Kill-Switch
Inital Kill Switch
Our project requires the deployment of genetically engineered organisms in environments outside of a contained laboratory, which may rise concerns about unchecked microbial proliferation and/or unwanted spread of synthetic genes. While it might not be an immediate priority, designing a kill switch, or programmed cell death (PCD), is necessary in order to introduce safety into our project for when we reach the phase to take our bacteria outside of the laboratory. In bacteria PCD can act as a defence mechanism against phage infection, provide benefits such as recycling nutrients during severe nutritional stress, and preserve genomic stability by eliminating cells with defects.
Our initial approach consisted of employing the Holin-Endolysin system, which, when active, involves bacteriophage-derived holin proteins that, if present in a large enough amount in the cell, tear through the bacterial inner membrane, allowing endolysins to enter the periplasm. Endolysins are proteins causing bacterial cell lysis by breaking down the cell wall, but they cannot reach it without holins, making the death of the cell dependent on the controllable holin levels.
The alternative solution
However, after careful consideration the PCD of the project got slightly modified. Instead of using the bacteriophage inspired system, we decided to follow ZJU China’s Story Light’s (2022)[1] steps by using the MazEF toxin-antitoxin (TA) system documented in the iGEM parts website as "BBa_K2292006".
Since Story Light used Bacillus subtilis, their design got adapted specifically for that bacteria and their project. They used a sucrose-inducible promoter for their dual system.
Our project uses Escherichia Coli, which is the bacteria where this TA mechanism was originally found in. The regulation of this system will be done by the presence of arabinose in the engineered organism’s medium, just like the documented iGEM part. The system is made of MazF, a gene encoding a stable toxin, and MazE encoding, an unstable antitoxin.
As long as there is a high enough antitoxin to toxin ratio, MazE antagonizes MazF and inactivates it by forming a dimer. However, MazE is an unstable molecule that gets rapidly degraded by proteases in the cell; if its production is interrupted by a lack of arabinose in the environment, the remaining MazF will randomly cleave the cell’s mRNA in a ribosome-independent manner at ACA sequences, ultimately leading to cell lysis. Stressful conditions such as nutrient starvation trigger the mazEF-mediated PCD system (Engelberg-Kulka et. al, 2005)[2:1].
The set up of this system will allow cultures to slowly undergo apoptosis the longer they feed on the provided arabinose medium, giving us power over their lysis by controlling concentrations of arabinose in the medium.
References
ZJU-China - IGEM 2022. (2022). https://2022.igem.wiki/zju-china/ ↩︎
Engelberg‐Kulka, H., Hazan, R., & Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of Cell Science, 118(19), 4327–4332. https://doi.org/10.1242/jcs.02619 ↩︎ ↩︎