An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system
- Subject Areas
- Bioengineering, Biotechnology, Environmental Sciences, Microbiology
- biological containment, colicin, mutation rate, toxin-antitoxin system, unnatural amino acids, auxotrophy
- © 2015 Kato
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2015. An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system. PeerJ PrePrints 3:e1001v1 https://doi.org/10.7287/peerj.preprints.1001v1
Biological containment is a genetic technique to program dangerous organisms to grow only in the laboratory and to die in the natural environment. Auxotropy for a substance not found in the natural environment is an ideal biological containment. Here, we constructed an Escherichia coli strain that cannot survive in the absence of the unnatural amino acid 3-iodo-L-tyrosine. This synthetic auxotrophy was achieved by conditional production of the antidote protein against the highly toxic enzyme colicin E3. An amber stop codon was inserted in the antidote gene. The translation of the antidote mRNA was controlled by a translational switch using amber-specific 3-iodo-L-tyrosine incorporation. The antidote is synthesized only when 3-iodo-L-tyrosine is present in the culture medium. The viability of this strain rapidly decreased with less than a 1 h half-life after removal of 3-iodo-L-tyrosine, suggesting that the decay of the antidote causes the host killing by activated colicin E3 in the absence of this unnatural amino acid. This containment system can be constructed by only plasmid introduction without genome editing, suggesting that this system may be applicable to other microbes carrying toxin-antidote systems similar to that of colicin E3.
This is a submission to PeerJ for review.
Figure S1. Estimation of the half-life
The half-life of the viability for BL21-AI(IY,1amb-immE3) was estimated in the absence of IY. A single fitted curve was generated using an exponential decrease model. The half-life was calculated from the equation for the fitted curve.
Figure S2. Estimation of the growth rates
The growth rates were estimated for BL21-AI(IY,1amb-immE3), the parent strain carrying pSH350, and for a vector control carrying pBR322 instead of pSH350. A single fitted curve was generated for each strain using an exponential increase model. The growth rates were calculated from the equations for the fitted curves.