An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system

Biological containment is a genetic technique that programs dangerous organisms to grow only in the laboratory and to die in the natural environment. Auxotrophy 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. The contained strain grew 1.5 times more slowly than the parent strains. The escaper frequency was estimated to be 1.4 mutations (95% highest posterior density 1.1–1.8) per 10(5) cell divisions. 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.