When we first designed our project, it soon came to our mind that we had to ensure the containment of our bacteria from the environment. The best way to do that, apart from the physical containment performed by the porous membrane, was to implement our bacterial interface with a kill-switch.

A kill-switch is, literally, something that tells a bacterium whether to live or die, depending on specific environmental conditions. With the rapid development of synthetic biology, these switches are of many kinds and have been increasingly used in recent years [1]. They mainly work by introducing in the bacteria gene constructs that block important genes or express toxins when exposed to a specific environmental condition. We realized an educational video destined to explain to the public audience the principle of a kill-switch. This video can be found here.

Our interface is eventually destined to “cohabit” in a human body. We want to make sure that the bacteria we create cannot develop outside of the space we put them in. Thus, our kill-switch makes sure that when the environmental conditions of the inner human body are not met, the bacteria die.

Reviewing the literature led us to come across a very interesting article presenting the cryodeath kill-switch, invented by Finn Stirling in 2017, which is triggered by temperatures under 37°C [2]. For obvious reasons, this kill switch sounded like a perfect fit to our application. Contacting Finn comforted us in that choice, and we had the chance to exchange a lot, not only concerning the kill switch but on our project in general. After reviewing the original sequence, we modified it so that it would become a biobrick, and we removed what was unnecessary to us, leading to the making of our biobrick BBa_K2616002.

The kill-switch functions by the means of the toxin/antitoxin couple CcdB/CcdA. The toxin targets and inhibits the GyrA subunit of DNA gyrase, an essential bacterial enzyme that catalyzes the super-coiling of double-stranded closed circular DNA [3].

In the kill-switch, the transcription of CcdB and CcdA are regulated by two different promoters. CcdA is expressed by the constitutive promoter P_lac, which ensures a constant low production of antitoxin [4]. On the contrary, the toxin CcdB is expressed by the promoter PcspA. This promoter was originally discovered upstream of the Cold Shock Protein A in many bacteria, as a way to cope with environmental stress [5]. This promoter ensures a very low transcription rate at 37°C, but it increases drastically below 22°C.

Overall, at 37°C, the quantity of antitoxin CcdA is high enough to cope with the leaky low level of toxin produced. However, if the bacteria happen to be in an environment at a lower temperature, the toxin promoter is not repressed anymore, the quantity of toxin becomes too important, and the bacteria is not able to grow (see figure 1). This allows us to make sure that our genetically modified organisms will not spread out in an open environment.

To test the efficiency of our kill-switch, we decided to culture BL21 pLysS Escherichia coli transformed with it at several temperatures (15°C, 20°C, 25°C and 37°C). The growth was followed by measuring the optical density at 600 nm every 30 minutes for 6 hours, followed by two additional points at 18 hours and at 72 hours. Each experiment was done in a triplicate and the standard deviations were calculated for every point. On figure 2, we show that the bacteria transformed with the kill-switch showed no measurable growth at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally.

At 25°C, the kill-switch population grew more slowly than the control for the first 18 hours, but the growth eventually started to reach normal values at 72 hours.

Finally, at 37°C there was no difference in the growth of the kill-switch population compared to the control bacteria.

To conclude, the cryodeath kill-switch is a highly efficient way to prevent bacterial growth below 22°C. In the original design by Finn Sterling, the kill-switch was introduced in the genome of E. coli bacteria using lambda red recombineering. We successfully managed to modify this design, clone it into a plasmid and making it a novel composite biobrick, while keeping its efficiency.

• “Kill switches limit modified microbes,” Nature, vol. 528, no. 7581, pp. 166–167, Dec. 2015.
  
  
• F. Stirling et al., “Rational Design of Evolutionarily Stable Microbial Kill Switches,” Mol. Cell, vol. 68, no. 4, p. 686–697.e3, Nov. 2017.
  
  
• R. J. Reece and A. Maxwell, “DNA Gyrase: Structure and Function,” Crit. Rev. Biochem. Mol. Biol., vol. 26, no. 3–4, pp. 335–375, Jan. 1991.
  
  
• T. P. Malan and W. R. McClure, “Dual promoter control of the E. coli lactose operon.,” Cell, vol. 39, no. 1, pp. 173–80, Nov. 1984.
  
  
• R. Keto-Timonen, N. Hietala, E. Palonen, A. Hakakorpi, M. Lindström, and H. Korkeala, “Cold Shock Proteins: A Minireview with Special Emphasis on Csp-family of Enteropathogenic Yersinia,” Front. Microbiol., vol. 7, Jul. 2016.