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 activity. 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.
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.

Figure 1: Design of our killswitch, including constitutive anderson promoter systems for the DNase miniColicin and the Im2 immunity protein.

Results

Expression

For the constructs we assembled containing corresponding Anderson promoters, we were able to show expression, as you can see in figure three and four.

Figure 3: SDS-PAGE of the Im2 immunity protein, the blue arrow marks the band of the expression. From left to right: Im2 without the Amber-mutation and induced with IPTG, with the Amber-mutation and induced with IPTG, with and without the Amber-mutation uninduced.

Figure 4: SDS-PAGE of miniColicin, the blue arrow marks the band of the expression. From left to right: Top10 E. coli, Top10 E. coli transformed with BBa_K1976052 and Top10 E. coli transformed with BBa_K1976053.

Functionality of DNase

A first hint given at the functionality of our proteins was by transforming Top10 E. coli with BBa_K1976055 and BBa_K1976056 to show expression by inducing with IPTG. As you can see in Figure 5, the leakage of the T7 promoter alone seemed to show significant growth inhibition, even without induction.

Figure 5: growth of BL21 E. coli transformed with a): BBa_K1976054 and b): BBa_K1976055.