Wild-type E. coli MG1655 has 321 known UAG codons that are decoded as translation stops by RF1 (for UAG and UAA). (1) Remove codons: converted all known UAG codons to UAA, relieving dependence on RF1 for termination. (2) Eliminate natural codon function: abolished UAG translational termination by deleting RF1, creating a blank codon. (3) Expand the genetic code: introduced an orthogonal aminoacyl–tRNA synthetase (aaRS) and tRNA to reassign UAG as a dedicated sense codon capable of incorporating nonstandard amino acids (NSAAs) with new chemical properties.

Ratios of maximum cell densities (horizontal axis) and doubling times (vertical axis) were determined for RF1⁺ strains versus their corresponding RF1⁻ strains (n = 3) in the presence or absence of UAG suppression. Symbol color specifies genotype: UAA is the number of UAG→UAA mutations, and RF2 is “WT” (wild type) or “sup” [RF2 variant that can compensate for RF1 deletion ()]. Symbol shape specifies NSAA expression: aaRS (aminoacyl–tRNA synthetase) is “none” (genes for UAG reassignment were absent), “−” [pEVOL-pAcF () is present but not induced, so only the constitutive aaRS and tRNA are expressed], or “+” (pEVOL-pAcF is fully induced using L-arabinose), and pAcF is “−” (excluded) or “+” (supplemented). Strains that do not rely on RF1 are expected to have a RF1⁺/RF1⁻ ratio at (,). RF1⁻ strains exhibiting slower growth are below the horizontal gray line, and RF1⁻ strains exhibiting lower maximum cell density are to the right of the vertical gray line. The doubling-time error bars are too small to visualize.

(A) Western blots demonstrate that C0.B*.ΔA::S terminates at UAG in the absence of RF1 and that C7.ΔA::S and C13.ΔA::S have acquired natural suppressors that allow strong NSAA-independent read-through of three UAG codons. When pAcF was omitted, one UAG reduced the production of full-length GFP, and three UAGs reduced production to undetectable levels for all strains except C7.ΔA::S and C13.ΔA::S, demonstrating that undesired near-cognate suppression () is weak for most strains even when RF1 is inactivated. However, all strains show efficient translation through three UAG codons when pAcF is incorporated. Western blots were probed with an antibody to GFP that recognizes an N-terminal epitope. UAA is the number of UAG→UAA mutations; RF2 is “WT” (wild type) or “sup” [RF2 variant that can compensate for RF1 deletion ()]; RF1 is “WT” (wild type) or “S” (ΔprfA::spec^R). “GFP” is full-length GFP; “trunc” is truncated GFP from UAG termination and is enriched in the insoluble fraction; “ns” indicates a nonspecific band. (B) Venn diagram representing NSAA-containing peptides detected by mass spectrometry in C0.B*.ΔA::S when UAG was reassigned to incorporate p-acetylphenylalanine (pAcF, red) or phosphoserine (Sep, blue). No NSAA-containing peptides were identified in C321.ΔA::S. Asterisk (*) indicates coding DNA sequence possessing two tandem UAG codons. (C) Extracted ion chromatograms are shown for UAG suppression of the SpeG peptide to investigate Sep incorporation in natural proteins. Peptides containing Sep were only observed in C0.B*.ΔA::S, C7.ΔA::S, and C13.ΔA::S, as Sep incorporation was below the detection limit in EcNR2 (RF1⁺), and speG was recoded in C321.ΔA::S.

RF1 (prfA) status is denoted by symbol shape: (■) wt prfA (WT); (★) ΔprfA::spec^R (ΔA::S); ( ) ΔprfA::tolC (ΔA::T); and (×) a clean deletion of prfA (ΔA). (A) RF1 status affects plaque area (Kruskal-Wallis one-way analysis of variance, P < 0.001), but strain doubling time does not (Pearson correlation, P = 0.49). Plaque areas (mm²) were calculated with ImageJ, and means ± 95% confidence intervals are reported (n > 12 for each strain). In the absence of RF1, all strains except C0.B*.ΔA::S yielded significantly smaller plaques, indicating that the RF2 variant () can terminate UAG adequately to maintain T7 fitness. A statistical summary can be found in . (B) T7 fitness (doublings/hour) () is impaired (P = 0.002) and mean lysis time (min) is increased (P < 0.0001) in C321.ΔA compared to C321. Significance was assessed for each metric by using an unpaired t test with Welch’s correction.