Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals1. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems2,3, which include bioremediation4 and probiotics5. Although safeguards have been designed to control cell growth by essential gene regulation6, inducible toxin switches7 and engineered auxotrophies8, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations9,10. Here we describe the construction of a series of genomically recoded organisms (GROs)11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering12, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6,7,8,9,10. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.
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We are grateful to D. Söll and Y. S. Wang for discussion and for providing pTech-supU. We thank N. Carriero and R. Bjornson at the Yale Biomedical High Performance Computing Cluster for assistance with SIFT. Funding received was from the Defense Advanced Research Projects Agency (N66001-12-C-4020, N66001-12-C-4211); NIH-MSTP-TG-T32GM07205 (A.D.H.); DuPont Inc. and the Arnold and Mabel Beckman Foundation (F.J.I.).
A.J.R. and F.J.I. have filed a provisional application with the US Patent and Trademark Office on this work. F.J.I. is a founder of enEvolv, Inc.
Extended data figures and tables
Circos plot summarizing synthetic auxotrophs generated in this study. Red and green genes reflect knockouts and insertions, respectively. Outermost ticks indicate genomic location, inner blue ticks indicate locations where TAG codons were converted to TAA in the GRO, and green ticks reflect the locations of 303 E. coli essential genes. The shaded grey inner circle contains essential TAG loci in synthetic auxotrophs, where yellow ticks represent amino-terminal insertions, blue ticks represent tolerant substitutions, and red ticks represent functional-site substitutions. Innermost links represent unique combinations of TAGs in higher-order synthetic auxotrophs. Links of a single colour correspond to a single strain.
a, Plate map with genotypes of strains shown in b and c. On the top half of the plate SecY.Y122α.E1 (top right quadrant) contains the rpsD.Q54R mutation and is an escape mutant of pAcF-auxotroph, SecY.Y122α (top left quadrant). On the bottom half of the plate the rpsD.Q54R mutation was introduced into SecY.Y122α (bottom right quadrant), resulting in a loss of pAcF-dependence, and reverted to wild type in SecY.Y122α.E1 (bottom left quadrant), restoring pAcF-dependence. The amino acid present at residue 54 within RpsD is indicated at the perimeter of the plate, where red signifies that the given mutation was introduced into the genotype by MAGE to demonstrate the causal mechanism of escape. b, Growth on solid permissive media demonstrates growth of all strains. c, Growth on solid non-permissive media. Introduction of the rpsD.Q54R mutation into the synthetic auxotroph SecY.Y122α results in loss of containment (bottom right quadrant). Reverting the mutation to wild type in SecY.Y122α.E1 results in restoration of containment (bottom left quadrant). Together, these data demonstrate that the rpsD.Q54R mutation is sufficient for loss of pAcF-dependence in SecY.Y122α.
Extended Data Figure 3 Quantitative assessment of amino acid tolerance in higher-order pIF auxotrophs.
Representative assay surveying tolerance of one of three essential TAG loci to the twenty amino acids in different synthetic auxotrophs and expressed as log10 of total cell survival. The + symbol indicates the presence of a TAG codon at the specified locus in the background strain and – indicates the wild-type codon. Blue and yellow indicate high and low tolerance to substitution, respectively. Substitutions DnaX.Y113W and SecY.Y122Q are tolerated but yielded a lower percentage of survival on non-permissive media in a background with two TAGs, an effect that was pronounced in a background with three TAGs. While DnaX.Y113, SecY.Y122 and LspA.Y54 are permissive for most natural amino acids, strains with more than one of these essential TAGs are less prone to survive in the event that any one TAG is compromised. SecY.Y122Q and DnaX.Y113W were tolerated substitutions also observed in real escape mutants of these strains (Supplementary Table 7). Reported results repeated at least three times in independent experiments. Refer to the Methods for a complete description of this experiment.
Extended Data Figure 4 Deletion of tyrT and tyrV restores pIF-dependence and fitness of rEc.β.dC.12′.E7.
a, Plate map with genotypes of strains shown in b and c. rEc.β.dC.12′.E7 is an escape mutant of its sAA-dependent ancestor (rEc.β.dC.12′) and contains a tyrT ochre suppressor mutation (supC). The fitness of rEc.β.dC.12′.E7 in permissive media is impaired relative to rEc.β.dC.12′, with doubling times of 91.74 (±1.49) and 61.81 (±0.65) minutes, respectively. Tyrosine tRNA redundancy was eliminated (ΔtY) in both strains by λ-Red mediated replacement of tyrT and tyrV with chloramphenicol acetyltransferase (cat), rendering the resulting strains (rEc.β.dC.12′.ΔtY and rEc.β.dC.12′.E7.ΔtY) dependent on tyrU for tyrosine incorporation during normal protein synthesis. Elimination of tyrosine redundancy reduced the escape frequency of rEc.β.dC.12′ from 2.17 × 10−9 (Fig. 2e) to <4.85 × 10−12 (no escape mutants were observed upon plating 2.06 × 1011 cells) and restored pIF-dependence in rEc.β.dC.12′.E7 to <4.73 × 10−12 (no escape mutants were observed upon plating 2.12 × 1011 cells). Escape mutants were not detected for either strain up to 7 days after plating on non-permissive media (Fig. 3d and Supplementary Table 11). Tyrosine tRNA deletion also restored the fitness of the escape mutant to approximately that of its sAA-dependent ancestor (60.66 ± 0.12 min). Taken together, these results establish tyrT as the causal mechanism of escape in rEc.β.dC.12′.E7. b, Growth on solid permissive LB media. c, Growth on solid non-permissive LB media. All reported doubling times are averages, where n = 3 technical replicates, and error bars represent ±s.d. Refer to the Methods for a complete description of escape frequencies.
Extended Data Figure 5 Growth profiles of strains expressing phenylalanine or tryptophan amber-suppressor tRNAs.
a–d, Growth was assessed for rEc.γ.dC.46′.ΔtY and rEc.β.dC.12′.ΔtY in the presence of amber suppression by either pTech-supU (blue), pTech-supPhe (red), or in the absence of plasmid-based amber suppression (black). Cells were washed twice with dH2O and re-suspended in the same volume of 1× PBS. Washed cells were normalized by OD600 to inoculate roughly equal numbers of cells per well. Growth profiles are shown for rEc.γ.dC.46′.ΔtY (a, b) and rEc.β.dC.12′.ΔtY (c, d) in permissive (+sAA/+l-arabinose, solid lines) and non-permissive (−sAA/−l-arabinose, dashed lines) LB liquid media. Doubling times are shown for the ancestral strain (black) in permissive media and suppressor-containing strains (red and blue) in non-permissive media where growth was observed. Plasmid containing strains were always grown in the presence of zeocin for plasmid maintenance. Growth was never observed for the contained ancestors in non-permissive media (black, dashed lines). In the presence of tryptophan suppression, growth of rEc.γ.dC.46′.ΔtY was not observed and growth of rEc.β.dC.12′.ΔtY was severely impaired (380 min doubling time), with a 6.24-fold increase in doubling time relative to the contained ancestor grown in permissive media. In the presence of phenylalanine suppression, growth of rEc.β.dC.12′.ΔtY was not observed and growth of rEc.γ.dC.46′.ΔtY was severely impaired (252 min doubling time), with a 3.90-fold increase in doubling time relative to the contained ancestor grown in permissive media. Representative growth profiles and doubling times are reported. These results repeated at least three times in individual experiments.
a–c, Approximately 1011 cells of strain rEc.γ.dC.46′.ΔtY (indicated with triangles) were inoculated into 1 l of permissive (+sAA/+l-arabinose, blue) or non-permissive (−sAA/−l-arabinose, red) LB media and incubated with agitation at 34 °C for 20 days. Results from the equivalent experiment with the non-contained ancestor rEc.γ (indicated with diamonds) are also shown. Cultures were frequently monitored by OD600 (a) and quantification of c.f.u. on solid permissive (+sAA/+l-arabinose) (b) and non-permissive (−sAA/−l-arabinose) (c) LB media. C.f.u. are plotted as the average of three replicates. Open symbols indicate that no c.f.u. were observed. Symbols for rEc.γ.dC.46′.ΔtY are not visible because c.f.u. were never observed from either permissive or non-permissive liquid cultures plated on non-permissive solid media. At the end of the 20-day growth period, both cultures containing rEc.γ.dC.46′.ΔtY were interrogated for the presence of a single escape mutant by plating each 1 l of culture across 30 non-permissive solid media plates. C.f.u. were not observed and remained below the limit of detection for the following 7-day observation period. We hypothesize that the decrease in c.f.u. counts obtained on permissive solid media for the permissive culture of rEc.γ.dC.46′.ΔtY reflects pAzF degradation at ≥6 days. Reported c.f.u. values are averages, where n = 3 technical replicates, and error bars are ±s.d. Reported results repeated at least three times in independent experiments. Refer to the Methods for a complete description of this experiment.
Extended Data Figure 7 Dose-dependent growth of rEc.γ.dC.46′.ΔtY in pAzF and l-arabinose compared to the non-contained ancestor.
Growth in LB media supplemented with different concentrations of pAzF and l-arabinose. a–d, Growth profiles for rEc.γ across a gradient of pAzF concentrations in the presence of 0% (a), 0.002% (b), 0.02% (c) and 0.2% (d) l-arabinose. f–i, Growth profiles for rEc.γ.dC.46′.ΔtY across a gradient of pAzF concentrations in the presence of 0% (f), 0.002% (g), 0.02% (h) and 0.2% (i) l-arabinose. e, j, Growth profiles illustrated in a–d and f–i are depicted as heat maps in e and j, respectively, where the maximum OD600 was obtained from the average of three replicates and plotted in MATLAB. Reported growth profiles and heat map values are averages, where n = 3 technical replicates. Reported results repeated at least three times in independent experiments.
Growth in LB media supplemented with different concentrations of pIF and l-arabinose. a–d, Growth profiles for rEc.β.dC.12′.ΔtY across a gradient of pIF concentrations in the presence of 0% (a), 0.002% (b), 0.02% (c) and 0.2% (d) l-arabinose. e, Growth profiles illustrated in parts a–d are depicted as a heat map, where the maximum OD600 was obtained from the average of three replicates and plotted in MATLAB. Reported growth profiles and heat map values are averages, where n = 3 technical replicates. Reported results repeated at least three times in independent experiments.
Wild-type E. coli K-12 substr. MG1655 and three strains auxotrophic for biotin, EcNR2, rEc.γ (a non-contained GRO with an integrated pAzF OTS) and rEc.γ.dC.46′ (also a synthetic auxotroph) were grown either adjacent or separately on rich-defined solid media. EcNR2 grew on biotin-deficient media when plated in close proximity to wild-type E. coli, suggesting cross-feeding of the essential metabolite. The pAzF auxotroph only grew on media supplemented with biotin, pAzF and l-arabinose.
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Rovner, A., Haimovich, A., Katz, S. et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015). https://doi.org/10.1038/nature14095
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