT. By designing our killswitch like this, we aimed to enable the user to not only create genetically modified organisms safely, but also to minimize the risk of their vectors to propagate freely throughout ecosystems and to protect the users intellectual property from being stolen or recreated. The activity of our toxin even after the death of the host assures, that all DNA in the vicinity of the host gets degraded. You can find out more about the design of our killswitch here.
Design
Killswitch
Our design approach for the killswitch was to use consitutive promoters for our DNase as well as the immunity protein. The immunity protein needs to be expressed in a higher amount to ensure its' function as a suppressor, as it needs to be at least equimolar to the colicin to suppress it properly. A higher than equimolar expression is optimal. The activation of our Killswitch happens as soon as the immunity protein is suppressed by a lack of the non-natural amino acid. Thus only part of it is synthesized and the DNase is not inhibited anymore. The pathway is designed as seen in figure two.MiniColicin
Colicin E2 is comprised of 3 domains which represents a major metabolic burden. Two of the three domains are import and export signals and thus not necessary for its activity as a DNase. To minimalize the metabolic stress put on our cells by the incorporation of our killswitch, we designed a rational approach for the expression of our colicin as only its DNase domain. By removing the import and export signals of the colicin on a DNA level, we shortened it by more than two thirds. We modeled its stability, you can find more on that here. You can find the pathway design we chose in figure two.
Figure 1: Complex of colicin E2 (black: export domain; blue: import domain; yellow: DNase) and Im2 (green)
Figure 2: Design of our Pathway, anderson promoters for our DNase as well as the Im2 immunity protein.Results
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References
- [1] Kong, W. et al. Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment. Proc. Natl Acad. Sci. USA105, 9361-9366 (2008)
- [2] Steidler, L. et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nature Biotechnol. 21, 785-789 (2003)
- [3] Szafranski, P. et al. A new approach for containment of microorganisms: dual control of streptavidin expression by antisense RNA and the T7 transcription system. Proc. Natl Acad. Sci. USA 94, 1059-1063 (1997)
- [4] Schaller, K. and Nomura, M. Colicin E2 is DNA endonuclease. Proc. Natl Acad. Sci. USA 73, No. 11, pp. 3989-S993 November (1976) Biochemistry
- [5] Lang, K. and Chin, J. W., Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins, 114, 4764-4806 Chem. Rev. (2014)
