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 regulating mechanism and a host killing toxin or different kinds of inhibitation of metabolic pathways. However, these mechanisms most often do not tackle the problem of synthetic DNA surviving the death of host cells.
Here we show a possible design for a simple synthetic killswitch based on the endonuclease Colicin E2 and its corresponding suppressing protein Im2. Endonuclease and suppression protein expression is regulated by amber suppression. Therefore, an amber stop codon coding for the non-natural amino acid O-methyl-l-tyrosine is implemented into the gene. The aim of the system is not to simply kill the host, but also to degrade all DNA within the cell and its surroundings, 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). That represent a major burden 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 working hard to implement safeguards in their genetically modified organisms. They were making use of many different designs such as controlled essential gene regulations [1], engineered auxotrophies [2] and inducible toxin switches [3]. One problem that arises with most of these systems is not tackling the problem of bioorthogonal DNA escaping into the environment after cell death. That DNA cannot only be used for industrial espionage, but bacteria in the environment could also get transformed with synthetic plasmids and thus propagate synthetic pathways throughout ecosystems. The general aim of a killswitch is to kill its host, most often bearer of plasmids or other forms of synthetic DNA. They are generally designed with some sort of regulatory mechanism connected to a host-killing element. There is a high variability of host-killing mechanisms. The toxin we chose for the design of our killswitch is colicin E2, a nicking endonuclease [4]. 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 regulation of the toxin release. Colicin E2 has a natural counterpart, the immunity protein Im2, which inhibits Colicin E2 by tightly binding to it. It can be used as a suppression mechanism for our toxin to protect the bearer of the Colicin E2 from its martial effects. We chose to use the amber suppression [5] for regulating the expression of the immunity protein. By introducing a bioorthogonal pair of a tRNA and an aminoacyl RNA synthetase into the cells, we can insert a non-natural amino acid into the sequence of the immunity protein. The rational design approach was validated by molecular dynamic simulation to ensure a functional suppression system suppression system. In the design of our killswitch, we aimed for enabling the user to not simply 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 after cell death assures DNA degradation in the vicinity of the host.
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), rendered with PyMol v1.7.2.1
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)
