Genomically recoded organisms and methods of using the same

Abstract

Genomically recoded organisms (GRO) are provided. In some forms, the GRO include no more than 25 genomic instances of the TGA codon. In some forms, genomically recoded organisms include two or more of single nucleotide substitutions, TAA insertions, and deletions to eliminate most or all of the TGA codons relative to a progenitor cell. In some forms, the GRO have a genome having stops codons, wherein the stop codons consisting of a single stop codon sequence. In some forms, the GRO provides translation termination only at the single stop codon sequence. Also provided are (i) a mutant release factor 2 (RF2) with reduced ability to terminate translation at UGA and / or a nucleic acid encoding the same and (ii) a mutant tRNATrp with reduced ability to decode UGA and / or a nucleic acid encoding the same, relative to a progenitor cell. GRO's expressing (i) and / or (ii) are also provided.

Description

[0001]YU 8909 PCT GENOMICALLY RECODED ORGANISMS AND METHODS OF USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 5 63 / 663,593 filed June 24, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under N66001-12-C-4211 awarded by Defense Advanced Research Projects Agency and GM117230 awarded by the National Institutes 10 of Health. The government has certain rights in the invention. REFERENCE TO THE SEQUENCE LISTING The Sequence Listing XML submitted as a file named “YU_8909_PCT_ST26.xml”, created on June 24, 2025, and having a size of 55,899 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1). 15 FIELD OF THE INVENTION The field of the invention generally relates to genomically recoded organisms and methods of using the same. BACKGROUND OF THE INVENTION Genomically recoded organisms (GROs) are organisms with alternative genetic codes20 wherein redundant codons are reassigned to new functions. GROs can be utilized for site- specific incorporation of nonstandard amino acids (nsAAs) into proteins. However, improved GROs nonetheless remain desirable. Thus, it is an object of the invention to provide improved GROs and methods of making and using the same. 25 SUMMARY OF THE INVENTION The experiments below illustrate the first genomically recorded organisms (GRO) that fully compresses a redundant codon functionality into a single codon, liberating two essential stop codons for reassignment. This work advances whole-genome recoding efforts by eliminating 1,195 instances of the stop codon TGA within C321.ΔA (∆TAG), accompanied by 30 engineering of cognate release factor 2 (RF2) and tRNATrp to attenuate native UGA recognition, thereby rendering TAA as the sole stop codon while freeing TGA as a second open codon available for functional reassignment. This effort renders four codons - TAG, TGA, TAA, TGG – nondegenerate within the canonical code, decoupling translational crosstalk to allow each 1 45741195.1 YU 8909 PCT to serve unique functions, demonstrated by the multisite incorporation of two distinct nsAAs into single proteins with ~99% accuracy. In doing so, this work provides a new genomic context to interrogate bacterial translation mechanisms more deeply and to evolve new translation factors and configurations with applications in biocontainment, genetic isolation, and precision 5 production of synthetic proteins and biomaterials with expanded chemistries. In one aspect, the present disclosure provides GROs capable of incorporating two distinct nsAA into a peptide. As a non-limiting example, the GRO is a bacterial strain lacking both native TAG and TGA codons that, when complemented by a noncompetitive mutant RF, are freed for reassignment as sense codons for the two nsAA. 10 Thus, genomically recorded organisms (GRO) are provided. In some forms, the genomically recoded organisms include no more than 25 genomic instances of the TGA codon. In some forms, genomically recoded organisms include two or more of single nucleotide substitutions, TAA and / or TAG insertions, and deletions to eliminate most or all of the TGA codons relative to a progenitor cell. 15 In some forms, the genomically recoded organisms include no more than 25 genomic instances of the TAA codon. In other forms, the genomically recoded organisms include two or more of single nucleotide substitutions, TAG and / or TGA insertions, and deletions to eliminate most or all of the TAA codons relative to a progenitor cell. Thus provided are GRO having a genome having stops codons, wherein the stop codons 20 consisting of a single stop codon sequence. In some forms, the GRO includes one or more, optionally all, of the features of Table 9. In some forms, the GRO is the E. coli strain referred to herein as rEc∆2.∆A.mB- oKP.tW* (Ochre.tW*). In some forms, the release factor(s) of the GRO provides translation termination only at 25 the single stop codon sequence. In some forms, the GRO includes one or both of (i) a mutant release factor 2 (RF2) with reduced ability to terminate translation at UGA and / or a nucleic acid encoding the same and (ii) a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same, relative to a progenitor cell. 30 In some forms, greater than 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% of TGA codons eliminated in the GRO relative to the progenitor cell. In some forms, less than 100% of the TGA codons are eliminated. In some forms, TGA codon(s), if present, are only present in pseudogenes, ins, and / or selenocystine- encoding codons. Thus, the TGA codon can be a functionally open codon that can be 2 45741195.1 YU 8909 PCT reassigned. In other forms, TAG is absent from the genome. Thus, TAA codon can be a functionally open codon that can be reassigned. Crosstalk between four codons TAG, TGA, TAA, and TGG can attenuated rendering each codon a unique translational function: open, open, stop, and standard amino acid encoding 5 optionally wherein the standard amino acid is Trp; or open, stop, open, and standard amino acid encoding optionally wherein the standard amino acid is Trp. In some forms, the GRO includes a mutant RF2 and / or nucleic acid encoding the same. Thus, also provided are mutant RF2, and nucleic acids encoding the same. In some forms, the mutant RF2 includes (i) one or more of N26, K170, P205, and A246 relative to SEQ ID NO:3 10 and optionally has an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% to the amino acid sequence of SEQ ID NO:3, or (ii) the corresponding amino acids in a homolog thereof, optionally wherein the homolog includes an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity thereto. For example, 15 the mutant RF2 can include at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, preferably including all of N26, K170, P205, and A246 relative to SEQ ID NO:3. In some forms, the GRO includes a mutant prfB gene having one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the 20 terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to SEQ ID NO:1. For example, the mutant prfB gene can have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 25 98%, or 99% sequence identity to SEQ ID NO:2. Thus, mutant prfB genes are also provided. In some forms, the GRO includes a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same. Thus, mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same and nucleic acids encoding the same are also provided. In some forms, the mutant tRNATrpis encoded by nucleic acid sequence including a 30 A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:5. For example, the mutant tRNATrpcan be encoded by a nucleic acid sequence including at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6. 3 45741195.1 YU 8909 PCT In some forms, the GRO includes a mutant trpT gene including a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:5. For example, the mutant trpT gene can have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6. Thus, mutant trpT genes are also 5 provided. In some forms, the GRO includes a first heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate tRNA capable of decoding TGA (where TGA has been reduced or removed as stop codon) and TAA (where TAA has been reduced or removed as a stop codon). In some forms, the includes a second heterologous orthogonal aminoacyl tRNA 10 synthetase (AARS) and cognate tRNA capable of decoding TAG. In some forms, the GRO includes a heterologous mRNA or nucleic acid encoding the same including one or more TGA codons (where TGA has been reduced or removed as stop codon) and TAA codons (where TAA has been reduced or removed as a stop codon). In some forms, the heterologous mRNA or nucleic acid encoding the same includes one 15 or more TAG codons. In some forms, the GRO further includes a heterologous mRNA or nucleic acid encoding the same including one or more TGA codons and one or more TAG codons. GRO can be, for example, bacteria, yeast, fungi, insect, plant, or animal cell(s). In a particular form, the GRO is E. coli. 20 Cultures and lysates thereof including a plurality of the one or more GRO are also provided. Method of use and methods of making are also provided. A method of making a polypeptide including one or more instances of a non-standard amino acid can include expressing a first messenger RNA (mRNA) encoding the target protein 25 in a system including: a first orthogonal translation system (OTS) including a nucleic acid sequence encoding a first orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the genomically recoded organism (GRO), and a plurality of a first non-standard amino acid, wherein the first mRNA includes a nucleic acid sequence including at least one instance of a first codon reduce or absent30 in the GRO, wherein the first orthogonal AARS can charge its cognate tRNA with the first non- standard amino acid, and wherein the first cognate tRNA includes an anticodon that can bind to the codon reduced or absent from the GRO. The GRO can also include a second orthogonal translation system (OTS) include a nucleic acid sequence encoding a second orthogonal AARS and its cognate tRNA operably 4 45741195.1 YU 8909 PCT linked to expression control sequences and transformed, transfected, or integrated into the GRO, and a plurality of as second non-standard amino acid, wherein the first mRNA or a second heterologous mRNA includes a nucleic acid sequence including at least one instance of a second codon reduced or absent in the GRO, wherein the second orthogonal AARS can charge its 5 cognate tRNA with the second non-standard amino acid, and wherein the second cognate tRNA includes an anticodon that can bind to a second codon reduced or absent from the GRO. Thus provided is a method of making a polypeptide including one or more instances of a non-standard amino acid including expressing a first messenger RNA (mRNA) encoding the target protein in a system including: a first orthogonal translation system (OTS) including a 10 nucleic acid sequence encoding a first orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the genomically recoded organism (GRO), a second orthogonal translation system (OTS) including a nucleic acid sequence encoding a second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the GRO and a 15 plurality of first and second non-standard amino acids, wherein the first mRNA includes a nucleic acid sequence including one or more instances of a first codon and / or one or more instances of a second codon of a second codon reduced or absent in the GRO, wherein the first and second orthogonal AARS’s can charge their cognate tRNA with first and second non- standard amino acids, respectively, and wherein the first and second cognate tRNA includes 20 anticodons that can bind to different codons reduced or absent from the GRO. In some forms, the first and / or second mRNA include between 1-100 instances includes, or any specific integer or subrange in between, of the first codon that is reduced or eliminated from the GRO, the second codon that is reduced or eliminated from the GRO, or a combination thereof. 25 In some forms, the target polypeptide is made in greater yield and / or purity compared to making the polypeptide using the same system in the GRO’s progenitor cell. A method of making a GRO can include, for example, a genome consisting of a single stop codon sequence, the method including recoding other stop codon sequences to the stop codon sequence, optionally further including altering one or more release factors to provide 30 translation termination only at the single stop codon sequence. Also provided are nucleic acids encoding a polypeptide, wherein the nucleic acid sequence includes one or more UAG / TAG codons and one or more UGA / TGA codons, wherein the polypeptide including nsAAs and the UAG / TAG codons and UGA / TGA codons encode for the nsAAs. 5 45741195.1 YU 8909 PCT BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a schematic summary of translation decoding of the three canonical stop codons – UAG, UGA, UAA – and the Tryptophan UGG codon alongside their native cognate translation factors across three strains of E. coli: MG1655 (wt), rEc∆1.∆A (∆UAG, ∆RF1), and 5 Ochre.tW* (∆UAG, ∆RF1, mRF2, tW*). Codons within multiple iterations of genomic recoding from WT E. coli to Ochre.tW*, a ∆TAG-∆TGA recoded strain containing a mutant release factor 2 and a mutant tRNATrpboth engineered to discriminate against UGA. Orthogonal translation system components (e.g. o-aaRS and o-tRNA) within recoded strains make possible reassignment of UAG and UGA to sense codons incorporating non-standard amino acids 10 (nsAAs). Figure 1B is a diagram of ∆TAG-∆TGA recoded genome (rEc∆2.∆A) depicting relative locations of important designed mutagenic events in the genome-wide removal of TGA. Innermost sections of circles depict genomic subdivisions for targeted recoding of rEc∆1.∆A to acquire rEc∆2E.∆A (A' and B') and subsequently to acquire fully recoded rEc∆2.∆A (Figs.1A- 1H). Figure 1C is: Top: representations of common terminal TGA codons overlapping genes. 15 Bottom: common mutational means of resolving codon conversions with minimal genetic deviation. Figure 1D is a dot plot of predicted translation initiation rates for genes overlapped by those TGA-ending genes denoted in subsection B recoded from TGA to TAA (Y-axis), plotted against the predicted translation initiation rates of WT overlapped genes. The diagonal corresponds to recoding events that do not alter the projected translation rate. R2is the Pearson20 correlation coefficient. Figure 1E is a schematic overview depicting iterative steps of MAGE- mediated genomic recoding and CAGE-mediated hierarchical assembly of recoded subdomains into a final fully recoded strain (rEc∆2) lacking terminal TGA codons. Figure 2A is a comparison of mRNA transcript and protein diagrams displaying key nucleotides and amino acids in WT-RF2 and mRF2-oKP. Figure 2B is: Top: AlphaFold 25 structural prediction of mRF2-oKP with residue deviations from WT-RF2 marked in purple. Bottom: magnified views of WT-RF2 SPF (left) and mRF2-oKP PPF (right) codon recognition loops, highlighting interactions with amino acid residue 205 as either WT Ser or mutant Pro, respectively. AlphaFold structural prediction indicates wild-type S205 presents up to 4 hydrogen bonds within the RF2 codon recognition loop, while P205 presents one, likely altering loop 30 conformation and stability. Shown are the mRNA sequences including partial sequences for prfB AGGGGGUAUCUUUGACUAC (SEQ ID NO:23) and mprfB AGGGGUAUCUUUAACUAC (SEQ ID NO:24), and mRF2-oKP fragments: RGYLBY (SEQ ID NO:25), EESXGE (SEQ ID NO:26, X= E or K), and XSGAGGQ (SEQ ID NO:27; X = T or A). 6 45741195.1 YU 8909 PCT Figure 2C is a schematic representation of YFP:mCherry fluorescent reporter used to assess release activity at one of four target codons (X = GCG, UAG, UGA, or UAA) within a peptide linker between mCherry and YFP. Release activity at codon X prevents downstream YFP expression and fluorescence without compromising mCherry. High YFP fluorescence indicates 5 readthrough. A color key for sections 2C-2E is located to the right. Figure 2D-2E are bar graphs of mCherry (above) and YFP fluorescence (below) expressed as a function of translation termination or readthrough at target codon X during reporter translation within variant strains with release factors. Fluorescence is normalized to GCG in Fig.2D, absence, or Fig.2E presence, of UAG- or UGA-suppressing SupD tRNAs. Statistical significance is displayed 10 compared to rEc∆1.∆A. Unpaired t-tests (n = 3), * = p-value < 0.05, ** = p-value < 0.01. Error bars display standard errors of the mean. Figure 3A is a bar graph of RF variant strains challenged with λ phage to assess phage infectivity with genes terminating with TAG, TAA, and TGA. Figure 3B is a bar graph of RF variant strains challenged with µ phage to assess phage infectivity with genes terminating at 15 TAA and TGA only. Statistics for Figs.3A-3B were based on an unpaired t-test in relation to E. coli MG1655. Error bars display confidence interval 0.95 of n = 3. Unpaired t-tests (n = 3), where * = p-value < 0.05, ** = p-value < 0.01, n.s. = not significant. Figure 4A is a schematic comparison of WT and mutant (A37G) tRNATrpanticodon loops between S. cerevisiae and E. coli, depicting loss of anticodon modification and codon 20 recognition in mutant forms. Figure 4B is a bar graph of L-arabinose (Ara)-inducible TyrOTS suppression of UGA within a YFP:mCherry fluorescent reporter expressed within rEc∆2.mB-oP strains with WT- or mutant tRNATrp. Statistical significance is displayed compared to no TyrOTS and no Ara (inducer) for each strain. Unpaired t-tests (n = 3), * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001. Error bars display standard errors of the mean. Figure 4C 25 is a bar graph of stacked mass spectrometry data depicting identities of incorporated residues at UGA from MS-Read analysis of rEc∆2.mB-oP strains with WT- or mutant tRNATrp. Figure 5 is a set of bar graphs of maximum cellular density measured at OD600 (MaxOD) and doubling times (DT) of RF variant strains within nutrient rich LB (top) and nutrient poor M9 (bottom) liquid media. Statistical significance is displayed compared to 30 rEc∆1.∆A. Error bars display confidence interval 0.95 of n = 7-8 replicates. Mann-Whitney U tests, * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, n.s. = not significant. Figure 6A is a schematic depicting dual OTS and fluorescent reporter expression system. L-arabinose inducible aaRSs and accompanying constitutively expressed UAG- and UGA- suppressing tRNAs coordinate charging and incorporation of nsAAs Bock and pAcF at UAG 7 45741195.1 YU 8909 PCT and UGA, respectively, within one of three aTc-inducible fluorescent ELP-GFP reporters. Each reporter contains either 3 instances of TAG, TGA, or TAG and TGA within a central ELP-GFP linker to study single or dual nsAA encoding. Diagrammatic key presenting nsAA incorporation at UAG, UGA, or both within four variant recoded strains. To the right is a color key for Figs. 5 6B-6D. Figure 6B-6D are bar graphs of fluorescent expression of ELP-GFP reporter containing: Fig.6B, 3 TAG codons, Fig.6C, 3 TGA codons, or Fig.6D, 3 TAG and 3 TGA codons within variant strains in the absence or presence of one or both: BocK and pAcF. Statistical significance is displayed compared to rEc∆1.∆A. Error bars display confidence interval 0.95 of n = 3. Unpaired t-tests (n = 3), * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, **** =10 p-value < 0.0001. Figure 6E is a diagrammatic depiction of dual reporter ELP-3x(TGA-TAG)- GFP with TAG (red) and TGA (blue) stop codon positions and associated nsAAs. Figure 6F is chromatogram of mass spectrometry data revealing incorporation of BocK and pAcF within Ochre-expressed ELP-3x(TGA-TAG)-GFP reporter proteins. Figure 6G is a bar graph showing MS-READ % peptide intensity for incorporations of projected nsAAs BocK at 3 UAGs, pAcF at 15 3 UGAs, or misincorporations found within all digested peptides examined. Figure 6H is a zoom in bar graph of misincorporations by % peptide intensity from Fig.5G MS-READ data. Figure 7 is a TGA-conversion summary table. Genes and pseudogenes containing TGA codons and those deliberately converted to TAA, deleted, or deactivated (i.e. garK). Release factor 2 gene prfB contains 2 TGAs converted to TAA, counted as one converted gene. Over 300 20 open reading frames (ORFs) overlapping TGA codons were intentionally mutated as a result of targeted TGA-to-TAA conversions via MAGE. Twenty-one ORFs remain unrecoded, including 3 selenoproteins that contain UGA-specific SECIS elements, 8 transposons, and 10 predicted pseudogenes leftover from extra and unnecessary TGA conversion targeting. See Table 9 for full list of TGA conversions. Unrecoded ORFs wither terminal TGAs include: agaA, agaW, pbI, 25 peaD, yrdE, yeeP, ylbI, yjhZ, yibZ, yncI, insQ, insI1(2-3), insE1(1-5). Figure 8 is a figure displaying all insertions and deletions greater than 50bp identified through breseq1compared to progenitor strain rEc∆1.∆A2as a reference genome. Details on deliberate mutations can be found in Table 2. Figure 9 is a diagrammatic sample of CAGE-mediated assembly of partially recoded 30 ∆TGA regions within rEc∆2E.∆A(EF) & (D). Selectable markers are depicted by rectangles (brown = hygR; green = tolC; blue = specR; yellow = gentR; orange = tetA). RK24 plasmid and an OriT-kanR(Origin of Transfer (OriT) adjacent to a kanamycin selectable marker (kanR) – depicted by black triangle) are present in all donor strains. Point of OriT-kanRtriangle conveys counter-clockwise directional orientation. Selectable marker (e.g. tolC) is placed 3-5kb 8 45741195.1 YU 8909 PCT downstream of the OriT. Two selectable markers are placed flanking recoded regions to select for and preserve these regions within final strains. To select for multiple recoded regions with greater than two antibiotics, subsequent rounds of selection with additional antibiotics are performed. Marker gentRwas overwritten by conjugal transfer and homologous recombination, 5 while kanRis excluded by its position upstream of OriT. Offspring strain is confirmed via MASC-PCR of known recoded sites, followed by whole genome sequencing if all MASC-PCR sites are positive for TGA-to-TAA conversions. Graphs and table to the right depict comparative kinetic growth curve data (OD600 over time in hours) in nutrient rich (LB) and minimal media (M9) for donor, recipient, and offspring strains. Grey lines depict the phenotypic variation in 10 alternative offspring strains, resulting from the same conjugation experiment, that revealed undesirable phenotypic or genotypic results (e.g. containing unrecoded TGA sites in region EF). Graph display of MaxOD and DT readings are single well n =1 samples corresponding to example well numbers in black boxes. Figure data is real and representative of typical experimental results. 15 Figure 10 are a set of bar graphs of growth data, MaxOD and Doubling Time (hours), for recoded strains with release factor variants after 36 hours incubation at 37°C in variable media: Lysogeny Broth (LB), 2x Yeast Tryptone (2xYT), and Terrific Broth (TB). Errors bars display confidence interval 0.95 for growth replicates n = 4. Figure 11 is a table mutation frequencies resulting from six MAGE cycles with nine 20 oligos targeting prfB within rEc∆2.∆A produced eleven unique genotypes. The final MAGE cycle culture was streaked onto LB agar and grown overnight at 34°C. Colonies were picked into a 96-well plate and PCR screened via MASC primers. Conversion frequencies are listed. One well yielded the desired S205P mutation alongside E170K. Following the same protocol, multiple attempts to revert E170K, leaving S205P alone, failed. 1 = presence of mutant band in 25 MASC gel; 0 = presence of WT band. Strains from wells F5 and H4 presented both Mut and WT genotypes, but resolved into two distinct genotypes, designated .1 and .2, when streaked onto agar plates and rescreened. Mutations not listed as columns had 0 conversion frequency. Figure 12 is a set of bar graphs of non-normalized mCherry (top) and YFP (middle) fluorescence (corresponds to Fig.2C-2E), along with strain OD at time of measurement 30 (bottom) resulting from YFP:mCherry fluorescent assay presenting readthrough and Ser suppression at GCG, UAG, UGA, and UAA, normalized to GCG (+ control), in the absence and presence of UAG- or UGA-suppressing SupD tRNAs. Error bars display standard errors of the mean, n = 3. 9 45741195.1 YU 8909 PCT Figure 13 is a set of bar graphs of normalized mCherry and YFP readthrough fluorescence (same assay as Fig.2D – “No Suppressor”) interrogating the impacts of E170K or S205P mutations in mRF2 release activity. mCherry (top) and YFP (bottom) fluorescence 5 resulting from YFP:mCherry fluorescent assay presenting translation termination or native suppression (readthrough) at GCG, UAG, UGA, and UAA, normalized to GCG (+ control) mCherry or YFP fluorescence, respectively. Statistical significance is displayed compared to rEc∆1.∆A. Unpaired t-tests (n = 3), * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001. Error bars display standard errors of the mean, n = 3. 10 Figure 14A is a set of close-up images from viral relative titer assays for^λ phage.^ Figure 14B is a set of close-up images from viral relative titer assays for µ ^phage.^Spots withphage λ^or µ at high multiplicity of infection are clear of bacterial^growth for all strains.^ Figure 15 is a set of images depicting fully recoded strain cell morphology with wildtype and mutant RF2 imaged via differential interference contrast (DIC) brightfield Leica DMi8 S 15 Wide-Field microscopy using a 100x oil immersion objective, 1.6x internal lens. Recoding and RF2 mutations do not cause cell aggregation or a filamentous phenotype, which are indicators of cell stress. Scale bars are 16.6µm. Figure 16 is a set of illustrated structures of BocK & pAcF and purified protein yields. Non-standard amino acids (nsAAs) Nε-Boc-L-lysine (BocK) and para-acetyl-L-phenylalanine 20 (pAcF) incorporated at UAG and UGA, respectively. Yields of purified ELP-GFP containing BocK and pAcF incorporations at one and three pairs of interspersed TGA and TAG codons expressed within dual recoded Ochre.tW* and unrecoded BL21. DETAILED DESCRIPTION OF THE INVENTION I. Definitions 25 As used herein, the terms “transfer RNA” and “tRNA” refers to a set of genetically encoded RNAs that act during protein synthesis as adaptor molecules, matching individual amino acids to their corresponding codon on a messenger RNA (mRNA). tRNAs assume a secondary structure with four base paired stems known as the cloverleaf structure. The tRNA contains a stem-loop with an anticodon. The anticodon is complementary to the codon 30 specifying the tRNA’s corresponding amino acid. The anticodon is in the loop that is opposite of the stem (acceptor stem) containing the terminal nucleotides. The 3' end of a tRNA acceptor stem is aminoacylated by a tRNA synthetase so that an amino acid is attached to the 3’end of the tRNA. This amino acid is delivered to a growing polypeptide chain as the anticodon sequence of the tRNA reads a codon triplet in an mRNA. 10 45741195.1 YU 8909 PCT As used herein, the term “anticodon” refers to a unit made up of typically three nucleotides that correspond to the three bases of a codon on the mRNA. Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid or “stop codon.” Known “stop codons” include, but are not limited to, the three codon bases, UAA 5 known as ochre, UAG known as amber and UGA known as opal, which do not code for an amino acid but act as signals for the termination of protein synthesis. tRNAs do not decode stop codons robustly under natural conditions, but can and have been engineered to do so. Stop codons are usually recognized by enzymes (release factors) that cleave the polypeptide as opposed to encode an AA via a tRNA. 10 As used herein, the term “suppressor tRNA” refers to a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system. For example, a nonsense suppressor tRNA can read through a stop codon. As used herein, the term “aminoacyl tRNA synthetase (AARS)” refers to an enzyme that catalyzes the esterification of a specific amino acid or its precursor to one of all its compatible 15 cognate tRNAs to form an aminoacyl-tRNA. These charged aminoacyl tRNAs then participate in mRNA translation and protein synthesis. The AARS show high specificity for charging a specific tRNA with the appropriate amino acid. In general, there is at least one AARS for each of the twenty amino acids. As used herein, the term “residue” refers to an amino acid that is incorporated into a 20 protein. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids. As used herein, the terms “polynucleotide” and “nucleic acid sequence” refers to a natural or synthetic molecule including two or more nucleotides linked by a phosphate group at 25 the 3’ position of one nucleotide to the 5’ end of another nucleotide. The polynucleotide is not limited by length, and the polynucleotide can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As used herein, the terms “transformation” and “transfection” refer to the introduction of a polynucleotide, e.g., an expression vector, into a recipient cell or introduction of a 30 polynucleotide to the chromosomal DNA of the cell. As used herein, the term “transgenic organism” refers to any organism, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way 11 45741195.1 YU 8909 PCT of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. Suitable transgenic organisms include, but are not limited to, bacteria, cyanobacteria, fungi, plants and animals. The nucleic acids described herein can be introduced into the host by methods known in the art, for example infection, transfection, transformation or 5 transconjugation. As used herein, the term “eukaryote” or “eukaryotic” refers to organisms or cells or tissues derived from these organisms belonging to the phylogenetic domain Eukarya such as animals (e.g., mammals, insects, reptiles, and birds), ciliates, plants (e.g., monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists. 10 As used herein, the term “prokaryote” or “prokaryotic” refers to organisms including, but not limited to, organisms of the Eubacteria phylogenetic domain, such as Escherichia coli, Thermus thermophilus, and Bacillus stearothermophilus, or organisms of the Archaea phylogenetic domain such as, Methanocaldococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species 15 NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, and Aeuropyrum pernix. As used herein, the term “isolated” is meant to describe a compound of interest (e.g., nucleic acids) that is in an environment different from that in which the compound naturally occurs, e.g., separated from its natural milieu such as by concentrating a peptide to a 20 concentration at which it is not found in nature. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and / or in which the compound of interest is partially or substantially purified. Isolated nucleic acids are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components. As used herein, the term “purified” and like terms relate to the isolation of a molecule or 25 compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in a native environment. As used herein, the term “translation system” refers to the components that facilitate incorporation of an amino acid into a growing polypeptide chain (protein). Key components of a 30 translation system generally include at least AARS and tRNA, and may also include amino acids, ribosomes, AARS, EF-Tu, RFs, and mRNA. As used herein, the term “orthogonal translation system (OTS)” refers to at least an AARS and paired tRNA that are both heterologous to a host or translational system in which 12 45741195.1 YU 8909 PCT they can participate in translation of an mRNA including at least one codon that can hybridize to the anticodon of the tRNA. As used herein, the terms “recoded organism” and “genomically recoded organism (GRO)” in the context of codons refer to an organism in which the genetic code of the organism 5 has been altered such that at least one codon has been eliminated from the genetic code by reassignment to a synonymous or nonsynonymous codon. As used herein, the term “polyspecific” refers to an AARS that can accept and incorporate two or more different non-standard amino acids. As used herein, the terms “protein,” “polypeptide,” and “peptide” refers to a natural or 10 synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The term polypeptide includes proteins and fragments thereof. The polypeptides can be “exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are 15 written left to right in the direction from the amino to the carboxy terminus. As used herein, “standard amino acid” and “canonical amino acid” refer to the twenty amino acids that are encoded directly by the codons of the universal genetic code denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), 20 Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V), and in rare cases Selenocysteine (Sec). As used herein, “non-standard amino acid (nsAA)” refers to any and all amino acids that 25 are not a standard amino acid. nsAA can be created by enzymes through posttranslational modifications; or those that are not found in nature and are entirely synthetic (e.g., synthetic amino acids (sAA)). In both classes, the nsAAs can be made synthetically. WO 2015 / 120287 provides a non-exhaustive list of exemplary non-standard and synthetic amino acids that are known in the art (see, e.g., Table 11 of WO 2015 / 120287). 30 As used herein, “genetically modified organism (GMO)” refers to any organism whose genetic material has been modified (e.g., altered, supplemented, etc.) using genetic engineering techniques. The modification can be extrachromasomal (e.g., an episome, plasmid, etc.), by insertion or modification of the organism’s genome, or a combination thereof. 13 45741195.1 YU 8909 PCT As used herein, the term “gene” refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term “gene” also refers to a DNA sequence that encodes an RNA product, for example a functional RNA that does not encode a protein or polypeptide (e.g., 5 miRNA, tRNA, etc.). The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’untranslated ends. The term gene as used herein with reference to recombinant expression constructs may, but need not, include intervening, non-coding regions, regulatory regions, and / or 5’ and 3’untranslated ends. Thus, with respect to a recombinant expression constructs, a gene 10 may be only an open reading frame (ORF). As used herein, the term “construct” refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism, also referred to “expression constructs”, include in the 5’-3’ direction, a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct 15 may also include selectable marker gene(s) and other regulatory elements for expression. As used herein, the term “vector” refers to a polynucleotide capable of transporting into a cell another polynucleotide to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control 20 element). “Plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector. As used herein, the term “operatively linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences 25 operatively linked to other sequences. For example, operative linkage of gene to a transcriptional control element refers to the physical and functional relationship between the gene and promoter such that the transcription of the gene is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA. As used herein, term “expression control sequence” refers to a DNA sequence that 30 controls and regulates the transcription and / or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. 14 45741195.1 YU 8909 PCT As used herein, the term “promoter” refers to a regulatory nucleic acid sequence, typically located upstream (5’) of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence. These include constitutive promoters, inducible promoters, tissue- and cell-specific 5 promoters and developmentally-regulated promoters. As used herein, the terms “transformed,” “transgenic,” “transfected” and “recombinant” refer to a host organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal 10 molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed,” “non-transgenic,” or “non-recombinant” host refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule. 15 As used herein, the term “endogenous” with regard to a nucleic acid refers to nucleic acids normally present in the host. As used here, the term “heterologous” refers to elements occurring where they are not normally found. For example, a promoter may be linked to a heterologous nucleic acid sequence, e.g., a sequence that is not normally found operably linked to the promoter. When 20 used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter sequence may be a control / regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter. The term “heterologous” thus can also encompass “exogenous” 25 and “non-native” elements. As used herein, the term “identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can also mean the degree of sequence relatedness 30 of a polypeptide compared to the full-length of a reference polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, 15 45741195.1 YU 8909 PCT H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). 5 Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 10 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure. By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. 15 Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference 20 sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide. Disclosed are materials, compositions, and components that can be used for, can be used 25 in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and 30 described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example 16 45741195.1 YU 8909 PCT of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. 5 Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any 10 group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that 15 each such combination is specifically contemplated and should be considered disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No 20 language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were 25 individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values can 30 range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. II. Genomically Recoded Organisms Genomically recoded organisms (GRO) and partially recoded intermediates thereof are provided. The GRO is a cell or cells, preferably a bacterial strain, for example, an E. coli 17 45741195.1 YU 8909 PCT bacterial strain, wherein one or more codons has been replaced by a synonymous or even a non- synonymous codon. Because there are 64 possible 3-base codons, but only 20 canonical amino acids (plus stop codons), some amino acids are coded for by 2, 3, 4, or 6 different codons (referred to herein as “synonymous codons”). In a GRO, most or all, of the instances of a 5 particular codon are replaced with a synonymous or non-synonymous codon. In some forms, the GRO is recoded such that at least one codon is completely absent from the genome (also referred to as an eliminated codon). In some forms, it is impractical or impossible to remove all iterations of codon. In such forms, the GRO is recoded such that at least one codon is nearly absent from the genome, and nonetheless creates a functionally “open” codon that can be 10 reassigned. In some forms, only one codon is recoded. In some forms, two, three, four, five, six, seven, eight, nine, ten, or more codons are recoded. Removal of a codon(s) from the precursor of the GRO allows reintroduction of the deleted codon in a heterologous mRNA of interest (also referred to as “reassignment”). As discussed in more detail below, the reintroduced codon can 15 be dedicated to a non-standard amino acid, which in the presence of the appropriate orthogonal translation machinery, can be incorporated in the nascent peptide chain of during translation of the mRNA. When a sense codon is eliminated, its elimination is preferably accompanied by mutation, or reduction or elimination of expression, of the cognate tRNA that decodes the codon during 20 translation, reducing or eliminating the recognition of the codon by the tRNA. For example, the tRNA can be deleted from the organism, the tRNA can be mutated to recognize fewer or different codons (e.g., from recognizing AUA and AUC to just recognizing AUC), etc. In preferred forms, tRNAs that decode a particular codon(s) are deleted, as in some instances (due to Wobble effect), one tRNA decodes >1 codon (e.g., AGG, AGA). 25 As discussed in more detail below, when a nonsense codon is reduced or eliminated, its reduction or elimination is preferably accompanied by mutation, reduction, or deletion of the endogenous factor or factors, for example, release factor(s), associated with terminating translation at the nonsense codon (e.g., to reduce or eliminate expression of the release factor or change the recognition specificity of codons for the release factor). 30 In some forms, wherein the organism does not have or use certain codon(s), the unused (i.e., reduced or eliminated) codon may not be strictly considered sense or nonsense codons, but can nonetheless be utilized in the strategies discussed herein. For example, a host organism can be created by taking a codon an organism does not have or use, but can still be recognized and 18 45741195.1 YU 8909 PCT mutating its translation machinery, e.g., tRNA and / or factors such release factors, to have a greater specificity, thus creating an unassigned codon. In some forms, a sense codon is reassigned as a nonsense codon. Typically, a release factor that recognizes the reassigned nonsense codon is also expressed by such organisms. 5 Different organisms often show particular preferences for one of the several codons that encode the same amino acid, and some codons are considered rare or infrequent. Preferably, the replaced codon is one that is rare or infrequent in the genome. The replaced codon can be one that codes for an amino acid (i.e., a sense codon) or a translation termination codon (i.e., a stop codon). 10 Prokaryotes useful as GRO cells include, but are not limited to, gram negative or gram positive organisms such as E. coli or Bacilli, and although the most preferred host organism is a bacterial GRO, it will be appreciated the methods and compositions disclosed herein can be adapted for use on other host GRO organisms, including, but not limited to, eukaryotic cells, including e.g., yeast, fungi, insect, plant, animal, human, etc. cells, and, viruses. 15 In some forms, the GRO is a bacterium preferably belonging to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus. The latter bacterium belonging to the phylum Proteobacteria belongs optionally to the family Enterobacteriaceae, optionally to the species Escherichia coli. The latter bacterium optionally relates to any strain belonging to the species Escherichia coli 20 such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. In some forms, the GRO is a cultivated Escherichia coli strains—designated as E. coli K12 strains—which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, 25 M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. In some forms the GRO is a bacterium belonging to the phylum Firmicutes optionally a Bacilli, optionally from the species Bacillus. The some forms the GRO is a fungus belonging optionally of the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. In some forms, the GRO strain belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of 30 the Deuteromycota or the phylum of the Zygomycetes. In some forms, the GRO strain belongs to the genus Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces. GRO can have two, three, or more codons replaced with a synonymous codon. Such GRO allow for reintroduction of the two, three, or more deleted codons in a heterologous mRNA 19 45741195.1 YU 8909 PCT of interest, each dedicated to a different non-standard amino acid. Such GRO can be used in combination with the appropriate orthogonal translation machinery to produce polypeptides having one or more instances each of two, three, or more different non-standard amino acids. The disclosed cells include partial or final recoding of TGA to form a final, mature GRO, 5 or a partially recoded intermediate thereof. In some forms, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 92, 94, 95, 96, 97, 98, 99, or 100 percent of the TGA are eliminated from the parental organism’s genome. In some forms, only an integer number or range of integers between 1-100, preferable 1-50, more preferable 1-25 of TGA remain. In some forms, no TGA remain. 10 Preferably, the cells are a ΔTGA GRO with most or all of the TGA removed or mutated to form a functionally “open” TGA codon. Thus, a ΔTGA GRO can be a bacteria genomically recoded from a parent bacteria such that at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of TGA codons present in the parent bacteria are eliminated. The parent GRO, e.g., bacteria, can be a native or genetically engineered strain, e.g., bacteria. The parent bacteria may be a native or 15 genetically modified bacteria such as those mentioned above and elsewhere herein. In a particular form, the parental bacteria is one within NCBI taxonomy ID 562 in genetically modified or unmodified form. For example, in the experiments below, the modified strains are derived from the MG1655 genome (www.ncbi.nlm.nih.gov / datasets / taxonomy / 511145 / ). Any of the ΔTGA GRO or partially recoded intermediates thereof can optionally further include 20 recoding of one or more additional codons, mutation or elimination of one or more release factors, mutation or elimination of one or more tRNAs, incorporation of one or more orthogonal tRNA-AARS pairs, or a combination thereof. Each of these aspects is discussed in more detail below. A. Recoded TGA GRO 25 ΔTGA GRO and partially recoded intermediates thereof are provided. Several strategies can be employed alone or in any combination to create a GRO with reduced iterations of TGA. In E. coli, all TGA codons, with few exceptions, occur at the end of a gene to terminate translation. According to GenBank, of the 1,216 annotated open reading frames (ORFs) terminating in TGA, 1,171 are annotated genes, while 45 are listed as pseudogenes. 30 In one strategy, nonessential genes and / or pseudogenes are partially or completely removed from the genome singularly or in blocks of two or more genes. This strategy removes the TGA codon(s) from the genome by removing one or more entire genes in which the TGA codon(s) occur. 20 45741195.1 YU 8909 PCT In another strategy, single-nucleotide substitution(s) (SNS) are introduced to convert TGA to another codon, most typically TAA. In most cases, this strategy can be employed to preserve a stop codon at the TGA location. 5 However, in E. coli, 380 terminal TGA codons overlap a neighboring gene and most cannot be converted in this way without compromising expression of neighboring genes. This overlap can fall into one of three categories (Fig.1C). In the first category (26 instances in E. coli), the A of TGA and ATG is shared, and thus mutating G to A in the TGA codon does not alter the ATG codon. 10 In the second category (257 instances in E. coli), both the T and G are shared between the ATG and TGA codons. Therefore, recoding TGA to TAA disrupts the start codon, which could preclude translation of the overlapped gene. To avoid this problem, another recoding strategy utilizes the insertion of three bases – TAA – which preserves both a new TAA codon and the ATG start codon, albeit with the ribosome binding site (RBS) shifted by three nucleotides from 15 the start codon. A third category of overlap (97 instances in E. coli) encompasses all instances where two genes overlap extensively so recoding inevitably introduces a mutation in the overlapped gene. In the latter two categories of TGA overlap, specialized oligos can be designed to recode the TGA-containing gene to minimize polar effects. Polar effects are when the translation and 20 expression of a downstream gene is compromised or altered by mutations within a target gene. Specialized oligos are designed to make the genetic changes listed in the above three categories. These changes permit mutation of the TGA stop codon to TAA of a target gene, while preserving start codon function and general translation capacity of the downstream overlapped genes. Figure 1C illustrates this concept. See, e.g. the middle panel of Fig 1C (“start codon”). 25 Thus, in some forms of a ΔTGA GRO, one or more TGA codons are not recoded. Non- recoded TGA codon(s) may be recalcitrant to recoding, present in pseudogenes, present in an insertion element, and / or present as an internal selenocycsteine codon. For example, three formate dehydrogenase genes (fdhF, fdoG, and fdnG) contained internal TGA codons and may be avoided for recoding due to their translational dependence on specialized selenocysteine 30 insertion sequence (SECIS) elements for UGA decoding. In another example from the experiments below, one terminal TGA within the nonessential glycerate kinase I gene, garK, was recalcitrant to conversion and ultimately deactivated by introducing a frameshift and premature nonsense mutation. 21 45741195.1 YU 8909 PCT In the exemplary GRO discussed in the experiments below, out of 1,216 predicted TGA codons in E. coli, 1,195 are abolished, leaving 10 predicted pseudogenes, 8 transposable elements, and 3 internal selenocysteine codons unrecoded. 76 nonessential genes and 3 pseudogenes were removed within 16 targeted genomic deletions. See, e.g., Figure 7. 5 These non-recoded TGA can be found as follows: Table 10: Exemplary Non-recoded TGA. Gene List: insE1 314,641 Not Recoded IS3 transposase A - copy 1 insE2 391653 Not Recoded IS3 trans osase A - co 2 ylbI 528,640 Not Recoded pseudogene - RHS element protein the 10 foregoing. 22 45741195.1 YU 8909 PCT In the experiments below, the modified strains are derived from the MG1655 genome (ncbi.nlm.nih.gov / datasets / taxonomy / 511145 / ). Recoding efforts can be accomplished by any single or combination of established methods such as DNA synthesis and genome engineering methods (e.g. multiplexed oligo-based 5 mutagenesis - MAGE - and conjugation-based assembly - CAGE), some of which are exemplified in the experiments below and / or are otherwise known in the art. The construction strategy can include two more phases. Cells in the construction phase, typically having less recoded TGA then needed to create a functionally open TGA codon, are referred to herein as ΔTGA GRO intermediates. 10 For example, in the experiments below, a first phase included construction of a preliminary ΔTGA-essentials strain that included recoding all 71 essential genes terminating in TGA to TAA. Second, construction of the final ΔTGA strain was accomplished by eliminating remaining TGA codons (Fig.1E). Both phases can employed iterative cycles of MAGE on distinct subdomains of the genome split amongst clonal progenitor strains. This step can be 15 followed by CAGE to assemble recoded subdomains into combined-genomic strains within each phase. For example, in a first phase, construction can involve the targeted conversion of 71 essential TGA-ending genes within two distinct genomic subdomains (i.e., A’ and B’). Additional oligos can be added to MAGE pools to converted proximal genes. Some TGA-ending genes can be deleted and include placing of selectable markers. Recoded subdomains A’ and B’ 20 can be assembled via CAGE. In a second phase, construction of fully ΔTGA-recoded GRO from ΔTGA-essentials strains generated in the first phase can include the conversion and / or deletion of most or all remaining nonessential ORFs. For example, in the experiments below, fully ΔTGA-recoded GRO from ΔTGA-essentials strains generated in the first phase included conversion of 25 nonessential ORFs (e.g., in the example below, 1,012 ORFs, 980 genes and 35 pseudogenes) via MAGE, alongside deletion of 229 nonessential ORFs (72 TGA genes and 3 TGA pseudogenes) using 15 additional targeted genomic deletions. Deletions often coincided with placement of selectable markers employed in CAGE, with markers and can be subsequently deleted via tolC placement-displacements. Conversions and deletions can be made sequentially or concurrently. 30 In the experiments below, conversions and deletions were made concurrently across eight distinct genomic subdomains each within a ΔTGA intermediate clone. Strains containing recoded subdomains can be hierarchically assembled via CAGE into the final ΔTGA GRO stain. As introduced above, the disclosed GRO includes recoding of the TGA codon, also referred to herein as ΔTGA to form a functionally open TGA codon. Exemplary ΔTGA GRO 23 45741195.1 YU 8909 PCT and numerous intermediate, partially recoded TGA strains used in the construction of a nearly fully recoded ΔTGA GRO are discussed in detail in the experiments below. All of the intermediate and final recoded ΔTGA disclosed in the experiments below, as well as similarly constructed intermediate and finally recoded ΔTGA, both with and without recoding of a second 5 or more codon (e.g., TAG), are expressly disclosed. Thus, intermediate and final recoded ΔTGA can include one or more of the features discussed herein (including those discussed in more detail in the experiments below), and can made according to one or more of the methods or strategies discussed herein. B. Recoded TGA+ GRO 10 In some forms, the ΔTGA GRO or partially recoded intermediate thereof include reduction or elimination of one or more additional codons (i.e., (“ΔTGA+”). Strategies for reducing or eliminate additional codons, including sense and non-sense codons, are introduce above. In some forms, the additional reduce or eliminated codon is one that codes for a rare stop codon. 15 Stop codons include TAG (UAG), TAA (UAA), and TGA (UGA). Although recoding to TGA (UGA) is discussed in more detail above, it will be appreciated that either another stop codon (or any sense codon) can be eliminated to form a second “open” codon, and optionally reintroduced encoding an amino acid such as a non-standard amino acid using the same strategy. Accordingly, in some forms, a sense codon is eliminated, e.g., AGG or AGA to CGG, CGA, 20 CGC, or CGG (arginine), e.g., as the principles can be extended to any set of synonymous or even non-synonymous codons, that are coding or non-coding. The foregoing is a non-limiting example, and in the same way, higher order recoded organisms having 3, 4, or more recoded codons can be constructed. In a particular form, the progenitor GRO of the TGA recoding is C321.ΔA [321 25 UAG→UAA conversions and deletion of prfA (encodes RF1)] (genome sequence at GenBank accession CP006698, also referred to herein as rEc∆1.∆A), or a further modified strain thereof. In this GRO the UAG is eliminated. That is, UAG has been transformed from a nonsense codon (terminates translation). UAG is a preferred codon for elimination or recoding because it is the rarest codon in Escherichia coli MG1655 (321 known instances) and a rich collection of 30 translation machinery capable of incorporating non-standard amino acids has been developed for UAG (Liu and Schultz, Annu. Rev. Biochem., 79:413-44 (2010), discussed in more detail below). Thus, in particular forms, the ΔTGA+ GRO is one in which most or all instances of the UAG (TAG) codon have been removed and replaced by another stop codon (e.g., TAA). Such GRO are also referred to herein as ΔTGA-ΔTAG and ΔTAG-ΔTGA GRO. In the experiments 24 45741195.1 YU 8909 PCT below C321.ΔA was used as the parental GRO for construction of a ΔTGA GRO, and thus the strains described in the experiments include recoding of both the TGA codon and the TAG codon, leaving TAA is a sole functional stop codon. For example, in some forms, the GRO are the exemplary ∆TGA and intermediates 5 thereof engineered from a progenitor ∆TAG genome (i.e., GRO rEc∆1.∆A) are provided in the Examples below. C. Mutation and / or Deletion of Release Factor(s) To establish an open TGA codon capable of reassignment as an amino acid encoding codon, the corresponding UGA-specific release factor is preferably altered to ensure the 10 reassigned codon is functional and not read as a nonsense codon during translation. As in most prokaryotes, E. coli release factor 1 (RF1), encoded by prfA, terminates translation at UAG and UAA, while release factor 2 (RF2), encoded by prfB, terminates translation at UGA and UAA. RF1 was previously deleted within ancestral rEcΔ1.∆A, confirming both the non-essential nature of RF1 in the absence of TAG codons and the ability of 15 RF2 to serve as the sole cellular release factor. Unlike RF1, E. coli RF2 serves central functions besides termination, including RF3-cooperative post-peptidyl transfer quality control and ArfA- cooperative codon-independent rescue of stalled ribosomal complexes from ‘non-stop’ mRNAs. The experiments below illustrate the inability to delete RF2 in the presence of RF1, despite an absence of genomic TGA codons. The experiments also show that the disclosed GRO is a 20 platform for the evolution of any RF that restricts STOP codon usage. Thus provided are methods of evolving and creating RF variants with changed properties such as different codon recognition. For example, provided are mutant RF2 proteins, and corresponding TGA-less genes encoding the same, and ΔTGA GRO and intermediates thereof expressing the same are 25 provided. In some forms, the mutant RF2 protein includes one or more of the following: N26, K170, P205, A246, or a combination thereof relative to B-wt amino acid sequence (SEQ ID NO:3). >RF2: B-wt peptide AA sequence 30 MFEINPVNNRIQDLTERSDVLRGYLDYDAKKERLEEVNAELEQPDVWNEPERAQALGKERSSLE AVVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFRRMFSGEYDSADC YLDIQAGSGGTEAQDWASMLERMYLRWAESRGFKTEIIEESEGEVAGIKSVTIKISGDYAYGWL RTETGVHRLVRKSPFDSGGRRHTSFSSAFVYPEVDDDIDIEINPADLRIDVYRTSGAGGQHVNR TESAVRITHIPTGIVTQCQNDRSQHKNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWG 25 45741195.1 YU 8909 PCT SQIRSYVLDDSRIKDLRTGVETRNTQAVLDGSLDQFIEASLKAGL* (SEQ ID NO:3). Thus provided are mutant RF2 proteins having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but not 100% sequence identity to SEQ ID NO:3, and preferably having one more of N26, K170, P205, 5 and A246, most preferably all four of N26, K170, P205, and A246, relative to SEQ ID NO:3. Typically, ΔTGA GRO and intermediates thereof expressing a nucleic acid encoding the mutant RF2 are viable and the mutant RF2 has reduced or no ability to recognize and terminate translation at UGA. Exemplary mutant RF2 amino acid sequence includes: 10 >mRF2: mB-oKP peptide AA sequence MFEINPVNNRIQDLTERSDVLRGYLNYDAKKERLEEVNAELEQPDVWNEPERAQALGKERSSLE AVVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFRRMFSGEYDSADC YLDIQAGSGGTEAQDWASMLERMYLRWAESRGFKTEIIEESKGEVAGIKSVTIKISGDYAYGWL RTETGVHRLVRKPPFDSGGRRHTSFSSAFVYPEVDDDIDIEINPADLRIDVYRASGAGGQHVNR 15 TESAVRITHIPTGIVTQCQNDRSQHKNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWG SQIRSYVLDDSRIKDLRTGVETRNTQAVLDGSLDQFIEASLKAGL* (SEQ ID NO:4), and variants thereof having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, and preferably having one more of N26, K170, P205, and A246, most preferably all four of N26, 20 K170, P205, and A246, relative to SEQ ID NO:4. Mutant prfB genes encoding mutant RF2 are also provided. In some forms, the mutant prfB includes one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the 25 adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to B-wt nucleotide sequence (SEQ ID NO:1). Thus provided are mutant prfB genes having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but not 30 100% sequence identity to SEQ ID NO:1, and preferably having one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a 26 45741195.1 YU 8909 PCT combination thereof relative to B-wt nucleotide sequence (SEQ ID NO:1). >RF2: B-wt nucleotide sequence atgtttgaaattaatccggtaaataatcgcattcaggacctcacggaacgctccgacgttctta gggggtatctttgactacgacgccaagaaagagcgtctggaagaagtaaacgccgagctggaac 5 agccggatgtctggaacgaacccgaacgcgcacaggcgctgggtaaagagcgttcctccctcga agccgttgtcgacaccctcgaccaaatgaaacaggggctggaagatgtttctggtctgctggaa ctggctgtagaagctgacgacgaagaaacctttaacgaagccgttgctgaactcgacgccctgg aagaaaaactggcgcagcttgagttccgccgtatgttctctggcgaatatgacagcgccgactg ctacctcgatattcaggcggggtctggcggtacggaagcacaggactgggcgagcatgcttgag10 cgtatgtatctgcgctgggcagaatcgcgtggtttcaaaactgaaatcatcgaagagtcggaag gtgaagtggcgggtattaaatccgtgacgatcaaaatctccggcgattacgcttacggctggct gcgtacagaaaccggcgttcaccgcctggtgcgtaaaagcccgtttgactccggcggtcgtcgc cacacgtcgttcagctccgcgtttgtttatccggaagttgatgatgatattgatatcgaaatca acccggcggatctgcgcattgacgtttatcgcacgtccggcgcgggcggtcagcacgttaaccg15 taccgaatctgcggtgcgtattacccacatcccgaccgggatcgtgacccagtgccagaacgac cgttcccagcacaagaacaaagatcaggccatgaagcagatgaaagcgaagctttatgaactgg agatgcagaagaaaaatgccgagaaacaggcgatggaagataacaaatccgacatcggctgggg cagccagattcgttcttatgtccttgatgactcccgcattaaagatctgcgcaccggggtagaa acccgcaacacgcaggccgtgctggacggcagcctggatcaatttatcgaagcaagtttgaaag 20 cagggttatga (SEQ ID NO:1) Exemplary mutant prfB gene nucleic acid sequences include >mRF2: mB-oKP nucleotide sequence atgtttgaaattaatccggtaaataatcgcattcaggacctcacggaacgctccgacgttctta gggggtatctttaactacgacgccaagaaagagcgtctggaagaagtaaacgccgagctggaac25 agccggatgtctggaacgaacccgaacgcgcacaggcgctgggtaaagagcgttcctccctcga agccgttgtcgacaccctcgaccaaatgaaacaggggctggaagatgtttctggtctgctggaa ctggctgtagaagctgacgacgaagaaacctttaacgaagccgttgctgaactcgacgccctgg aagaaaaactggcgcagcttgagttccgccgtatgttctctggcgaatatgacagcgccgactg ctacctcgatattcaggcggggtctggcggtacggaagcacaggactgggcgagcatgcttgag30 cgtatgtatctgcgctgggcagaatcgcgtggtttcaaaactgaaatcatcgaagagtcgaaag gtgaagtggcgggtattaaatccgtgacgatcaaaatctccggcgattacgcttacggctggct gcgtacagaaaccggcgttcaccgcctggtgcgtaaacctccgtttgactccggcggtcgtcgc cacacgtcgttcagctccgcgtttgtttatccggaagttgatgatgatattgatatcgaaatca acccggcggatctgcgcattgacgtttatcgcgcgtccggcgcgggcggtcagcacgttaaccg 27 45741195.1 YU 8909 PCT taccgaatctgcggtgcgtattacccacatcccgaccgggatcgtgacccagtgccagaacgac cgttcccagcacaagaacaaagatcaggccatgaagcagatgaaagcgaagctttatgaactgg agatgcagaagaaaaatgccgagaaacaggcgatggaagataacaaatccgacatcggctgggg cagccagattcgttcttatgtccttgatgactcccgcattaaagatctgcgcaccggggtagaa 5 acccgcaacacgcaggccgtgctggacggcagcctggatcaatttatcgaagcaagtttgaaag cagggttataatga (SEQ ID NO:2) and variants thereof having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2, and preferable having one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the 10 terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to mB-oKP nucleotide sequence (SEQ ID NO:2). Thus, in some forms, the disclosed ΔTGA GRO and partially recoded intermediates 15 thereof include a mutant RF2 that has reduced or no ability to recognize and terminate translation at UGA, optionally encoded be a mutant prfB gene. In some forms, the mutant RF2 optionally encoded by a mutant prfB gene, are those provided expressly above. The mutant prfB gene can be incorporated into the genome of the cell, expressed extrachromosomally, or a combination thereof. In some forms, the endogenous prfB loci of the progenitor ΔTGA GRO or 20 partially recoded intermediate thereof is mutated, and thus the genomic loci of the cell encodes the mutant prfB. In some forms, the ΔTGA GRO or partially recoded intermediate thereof does not encode a wildtype prfB such as SEQ ID NO:1. Similarly, when the disclosed ΔTGA GRO or partially recoded intermediate thereof further includes reduction or elimination of TAG codons, the cells can include removal, 25 mutation, or deletion of the gene encoding RF1 (i.e., prfA). D. Mutant tRNATrp(CCA) The experiments below revealed striking levels of Trp suppression including nearly 80% of residues at UGA, indicating significant suppression by tRNATrp(CCA) in ΔTGA GRO. This is because the native E. coli tRNATrp(Ec-tRNATrp) anticodon loop wobble pairs with UGA. 30 To establish an open TGA codon capable of reassignment to a desired amino acid encoding codon, and where the reassigned codon will be decoded by tRNA charged with desired amino acid with high efficiency and accuracy, Trp suppression should be reduced. In the experiments below, to reduce native Trp suppression, trpT, which encodes tRNATrp(CCA), was modified to reduce UGA recognition. In particular forms, A37 in native Ec-tRNATrpis 28 45741195.1 YU 8909 PCT substituted, preferably to G37 to disrupt the A36-A37-A38 recognition motif and prevent base modification leading to spurious decoding of UGA. The experimental results confirm that a single base substitution in Ec-tRNATrppreserves native Trp decoding while eliminating UGA suppression, removing the wobble effect and establishing an open UGA codon amenable for 5 reassignment. A sequence of the trpT gene is AGGGGCGTAGTTCAATTGGTAGAGCACCGGTCTCCAAAACCGGGTGTTGGGAGTTCGAGTCTCTCCGCCCCTGCCA (SEQ ID NO:5, Escherichia coli str. K-12 substr. MG1655, complete genome NCBI Reference Sequence: NC_000913.3), where the base targeted for mutation, A37, is illustrated in bold and 10 italics. Thus, in a specific example, a mutant trpT gene has the sequence AGGGGCGTAGTTCAATTGGTAGAGCACCGGTCTCCAGAACCGGGTGTTGGGAGTTCGAGTCTCTCCGCCCCTGCCA (SEQ ID NO:6), or a variant thereof with at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity 15 to SEQ ID NO:6, and having a mutation at A37, optionally the A37G mutation, preferably wherein the encoded tRNA preserves native Trp decoding while reducing or eliminating UGA suppression (i.e., removes wobble effect). Thus, in some forms, the disclosed ΔTGA GRO and partially recoded intermediates thereof include a mutant tRNATrp(CCA) optionally encoded by a mutant trpT gene, wherein the 20 tRNA preserves native Trp decoding while reducing or eliminating UGA suppression. In some forms, the mutant tRNATrp(CCA) optionally encoded by a mutant trpT gene, are those provided expressly above. The mutant trpT gene can be incorporated into the genome of the cell, expressed extrachromosomally, or a combination thereof. In some forms, the endogenous trpT loci of the progenitor ΔTGA GRO or partially recoded intermediate thereof is mutated, and thus 25 the genomic loci of the cell encodes the mutant trpT. In some forms, the ΔTGA GRO or partially recoded intermediate thereof does not encode a wildtype trpT such as SEQ ID NO:5. Similarly, when the disclosed ΔTGA GRO or partially recoded intermediate thereof further includes reassignment of one or sense codons, tRNA corresponding to an eliminated sense codon, etc. can be mutated or eliminated. 30 E. Additional Recombinant Constructs The GRO can be transformed or genetically engineered to express one or more additional recombinant constructs. For example, the GRO can be transformed or genetically engineered to express one or more orthogonal AARS-tRNA pairs and one or more mRNA of interest. As discussed in more detail below, the AARS-tRNA pair and mRNA of interest transformed or 29 45741195.1 YU 8909 PCT transfected into the host expressed extrachomasomally, for example by plasmid(s) or another vector(s) or an episome, or can be integrated into the host’s genome. The OTS system, particularly the antisense codon of the tRNA, can be designed to match a codon reintroduced by the mRNA(s), provided at least one codon remains eliminated. See, 5 e.g., Lajoie, et al., Science .2013 Oct 18;342(6156):357-60. doi: 10.1126 / science.1241459, and Chin, et al., Nature, 569(7757):514-518 (2019). doi: 10.1038 / s41586-019-1192-5, e.g., isoleucine, and Ostrov, et al., Science, 353(6301):819-822 (2016) DOI: 10.1126 / science.aaf3639, which describes reducing the number of codons in E. coli from 64 to 57 by removing instances of the UAG stop codon and 10 excising two arginine codons, two leucine codons, and two serine codons. See also F. Exemplary GRO Exemplary ∆TGA and intermediates thereof engineered from a progenitor ∆TAG genome (i.e., GRO rEc∆1.∆A) are provided. In some forms, the ∆TGA GRO or intermediate thereof includes elimination of any integer number or range of integers between 1 and 1,216 instances of 15 the stop codon TGA optionally wherein 1,195 are eliminated. In some forms, TGA codons that are not eliminated are left unrecoded (e.g., selenocystine encoding codons). In more specific forms, out of 1,216 predicted TGA codons in E. coli, 1,195 are abolished, leaving 10 predicted pseudogenes, 8 transposable elements, and 3 internal selenocysteine codons unrecoded. Thus, in some forms, at least 95, 96, 97, or 98 percent of the TGA are recoded. 20 Exemplary gene-specific modifications are outlined in Table 9. Each of these modifications is expressly disclosed alone and in all possible combinations. Alternatives to these specific modifications are also informed by the strategies provided herein, and thus, although they are expressly disclosed, the provided ∆TGA GRO are not limited to the specific combination of modifications outlined in Table 9, and encompass alternative means of 25 eliminating each of target TGA codons. In some forms, the disclosed GRO include engineering of associated translation machineries to free TGA as a second open codon for sense codon reassignment (e.g., RF2 and / or tRNATrpas discussed in more detail above). The resulting GRO can exhibit attenuated crosstalk between four codons within the 30 canonical code (TAG, TGA, TAA, and TGG), rendering each codon a unique translational function. Some forms include complete codon compression. As exemplified herein, the resulting GRO fully compresses a redundant codon (here illustrated with Stop codons) functionality into a single codon, liberating two other stop codons for reassignment. 30 45741195.1 YU 8909 PCT In preferred forms, a final recoded ΔTGA GRO includes a functionally open TGA codon that be used to code for an additional amino acid, such as a non-standard amino acid. Exemplary strains are discussed in more detail in the experiments and Tables below, all of which are expressly disclosed. 5 In particular preferred forms, the GRO is rEc∆2.∆A.mB-oKP.tW* (Ochre.tW*) of the experiments below, which includes the gene-specific modifications outlined in Table 9, the prfB of SEQ ID NO:2, and the trpT of SEQ ID NO:6. III. Compositions and Methods of Making Polypeptides with NSAAs The ∆TGA GRO and intermediates thereof can be used as host cells in methods of 10 making polypeptides containing one or more instances of one or more different non-standard amino acid. Thus, methods of making polypeptides containing one or more instances of one or a combination of two or more non-standard amino acids (nsAAs) are also provided. A. Systems Systems for making polypeptides including one or more non-standard amino acids are 15 provided. The systems typically include a host organism (i.e., a ∆TGA GRO or intermediate thereof) as well as one or more aminoacyl-tRNA synthetase (AARS) and paired transfer RNA (tRNA) pair (i.e., an orthogonal pair), and an mRNA encoding a polypeptide(s). As illustrated in the experiments below, ∆TAG-∆TGA GRO have two open codons, and can thereof accommodate incorporation of two different non-standard amino acid acids one or more user 20 defined locations in the same or separate mRNA. Thus, although typically discussed herein in the singular, it will be appreciated that two or more orthogonal pairs can be used in combination, particularly for GRO such as ∆TAG-∆TGA that contain two open codons. The AARS, tRNA, and mRNA are typically heterologous to the host GRO. The heterologous tRNA can include an anticodon that recognizes a reduced or missing codon. The25 heterologous AARS is typically one that can charge it’s paired heterologous tRNA with a non- standard amino acid. When a heterologous mRNA including at least one iteration of a GRO- deleted codon is expressed in the host in the presence of the non-standard amino acid, the non- standard amino acid is incorporated into the polypeptide by the heterologous tRNA during translation of the heterologous mRNA. 30 Nucleic acids encoding the orthogonal AARS and tRNA operably linked to one or more expression control sequences can be introduced or integrated into the GRO. The heterologous mRNA encoding the protein of interest can be introduced or integrated into the GRO, and can also be linked to an expression control sequence. 31 45741195.1 YU 8909 PCT B. Orthogonal Translation System Translation systems include most or all of the translation machinery of the host organism and additionally include a heterologous aminoacyl-tRNA synthetase (AARS)-rRNA pair (also referred to as an orthogonal translation system (OTS)) that can incorporate one or more non- 5 standard amino acids into a growing peptide during translation of the heterologous mRNA. AARS are enzymes that catalyze the esterification of a specific cognate amino acid or its precursor to one or all of its compatible cognate tRNAs to form an aminoacyl-tRNA. An AARS can be specific for a non-standard amino acid, or can be polyspecific for two or more non- standard amino acids, canonical amino acids, or a combination thereof. The heterologous 10 AARS used in the disclosed system can typically recognize, bind to, and transfer at least one non-standard amino acid to a cognate tRNA. Accordingly, the AARS can be selected by the practitioner based on the non-standard amino acid on interest. Some of the disclosed systems include two or more heterologous AARS. tRNA is an adaptor molecule composed of RNA, typically about 76 to about 90 15 nucleotides in length that carries an amino acid to the protein synthetic machinery. Typically, each type of tRNA molecule can be attached to only one type of amino acid, so each organism has many types of tRNA (in fact, because the genetic code contains multiple codons that specify the same amino acid, there are many tRNA molecules bearing different anticodons which also carry the same amino acid). The heterologous tRNA used in the disclosed systems is one that 20 can bind to the selected heterologous AARS and receive a non-standard amino acid to form an aminoacyl-tRNA. Because the transfer for the amino acid to the tRNA is dependent in-part on the binding of the tRNA to the AARS, these two components are typically selected by the practitioner based on their ability to interact with each other and participate in protein synthesis including the non-standard amino acid of choice in the host organism. Therefore, a selected25 heterologous AARS and tRNA are often referred to herein together as a heterologous AARS- tRNA pair, or an orthogonal translation system. Preferably, the heterologous AARS-tRNA pair does not cross-react with the existing host cell’s pool of synthetases and tRNAs, or does so only at a low or ineffecient level, but is recognized by the host ribosome. Therefore, preferably the heterologous AARS cannot charge an endogenous tRNA with a non-standard amino acid (or 30 does so a low frequency), and / or an endogenous AARS cannot charge the heterologous tRNA with a standard amino acid. Furthermore, preferably, the heterologous AARS cannot charge its paired heterologous tRNA with a standard amino acid (or does so at low frequency). The heterologous tRNA also includes an anticodon that recognizes the codon of the codon in the heterologous mRNA that encodes the non-standard amino acid of choice. In the 32 45741195.1 YU 8909 PCT most preferred form, the anticodon is one that hybridizes with a codon that is reduced or deleted in the host organism and reintroduced by the heterologous mRNA. In systems containing two or more open codons, the tRNA anticodon is designed or selected to decode the target open codon. For example, when the reduced or deleted target codon is UAG (TAG), the heterologous tRNA 5 anticodon is typically CUA, when the reduced or deleted target codon is UGA (TGA), the heterologous tRNA anticodon is typically UCA. The AARS-tRNA pair can be from an archaea, such as Methanococcus maripaludis, Methanocaldococcus jannaschii, Methanopyrus kandleri, Methanococcoides burtonii, Methanospirillum hungatei, Methanocorpusculum labreanum, Methanoregula boonei, 10 Methanococcus aeolicus, Methanococcus vannieli, Methanosarcina mazei, Methanosarcina barkeri, Methanosarcina acetivorans, Methanosaeta thermophila, Methanoculleus marisnigri, Methanocaldococcus vulcanius, Methanocaldococcus fervens, or Methanosphaerula palustris, for can be variant evolved therefrom. Suitable heterologous AARS-tRNA pairs for use in the disclosed systems and methods 15 are known in the art. For example, Table 7 and the electronic supplementary information provided in Dumas, et al., Chem. Sci., 6:50-69 (2015), provide non-natural amino acids that have been genetically encoded into proteins, the reported mutations in the AARS that permit their binding to the non-natural amino acid, the corresponding tRNA, and a host organism in which the translation system is operational. See also Liu and Schultz, Annu. Rev. Biochem., 79:413-44 20 (2010) and Davis and Chin, Nat. Rev. Mol. Cell Biol., 13:168-82 (2012), which provide additional examples of AARS-tRNA pairs which can be used in the disclosed systems and methods. Preferred AARS with improved activity and specificity for the specific non-naturally occurring amino acids are disclosed and described in WO 2015 / 120287, which is specifically incorporated by reference herein in its entirety. 25 The AARS and tRNA can be provided separately, or together, for example, as part of a single construct. Likewise, combinations of two or more AARS-tRNA pairs can be provided separately, or together, for example, as part of a single construct. In some forms, the construct is bi- or multicistronic. In some forms, the AARS is an evolved AARS. See, e.g., WO 2015 / 120287, which is 30 specifically incorporated by reference in its entirety. In a particular form, the AARS-tRNA pair is evolved from a Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (AARS) / suppressor tRNA pairs and suitable for use in an E. coli host organism. See, for example, Young, J. Mol. Biol., 395(2):361-74 (2010), which describes an OTS including constitutive and inducible promoters driving the transcription of two copies of a M. jannaschii 33 45741195.1 YU 8909 PCT AARS gene in combination with a suppressor tRNA(CUA)(opt) in a single-vector construct. During protein synthesis, tRNAs with attached amino acids are delivered to the ribosome by proteins called elongation factors (EF-Tu in bacteria, eEF-1 in eukaryotes), which aid in decoding the mRNA codon sequence. If the tRNA's anticodon matches the mRNA, another 5 tRNA already bound to the ribosome transfers the growing polypeptide chain from its 3’ end to the amino acid attached to the 3’ end of the newly delivered tRNA, a reaction catalyzed by the ribosome. Accordingly, the heterologous AARS-tRNA pair should be one that can be processed by the host organism’s elongation factor(s). Additional or alternatively, the system can include additional or alternative elongation factor variants or mutants that facilitate delivery of the 10 heterologous aminoacyl-tRNA to the ribosome. It will also be appreciated that methods of altering the anticodon of tRNA are known in the art. Any suitable tRNA selected for use in the disclosed systems and methods can be modified to hybridize to any desired codon. For example, although many of the heterologous tRNA disclosed here and elsewhere have a CUA anticodon, CUA can be substituted for another 15 stop anticodon (e.g., UUA or UCA), or anticodon for any desired sense codon. The tRNA anticodon can be selected based on the GRO and the sequence of the heterologous mRNA as discussed in more detail above. The OTS can also include mutated EF-Tu, in addition to AARS and tRNA, especially for bulky and / or highly charged NSAAs (e.g., phosphorylated amino acids) (Park, et al., Science, 20 333:1151-4 (2011)). C. Methods Making Polypeptides The methods typically involve using one or more orthogonal AARS-tRNA pair in the translation process for a target polypeptide from heterologous mRNA of interest. As discussed above, the AARS preferentially aminoacylates its cognate tRNA with a non-naturally occurring 25 amino acid. The resulting aminoacyl-tRNA recognizes at least one codon in the mRNA for the target protein, such as a reassigned stop codon. An elongation factor (such as EF-Tu in bacteria) mediates the entry of the amninoacyl-tRNA into a free site of the ribosome. If the codon- anticodon pairing is correct, the elongation factor hydrolyzes guanosine triphosphate (GTP) into guanosine diphosphate (GDP) and inorganic phosphate, and changes in conformation to 30 dissociate from the tRNA molecule. The aminoacyl-tRNA then fully enters the A site, where its non-standard amino acid is brought near the P site’s polypeptide and the ribosome catalyzes the covalent transfer of the pAzF onto the polypeptide. In some forms, the resulting polypeptides are treated with diazotransfer reaction, modified to include a further moiety or moieties using e.g., copper(I)-catalyzed azide-alkyne 34 45741195.1 YU 8909 PCT Huisgen cycloaddition (click-chemistry). See, e.g., WO 2022 / 235942, which is specifically incorporated by reference herein in its entirety. The resulting polypeptides can be isolated, purified, or otherwise enriched using methods known in the art, and discussed in more detail below. 5 In some forms, the heterologous AARS, its cognate tRNA, or more preferably both, are integrated into the host genome. Although suitable AARS are known in the art, in the most preferred forms, the AARS is a variant AARS that has improved binding to its cognate tRNA, its non-standard amino acid(s), or both compared to a known AARS. Exemplary variant AARS are discussed in more detail below. 10 The methods of making polypeptide are typically capable of producing polypeptides having a greater number of instances of non-standard amino acids and / or a greater yield of the desired polypeptide than the same or similar polypeptide made using conventional compositions, systems, and methods. The methods of making polypeptide are typically capable of producing polypeptides 15 having a greater number of iterations of non-standard amino acids and / or a greater yield of the desired polypeptide than the same or similar polypeptide made using conventional compositions, systems, and methods. Higher yield of the desired polypeptide can be measured as an increase in the amount of desired protein per total protein by weight or mass, or the amount of desired protein per culture 20 volume, relative to the same polypeptide made using conventional methods and reagents (i.e., in non-GRO host cells). For example, in some forms, the yield is increased by at least 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 300, 400, or 500 percent. In some forms, the yield is at least 5, 10, 15, 20, 25, 50, 75, or 100 mg / L. In an exemplary experiment illustrated in Figure 16, yield increased 40-fold and > 52-fold. 25 High purity of the desired polypeptide can be measured as at least 80%, 85%, 90%, 95%, 96%, 07%, 98%, or 99% correct non-standard amino acid incorporated at the desired residue(s) relative to an undesired amino acid at the same residue. The methods are able to produce biopolymers, optionally in high yields, with multiple nsAAs, and still maintain high purity. Purity can be determined using routine methods such as mass spectroscopy. Purity is largely 30 achieved from two areas: properties of OTS to encode the desired / cognate nsAA while eliminating other nsAAs or natural amino acids; and conducting such method in GRO background with a dedicated codon free from interference / competition from native biomolecular components. In preferred forms, the disclosed methods can achieve multi-site incorporation of one or more different nsAA with high yield and / or high purity of the desire polypeptide. 35 45741195.1 YU 8909 PCT In some forms, the polypeptides are ones that could not be made using conventional methods and reagents, or could not be made a sufficient yield and / or purity to serve a practical purpose using conventional methods and reagents. D. Compositions for Making Polypeptides with Nonstandard Amino Acids 5 1. Variant AARS Methods of making variant AARS are provided in WO 2015 / 120287, and variant AARS obtaining according to the method, including, but not limited to those provided in WO 2015 / 120287 are provided and can be used in the disclosed methods. DNA sequence(s) can also be deduced from the amino acid sequence of the variant. Accordingly, nucleic acid sequences 10 encoding variant AARS are also provided. The precise percentage of similarity between sequences that is useful in establishing sequence identity varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish sequence identity. Higher levels of sequence similarity, e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to 15 establish sequence identity. Therefore, in some forms, the variant includes at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more sequence identity with the parent AARS. Variant AARS of a parent M. jannaschii AARS referred to pAcF AARS (pAcFRS) (Young, et al., J Mol Biol, 395:361-74 (2010)) are provided. The amino acid sequence for pAcFRS is 20 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK RPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL 25 (SEQ ID NO:7). The nucleic acid sequence for a cognate tRNA of SEQ ID NO:7 is CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCAGGGGTTCAAATCCC CTCCGCCGGACCA (SEQ ID NO:8). This tRNA can also be a cognate tRNA for the variant AARS described in more detail below. 30 Variants of pAcFRS have one or more mutations relative to SEQ ID NO:1, and typically have altered specificity and / or activity toward one or more non-standard amino acids and / or altered specificity and / or activity toward a paired tRNA relative to the protein of SEQ ID NO:7. In some forms, the variant includes at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more sequence identity with the parent AARS, or a functional fragment thereof. 36 45741195.1 YU 8909 PCT The variants typically have one or more substitution mutations in the non-standard amino acid (amino acid ligand) binding pocket of SEQ ID NO:7, the tRNA anticodon recognition interface of SEQ ID NO:7, or a combination thereof. For example, the variants can have a substitution mutation at one or more of amino acid positions 65, 107, 108, 109, 158, 159, 162, 5 167, 257, and 261 of SEQ ID NO:7 relative to the N-terminal methionine of SEQ ID NO:7. Exemplary variants are provided below and have nsAA specificities at least as provided. The relative polyspecificities (or monospecificy) of each are discussed in more detail in WO 2015 / 120287. pAcFRS.1 (polyspecifity for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, 10 MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK 15 RPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:9); pAcFRS.t1 (polyspecifity for at least pAcF, pAzF, StyA): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIH 20 NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK GPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:10); pAcFRS.t2 (polyspecifity for at least pAcF, pAzF, StyA): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK 25 RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK CPEKEGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:11); pAcFRS.1.t1 (polyspecifity for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF): 30 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK GPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:12); 37 45741195.1 YU 8909 PCT pAcFRS.1.t2 (polyspecifity for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK 5 RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK CPEKEGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:13); pAcFRS.2 (polyspecifity for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF). 10 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII VLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK RPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:14); 15 pAcFRS.2.t1 (polyspecifity for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF) MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII VLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQRKIHMLARELLPKKVVCIH 20 NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK GPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:15); pAcFRS.2.t2 (polyspecifity for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaAPheF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII 25 VLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVDVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK CPEKEGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:16); pAzFRS.1 (specific for pAzF): 30 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNVMHYDGVDVYVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK RPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:17); 38 45741195.1 YU 8909 PCT pAzFRS.1.t1 (specific for pAzF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII 5 LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNVMHYDGVDVYVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMMEIAKYFLEYPLTI KGPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:18); pAzFRS.1.t2 (specific for pAzF): 10 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNVMHYDGVDVYVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMMEIAKYFLEYPLTI KCPEKEGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:19); 15 pAzRS.2 (polyspecific for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTYMLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIH 20 NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK RPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:20); pAzRS.2.t1(polyspecific for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII 25 LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTYMLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIH NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK GPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:21); and 30 pAzRS.2.t2 (polyspecific for at least pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PheF): MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIII LLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTYMLDKDYTLNVYRLALKTTLK RARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDVAVGGMEQRKIHMLARELLPKKVVCIH 39 45741195.1 YU 8909 PCT NPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIK CPEKEGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL (SEQ ID NO:22). The position and domain of the mutation in each of SEQ ID NO:9-22 relative to SEQ ID NO:1 is provided in Table 7 below. Variants having any combination of the mutations disclosed 5 in Table 7 are also specifically provided. Table 7: Annotations of specific mutations in AARS variants (mutations in evolved synthetases are annotated with respect to the progenitor pAcFRS variant) Annotation Mutant Evolved for nsAA / tRNA In some forms, the variant is a polypeptide including the amino acids of the non-standard 10 amino acid (amino acid ligand) binding pocket of any of SEQ ID NO:9-22; a polypeptide 40 45741195.1 YU 8909 PCT including the amino acids of the tRNA anticodon recognition interface of any of SEQ ID NO:9- 22; or a polypeptide including the non-standard amino acid (amino acid ligand) binding pocket and the amino acids of the tRNA anticodon recognition interface of any of SEQ ID NO:9-22. In some forms, the variant is a polypeptide including amino acids 65-261 of any of SEQ ID NO:9- 5 22. All of SEQ ID NOS:9-22 are also specifically provided both with and without the N- terminal methionine. Variants having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more sequence identity with any of SEQ ID NOS:9-22, with and without the N-terminal methionine, and functional fragments thereof are also provided. Other exemplary orthogonal AARS-tRNA pairs are discussed in the examples below and 10 can be used likewise used alone, together, or in combination with other AARS-tRNA as part of the provided methods. 2. Non-standard Amino Acids Standard and / or non-standard amino acids can be added to the GRO’s culture for incorporation of the non-standard amino acid into the recombinant non-standard amino acid- 15 containing proteins and polypeptide. Standard amino acids include, for example, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. The twenty-first and twenty second amino acids are selenocysteine and pyrrolysine. 20 Over 100 non-standard amino acids have been described containing diverse chemical groups, including post-translational modifications, photocaged amino acids, bioorthogonal reactive groups, and spectroscopic labels (Liu, et al., Annu Rev Biochem, 79:413-44 (2010); Johnson, et al., Curr Opin Chem Biol, 14:774-80 (2010), O'Donoghue, et al., Nat Chem Biol, 9:594-8 (2013), Chin, et al., Annu Rev Biochem, (2014), Seitchik, et al., J Am Chem Soc, 25 134:2898-901 (2012), Davis and Chin, Nature Reviews, 13:168-182 (2012)). Exemplary, non-limiting non-standard amino acids are provided in Table 8. Table 8: List of exemplary nonstandard amino acids Name NAA Name IUPAC Name CAS Number 41 45741195.1 YU 8909 PCT 245FF 2,4,5-Trifluoro-L- (S)-2-Amino-3-(2,4,5- 749847-57-2 phenylalanine trifluorophenyl)propan 42 45741195.1 YU 8909 PCT 2CNF L-2-Cyano (S)-2-Amino-3-(2- 263396-42-5 phenylalanine cyanophenyl)propanoi 45741195.1 YU 8909 PCT 34ClF 3,4-Dichloro-L- (S)-2-Amino-3-(3,4- 52794-99-7 phenylalanine dichlorophenyl)propan 44 45741195.1 YU 8909 PCT 3FY L-3-Fluorotyrosine (S)-2-Amino-3-(3-fluoro- 7423-96-3 4- 45741195.1 YU 8909 PCT 3ThiA 3-(3-Thienyl)-L- (S)-2-Amino-3-(thiophen- 3685-51-6 alanine 3-yl)propanoic acid 45741195.1 YU 8909 PCT 4MeW 4-Methyl-DL- 2-Amino-3-(4-methyl- 1954-45-6 tryptophan 1H-indol-3- 47 45741195.1 YU 8909 PCT 4tBuF L-4-tert-butyl (S)-2-amino-3-(4-(tert- 82372-74-5 phenylalanine butyl)phenyl)propanoi 48 45741195.1 YU 8909 PCT Abu L-2-Aminobutyric (S)-2-Aminobutanoic 1492-24-6 acid acid 45741195.1 YU 8909 PCT aMeE α-Methyl-L-glutamic (S)-2-Amino-2- 6208-95-3 acid methylpentanedioic 50 45741195.1 YU 8909 PCT BHOMeA α-Hydroxyisobutyric 2-Hydroxy-2- 209-848-8 acid methylpropanoic acid 45741195.1 YU 8909 PCT CF3L 5,5,5-Trifluoro-L- (2S)-2-amino-5,5,5- 372-22-5 leucine trifluoro-4- 45741195.1 YU 8909 PCT Eth L-Ethionine (S)-2-Amino-4- 13073-35-3 (ethylthio)butyric acid 53 45741195.1 YU 8909 PCT gBzP(R)-γ-(benzyl)-L- (2S,4R)-4- - proline Benzylpyrrolidine-2- 54 45741195.1 YU 8909 PCT MeRNω-methyl-L-arginine (2S)-2-amino-5-[(N'- 156706-47-7 hydrochloride methylcarbamimidoyl) 55 45741195.1 YU 8909 PCT NovF L-2-Amino-5- (S)-2-Amino-5- 62777-25-7 phenylpentanoic phenylpentanoic acid 56 45741195.1 YU 8909 PCT OtBuEL-Glutamic acid γ- (2S)-2-Amino-5-[(2- 2419-56-9 tert-butyl methylpropan-2- 57 45741195.1 YU 8909 PCT tBuY O-tert-Butyl-L- (S)-2-Amino-3-(4-(tert- 18822-59-8 tyrosine butoxy)phenyl)propan 58 45741195.1 YU 8909 PCT Z2RNω,N'ω-Di-(2S)-2-Amino-5- 4125-79-5 carbobenzyloxy-L- {[(benzyloxy)carbonyl] . The use of NSAA-containing polypeptides is typically based on the nature of the 5 polypeptide and the specific non-standard amino acid incorporated therein. Templates for polypeptides and methods of use thereof are known in the art. For example, site-specific incorporation of a non-standard amino acid at a single position facilitates engineering of protein- drug conjugates (Tian, et al., Proc Natl Acad Sci USA, 111:1766-71 (2014)), cross-linking proteins (Furman, et al., J Am Chem Soc, 136:8411-7 (2014)), and enzymes with altered or 10 improved function (Kang, et al., Chembiochem, 15:822-5 (2014), Wang, et al., Angew Chem Int Ed Engl, 51:10132-5 (2012)). Multi-site non-standard amino acid incorporation can further expand the function and properties of proteins and biomaterials by enabling synthesis of polypeptide polymers with programmable combinations of natural and non-standard amino acids. 15 The disclosed compositions and methods allow for site-specific non-standard amino acid incorporation where multiple identical non-standard amino acids provide the dominant physical and biophysical properties to biopolymers, proteins and peptides. Multi-site non-standard amino acid incorporation also facilitate design and production of post-translationally modified proteins (e.g., kinases) for the study and treatment of disease or of new biologics (e.g., antibodies) with 20 multiple instances of new chemical functionalities. Other biomolecules include, but are not limited to, tunable materials, nanostructures, polypeptide-based therapeutics with new properties, industrial enzymes with new chemistries and properties, bio-sensors, drug delivery vehicles, adhesives, stimuli (e.g., metals-responsvie materials), antimicrobials, synthetic peptides with enhanced pharmacokinetic properties, and 25 biologics. 59 45741195.1 YU 8909 PCT ELPs are biopolymers composed of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly (VPGXG) (SEQ ID NO:23), wherein “Xaa” and “X” can be any standard or non-standard amino acid. ELPs are discussed in U.S. Patent No.6,852,834, which is specifically incorporated by reference herein in its entirety, and Tang, et al., Angew Chem Int Ed Engl, 40:1494-1496 (2001), 5 Kothakota, Journal of the American Chemical Society, 117:536-537 (1995), and Wu, Chembiochem 14:968-78 (2013). They are monodisperse, stimuli-responsive, and biocompatible, making them attractive for applications like drug delivery and tissue engineering. Moreover, ELP properties can be precisely defined and genetically encoded, making them ideal candidates for expanded function via incorporation of multiple non-standard amino acids. 10 As with other polypeptides including one or more non-standard amino acids, uses for ELP include a wide range of medical and non-medical applications. The disclosed compositions and methods can be used to incorporate one or more non-standard amino acids, e.g., 1, 3, 5, 10, 15, 20, 25, 50, or more into protein polymers. Since ELPs can undergo a sharp soluble-to- insoluble phase transition at their transition temperature (Tt), which depends on the ELP 15 composition, ELP templates used for non-standard amino acid incorporation can be utilized as a scaffold for the design of smart biomaterials in which non-standard amino acid functionality can be translated to, for example, stimuli-responsiveness to light, electro-magnetic field, and various analytes. Multi-site nsAA incorporation into these and other protein-based biomaterials at high purity can modify and expand their chemical or physical properties to generate new materials. 20 V. Isolated Nucleic Acids The disclosed proteins and RNAs, and isolated nucleic acids encoding the disclosed proteins and RNAs, particularly the mutant RF2 and mutant tRNATrp(CCA), and orthogonal AARS and tRNA, are provided alone and as part of (i.e., transfected or integrated into) the disclosed ΔTGA GRO and partially recoded intermediates thereof 25 Thus, polynucleotides encoding the disclosed proteins and RNAs, including the engineered RF2 and tRNA, are provided. The polynucleotides can be isolated nucleic acids, incorporated into in a vector, or part of a host genome. The polynucleotides can also be part of a cassette including nucleic acids encoding other translational components such as a paired tRNA, selection marker, promoter and / or enhancer elements, integration sequences (e.g., homology 30 arms), etc. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a genome, including nucleic acids that normally flank one or both sides of the nucleic acid in the genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally-occurring 60 45741195.1 YU 8909 PCT nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. An isolated nucleic acid can be, for example, a DNA molecule or an RNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA 5 molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule or RNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA, or RNA, or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously 10 replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule or RNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing 15 a genomic DNA restriction digest, is not to be considered an isolated nucleic acid. Nucleic acids encoding the polypeptides and proteins disclosed herein may be optimized for expression in the expression host of choice. In the case of nucleic acids encoding expressed polypeptides, codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the organism from which the nucleic acid 20 sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons. Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence, for example, a sequence encoding the disclosed polypeptides and protein. Nucleic acids can be DNA, RNA, nucleic acid analogs, or combinations thereof. Nucleic acid 25 analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl- 2’-deoxycytidine or 5-bromo-2’-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 30 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev.7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 61 45741195.1 YU 8909 PCT 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. Isolated nucleic acid molecules can be produced by standard techniques, including, 5 without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding the disclosed polypeptides or proteins. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical 10 in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring 15 Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 20 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293. Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3’ to 5’ direction). For example, one or more pairs of long 25 oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also be 30 obtained by mutagenesis. Nucleic acids can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and / or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992. Examples of nucleic acid amino acid positions relative to a reference sequence that can be modified include those described herein. 62 45741195.1 YU 8909 PCT Constructs and vectors encoding the disclosed proteins and RNAs are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted 5 segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence. Nucleic acids in vectors can be operably linked to one or more expression control 10 sequences. Operably linked means the disclosed sequences are incorporated into a genetic construct so that expression control sequences effectively control expression of a sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which 15 transcription starts (generally near the initiation site for RNA polymerase II). A “promoter” as used herein is a DNA regulatory region capable of initiating transcription of a gene of interest. Some promoters are “constitutive,” and direct transcription in the absence of regulatory influences. Some promoters are “tissue specific,” and initiate transcription exclusively or selectively in one or a few tissue types. Some promoters are 20 “inducible,” and achieve gene transcription under the influence of an inducer. Induction can occur, e.g., as the result of a physiologic response, a response to outside signals, or as the result of artificial manipulation. Some promoters respond to the presence of tetracycline; “rtTA” is a reverse tetracycline controlled transactivator. Such promoters are well known to those of skill in the art. 25 To bring a coding sequence under the control of a promoter, it is advantageous to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be 30 located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence. 63 45741195.1 YU 8909 PCT Suitable promoters are generally obtained from viral genomes (e.g., polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus, and cytomegalovirus) or heterologous mammalian genes (e.g. beta actin promoter). Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be 5 either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and 10 insulin). However, an enhancer from a eukaryotic cell virus is preferably used for general expression. Suitable examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. In certain forms the promoter and / or enhancer region can act as a constitutive promoter 15 and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter. In other forms, the promoter and / or enhancer is tissue or cell specific. 20 In certain forms the promoter and / or enhancer region is inducible. Induction can occur, e.g., as the result of a physiologic response, a response to outside signals, or as the result of artificial manipulation. Such promoters are well known to those of skill in the art. For example, in some forms, the promotor and / or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such 25 as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as 30 polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3’ untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. 64 45741195.1 YU 8909 PCT The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. VI. GRO Cultures Cultures including a plurality of the disclosed GRO cells and lysates formed therefrom 5 are provided. The cells can be used, as discussed above, for preparation of recombinant polypeptides and proteins optionally, but preferably, including one or more instances of one or more non-standard amino acids. As discussed in more detail below, the cells themselves are also resistant to horizontal gene transfer, and thus can also be used in a variety of other applications, including, but not limited to conventional recombined protein production. 10 Hosts that lack machinery for decoding essential phage codons mistranslate viral proteins, halting propagation and conferring resistance to phage. Lambda (λ) phage genes containing TAG codons are targeted for degradation via tmRNA tagging when expressed in a strain lacking RF1 recognition of the UAG codon, preventing phage infection. Results presented in the experiments below show that abolition of UGA termination confers strains with resistance 15 to phage that employ essential genes ending in TGA. These strains exhibit reduced horizontal gene transfer (HGT) and therefore can be used for enhanced biocontainment. See, e.g., U.S. Patent No.11,149,280, which is specifically incorporated by reference in its entirety. To further reduce HGT, the disclosed GRO cells can also include one or more recombinant expression constructs for expression of one or more genes of one or more ribosomal20 rescue pathways. For example, in some forms, the gene or genes encode bacterial tmRNA- SmpB, ArfA, ArfB, or a combination thereof, or the corresponding gene or genes in another organism. In a preferred form, the cells include a recombinant expression construct for expression of the gene encoding bacterial tmRNA or the corresponding gene(s) in another organism. In a particular forms, gene is ssrA. 25 The GRO cells may also include one or more additional recombinant expression constructs, e.g., for production of a recombinant protein of interest, e.g., as discussed in more detail above. Any and / or all of the recombinant expression constructs can include, or lack one or more of the eliminated codon(s). In some forms the recombinant expression constructs lack at least 30 one of the open codons and / or the cells lack an orthogonal AARS-tRNA that can decode a reassigned codon. The GRO cells are typically resistant to complete transfer and / or expression and / or propagation of a horizontally transferred genetic element (HTGE) compared to a corresponding cell having a genome wherein the codon(s) reduce or eliminated from the GRO cell has not been 65 45741195.1 YU 8909 PCT reduced or eliminated. The transfer can be from another organism. In some forms, the organism from which the HTGE is being transferred is a bacterium or a virus. The HTGE can be, for example, an HTGE plasmid such as a conjugative plasmid, or an HTGE viral genome, or even a fragment of DNA. The virus can be a bacteriophage. 5 Cultures including a homogenous or heterogenous plurality of GRO cells are also provided. In some forms, the culture further includes one or more additional organisms capable of transferring a horizontally transferred genetic element (HTGE) to a corresponding non-HGT- resistant cell having a genome wherein a reduced or eliminated codon(s) has not been reduced or 10 eliminated. In some forms, the one or more additional organisms is one or more strains of bacteria, one or more viruses, or a combination thereof. The virus can be a bacteriophage. The HTGE can be or include, for example, an HTGE plasmid, such as a conjugative plasmid, or an HTGE viral genome. Compositions including a lysate of a plurality of the HGT-resistant cells are also provided. 15 The disclosed invention can be further understood by the following numbered paragraphs: 1. A genomically recoded organism including no more than 25 genomic instances of the TGA codon. 2. A genomically recoded organism including two or more of single nucleotide 20 substitutions, TAA and / or TAG insertions, and deletions to eliminate most or all of the TGA codons relative to a progenitor cell. 3. A genomically recoded organism including no more than 25 genomic instances of the TAA codon. 4. A genomically recoded organism including two or more of single nucleotide 25 substitutions, TAG and / or TGA insertions, and deletions to eliminate most or all of the TAA codons relative to a progenitor cell. 5. The genomically recoded organism of any one of paragraphs 1-4 further including one or both of (i) a mutant release factor 2 (RF2) with reduced ability to terminate translation at UGA and / or a nucleic acid encoding the same and (ii) a mutant tRNATrpwith reduced ability to 30 decode UGA and / or a nucleic acid encoding the same, relative to a progenitor cell. 6. A genomically recoded organism including one or both of (i) a mutant release factor 2 (RF2) with reduced ability to terminate translation at UGA and / or a nucleic acid encoding the same and (ii) a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same, relative to a progenitor cell. 66 45741195.1 YU 8909 PCT 7. The genomically recoded organism of any of paragraphs 2-6, wherein greater 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% of TGA codons eliminated relative to the progenitor cell. 8. The genomically recoded organism of paragraph 7, wherein less than 100% of the 5 TGA codons are eliminated. 9. The genomically recoded organism of one of paragraphs 1-8, wherein TGA codon(s), if present, are only present in pseudogenes, ins, and / or selenocystine-encoding codons. 10. The genomically recoded organism of one of paragraphs 1-9, wherein TAG is absent from the genome. 10 11. The genomically recoded organism of any one of paragraphs 1-10, wherein release factor 1 (RF1) has reduced activity or is absent. 12. The genomically recoded organisms of any one of paragraphs 1-2 or 5-11, wherein TAA is the only functional stop codon, or any one of paragraphs 3-11, wherein TGA is the only functional stop codon. 15 13. The genomically recoded organism of any one of paragraphs 1-2 or 5-12, wherein the TGA codon is a functionally open codon that can be reassigned, or any one of paragraphs 3- 12, wherein the TAA codon is a functionally open codon that can be reassigned. 14. The genomically recoded organism of any one of paragraphs 1-11, wherein the TAG codon is a functionally open codon that can be reassigned. 20 15. The genomically recoded organism of any one of paragraphs 1-14, wherein crosstalk between four codons TAG, TGA, TAA, and TGG is attenuated rendering each codon a unique translational function: open, open, stop, and standard amino acid encoding optionally wherein the standard amino acid is Trp; or open, stop, open, and standard amino acid encoding optionally wherein the standard amino acid is Trp. 25 16. The genomically recoded organism of any one of paragraphs 5-15 including a mutant RF2 and / or nucleic acid encoding the same. 17. The genomically recoded organism of paragraph 16, wherein the mutant RF2 includes (i) one or more of N26, K170, P205, and A246 relative to SEQ ID NO:3 and 30 optionally has an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% to the amino acid sequence of SEQ ID NO:3, or 67 45741195.1 YU 8909 PCT (ii) the corresponding amino acids in a homolog thereof, optionally wherein the homolog includes an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity thereto. 18. The genomically recoded organism of paragraphs 16, wherein the mutant RF2 5 includes at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, preferably including all of N26, K170, P205, and A246 relative to SEQ ID NO:3. 19. The genomically recoded organism of any one of paragraphs 5-18 including a mutant prfB gene having one or more of conversion of one or both the internal autoregulatory 10 TGA of the native sequence to TAA; conversion of the terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to SEQ ID NO:1. 15 20. The genomically recoded organism of paragraph 19, where the mutant prfB gene includes at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2. 21. The genomically recoded organism of any one of paragraphs 5-20, including a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same. 20 22. The genomically recoded organism of paragraph 21, wherein the mutant tRNATrpis encoded by nucleic acid sequence including a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:5. 23. The genomically recoded organism of paragraph 22, wherein the mutant tRNATrpis encoded by a nucleic acid sequence including at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 25 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6. 24. The genomically recoded organism of any one of paragraphs 5-20 including a mutant trpT gene including a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:5. 26. The genomically recoded organism of paragraph 24, wherein the mutant trpT 30 gene includes at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6. 26. The genomically recoded organism of any one of paragraphs 1-2 or 5-25 further including a first heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate tRNA capable of decoding TGA; or any one of paragraphs 3-25 further including a first 68 45741195.1 YU 8909 PCT heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate tRNA capable of decoding TAA. 27. The genomically recoded organism of any one of paragraphs 10-26 further including a second heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate 5 tRNA capable of decoding TGA where TGA has been reduced or removed as stop codon and TAA where TAA has been reduced or removed as a stop codon. 28. The genomically recoded organism of any one of paragraphs 1-27 further including a heterologous mRNA or nucleic acid encoding the same including one or more TGA codons where TGA has been reduced or removed as stop codon and TAA codons where TAA 10 has been reduced or removed as a stop codon. 29. The genomically recoded organism of any one of paragraphs 1-27 further including a heterologous mRNA or nucleic acid encoding the same including one or more TAG codons. 30. The genomically recoded organism of any one of paragraphs 1-27 further 15 including a heterologous mRNA or nucleic acid encoding the same including one or more TGA codons and one or more TAG codons. 31. The genomically recoded organism of any one of paragraphs 1-30, wherein the organism is a bacterium, yeast, fungi, insect, plant, or animal cell(s). 32. The genomically recoded organism of paragraph 31, wherein the bacterium is E. 20 coli. 33. A culture including a plurality of genomically recoded organisms of any one of paragraphs 1-32. 34. A method of making a polypeptide including one or more instances of a non- standard amino acid including expressing a first messenger RNA (mRNA) encoding the target 25 protein in a system including: a first orthogonal translation system (OTS) including a nucleic acid sequence encoding a first orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the genomically recoded organism (GRO) of any one of paragraphs 1-33, and 30 a plurality of a first non-standard amino acid, wherein the first mRNA includes a nucleic acid sequence including at least one instance of a first codon reduce or absent in the GRO, wherein the first orthogonal AARS can charge its cognate tRNA with the first non- standard amino acid, 69 45741195.1 YU 8909 PCT and wherein the first cognate tRNA includes an anticodon that can bind to the codon reduced or absent from the GRO. 35. The method of paragraph 34, further including a second orthogonal translation system (OTS) including a nucleic acid sequence 5 encoding a second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the GRO, and a plurality of as second non-standard amino acid, wherein the first mRNA or a second heterologous mRNA includes a nucleic acid sequence including at least one instance of a second codon reduced or absent in the GRO,10 wherein the second orthogonal AARS can charge its cognate tRNA with the second non- standard amino acid, and wherein the second cognate tRNA includes an anticodon that can bind to a second codon reduced or absent from the GRO. 36. A method of making a polypeptide including one or more instances of a non- 15 standard amino acid including expressing a first messenger RNA (mRNA) encoding the target protein in a system including: a first orthogonal translation system (OTS) including a nucleic acid sequence encoding a first orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the genomically recoded organism (GRO), 20 a second orthogonal translation system (OTS) including a nucleic acid sequence encoding a second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the GRO and a plurality of first and second non-standard amino acids, 25 wherein the first mRNA includes a nucleic acid sequence including one or more instances of a first codon and / or one or more instances of a second codon of a second codon reduced or absent in the GRO, wherein the first and second orthogonal AARS’s can charge their cognate tRNA with first and second non-standard amino acids, respectively, 30 and wherein the first and second cognate tRNA includes anticodons that can bind to different codons reduced or absent from the GRO. 37. The method of paragraphs 34, 35 or 36 wherein the first and / or second mRNA include between 1-100 instances includes, or any specific integer or subrange in between, of the 70 45741195.1 YU 8909 PCT first codon that is reduced or eliminated from the GRO, the second codon that is reduced or eliminated from the GRO, or a combination thereof. 38. The method of any one of paragraphs 34-37, wherein the target polypeptide is made in greater yield and / or purity compared to making the polypeptide using the same system 5 in the GRO’s progenitor cell. 39. A mutant RF2 including a reduced ability to terminate translation at UGA relative to wildtype RF2. 40. The mutant RF2 of paragraph 39 including one or more of N26, K170, P205, and A246, preferably all of N26, K170, P205, and A246, relative to SEQ ID NO:3. 10 41. The mutant RF2 of paragraphs 39 or 40 including at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, preferably including all of N26, K170, P205, and A246 relative to SEQ ID NO:3. 42. A nucleic acid encoding the mutant RF2 of any one of paragraphs 39-41. 15 43. The nucleic acid of paragraph 42 including an expression control sequence operably linked thereto. 44. A mutant prfB gene including one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and 20 minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to SEQ ID NO:1. 45. The mutant prfB of paragraph 44, wherein the mutant prfB gene includes at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 25 99% sequence identity to SEQ ID NO:2. 46. A nucleic acid including the mutant prfB of paragraphs 44 or 45 further including an expression control sequence operably linked thereto. 47. A mutant tRNATrpwith reduced ability to decode UGA. 48. The mutant tRNATrpincluding a sequence encoded by a nucleic acid sequence 30 including a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:5. 49. The mutant tRNATrpof paragraphs 47 or 48 including a nucleic acid sequence encoded by a nucleic acid sequence including at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6. 50. A nucleic acid encoding the mutant tRNATrpof any one of paragraphs 47-49. 71 45741195.1 YU 8909 PCT 51. The nucleic acid of paragraph 50 further including an expression control sequence. 52. A mutant trpT gene including a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:5. 5 53. The mutant trpT gene of paragraphs 52 including at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6. 54. A nucleic acid including the mutant trpT gene of paragraphs 52 or 53 further including an expression control sequence operably linked thereto. 10 55. A GRO including one or more, preferably all, of the features of Table 9. 56. The GRO referred to herein as rEc∆2.∆A.mB-oKP.tW* (Ochre.tW*). 57. A GRO including a genome including stop codons, wherein the stop codons consist of a single stop codon sequence. 58. The GRO of paragraph 57, wherein one or more release factors provide 15 translation termination only at the single stop codon sequence. 59. The GRO of paragraphs 57 or 58, wherein the GRO further include a heterologous translation system including one or two orthogonal translation systems (OTS) including a nucleic acid sequence encoding a first and optionally second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, 20 or integrated into the GRO, and a mRNA encoding a polypeptide, wherein the mRNA sequences includes one or both of reassigned stop codons as sense codons encoding nsAAs. wherein the first and second orthogonal AARS can charge their cognate tRNA with different non-standard amino acid, and wherein the first and second cognate tRNA includes different anticodons that can 25 bind to the reassigned stop codons. 60. A method of making a GRO including a genome consisting of a single stop codon sequence, the method including recoding other stop codon sequences to the stop codon sequence, optionally further including altering one or more release factors to provide translation termination only at the single stop codon sequence. 30 61. A nucleic acid encoding a polypeptide, wherein the nucleic acid sequence includes one or more UAG / TAG codons and one or more UGA / TGA codons, wherein the polypeptide includes nsAAs and the UAG / TAG codons and UGA / TGA codons encode for the nsAAs. 72 45741195.1 YU 8909 PCT 62. A GRO as described herein in the description, figures, tables, or combination thereof. 63. A method, composition, device, or process as described herein in the description, figures, tables, or combination thereof. 5 Examples Exemplary ∆TGA and intermediates thereof engineered from a progenitor ∆TAG genome (i.e., GRO rEc∆1.∆A) are provided. In some forms, the ∆TGA GRO includes elimination of 1,195 instances of the stop codon TGA, and engineering of associated translation machineries to free TGA as a second open codon for sense codon reassignment. The resulting genome 10 attenuates crosstalk between four codons within the canonical code (TAG, TGA, TAA, and TGG), rendering each codon a unique translational function. While past recoding efforts repurposed stop and sense codons via deletion of associated translation machineries (RF1 and tRNAs, respectively), the freeing of a second stop codon (TGA) has been limited by the need to engineer complex release factor proteins to precisely alter codon recognition without 15 compromising secondary RF functions. Related research targeting codon recognition by tRNAs and release factors demonstrated challenges in balancing codon specificity, the impacts from near-cognate suppression, and resulting competition between translational elements for codons, as well as adverse effects of disrupting the stoichiometries and interacting conformations of co- evolved, finely-tuned translational machineries. This disclosure establishes four key advances 20 toward the design and construction of a single-function-codon GRO: a recoded E. coli strain with a single TAA stop codon and highly efficient dual-incorporation of two distinct nsAAs into recombinant proteins at TAG and TGA. First, whole-genome recoding of TGA to TAA stop codons in E. coli were computationally analyzed and the functional impact predicted. Mutagenic oligos were constructed for multiplex automated genome engineering (MAGE) to recode to 25 TAA, or abolish, all instances of TGA stop codons, while minimizing unintended polar affects. Second, 1195 native terminal TGA stop codons were removed from all genes and a subset of pseudogenes in C321.ΔA. Third, translational compatibility was established by engineering native tRNATrpand release factor 2 to significantly attenuate their native recognition of UGA, while retaining tRNATrpspecificity for UGG, RF2 specificity for UAA, and orthogonality of 30 UAG. These changes eliminate natural suppression of TGA by tRNATrpwobble pairing and opened the TGA codon for reassignment. Fourth, The enhanced ability of this GRO to permit multi-site incorporation of two distinct nsAAs at six UAG and UGA codons via compatible orthogonal translation systems (OTSs) was characterized and shown to be ~99% accurate, greatly surpassing previous in vivo efforts. These results establish the first viable GRO with a 73 45741195.1 YU 8909 PCT single stop codon liberating two essential stop codons for sense codon reassignment. Hemez C, Mohler K, Radford F, Moen J, Rinehart J, Isaacs FJ. Genomically recoded Escherichia coli with optimized functional phenotypes. bioRxiv [Preprint].2024 Aug 29:2024.08.29.610322. doi: 10.1101 / 2024.08.29.610322. PMID: 39257802; PMCID: 5 PMC11383693; including all Associated Data and Supplementary Material associated therewith, is incorporated by reference in its entirety. Materials & Methods: DNA: Oligonucleotides were purchased from Integrated DNA Technologies with standard 10 purification and desalting. Media: Unless otherwise stated, all cultures were grown in Lysogeny Broth (LB)-Lennox medium (LB, 10 g / L bacto tryptone, 5 g / L sodium chloride, 5 g / L yeast extract). pH was adjusted to 7.4 via 10 M NaOH. LBLagar plates were composed of LBLplus 15 g / L bacto agar. 15 M9 minimal medium (12.8 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 3 mg / L CaCl2) was adjusted to pH 7.5 with 10 M NaOH. For phage experiments, Tryptone-KCl (TK) liquid medium comprised of 10 g / L Tryptone, 5 g / L KCl, and 0.5 ml / L of 1M CaCl2. TK Top agar (SLOPPY) contained 10 g / L Tryptone, 5 g / L NaCl, and 7 g / L agar. TK Bottom agar contained 10 g / L Tryptone, 2.5 g / L NaCl, 2.5 g / L KCl, 0.5 ml / L of 1M CaCl2, and 10 g / L agar. 20 Super Optimal broth with Catabolite repression (SOC) liquid medium was acquired through Thermo Scientific. Selective Agents: carbenicillin (50 µg / mL), chloramphenicol (40 µg / mL), gentamycin (10 µg / mL), kanamycin (30 µg / mL), sodium dodecyl sulfate (SDS) (0.005% w / v), spectinomycin 25 (190 µg / mL), tetracycline (15 µg / mL), hygromycin (150 µg / mL), zeocin (10 mg / mL), and Colicin E1 (ColE1; ~10 µg / mL). ColE1 was expressed in strain JC411 and purified. All other selective agents were purchased commercially. Promoter inducers: plasmid-induced pORTMAGE and Recombineering 30 L-arabinose (1.4 mg / ml – pBAD promoter), rhamnose (1.4 mg / ml – rhaB promoter). Plasmid maintenance employed anhydrotetracycline (5 µg / mL – tetA selectable marker). 74 45741195.1 YU 8909 PCT Promoter inducers: OTS Expression for nsAA Incorporation Assays L-arabinose (0.05% w / v – pBAD promoter) and anhydrotetracycline (100 ng / mL – pL tetO promoter) 5 Non-standard Amino Acids: Nε-Boc-L-lysine (BocK) was purchased from Chem-Impex (#00363) and dissolved in LB to a final concentration of 10 mM. Para-acetyl-L-phenylalanine (pAcF) was purchased from Chem-Impex (#24756), dissolved in sterile water to a concentration of 50 mM and used at a final concentration of 1 mM. 10 Strains: All strains were built upon rEc∆1.∆A - C321.ΔA (MG1655 dnaG.Q576A. exoX.K28TAA. ΔtolC. ΔprfA. ΔmutS::zeo.Δ (ybhB-bioAB)::[λcI857 N(cro-ea59)::tetR-bla]).12B.tolQRA); with all instances of TAG codons converted to TAA, and 1196 naturally occurring TGA codons. 15 Selectable Marker Prep: Selectable markers were amplified via PCR (40 µL per reaction) performed using Kapa HiFi HotStart ReadyMix according to the manufacturer’s protocols with annealing at 61 °C. Primers were designed via Benchling primer creation software, confirmed with Harvard’s Kun’s Oligonucleotide Tm Calculator. PCR products were purified using a Qiagen PCR purification 20 kit, eluted in 30 µL dH2O, and quantified either using an Eppendorf BioPhotometer plus or NanoDrop™ ND1000 spectrophotometer. For analysis, amplicons were run on a 1.5% agarose gel stained with ethidium bromide to confirm the projected band sizes. TGA-to-TAA Recoding: 25 The starting strain was a standard rEc∆1.∆A (C321.∆A) with a zeocin marker cassette replacing a chloramphenicol marker at the mutS locus. The ybgC-tolQRA locus was duplicated between the ycgV and ychF genes as previously described to increase the fidelity of colicin selections. The previously reported mutations to dnaG and exoX to improve multiplex automated genomic engineering (MAGE)-efficiency were also present in the starting strain. All genes containing 30 TGA codons were identified from whole genome sequencing (WGS) (Table 9, see sequencing method below). Eliminating TGA codons required a combined strategy of MAGE-mediated conversions of TGA to TAA and non-essential gene deletions (Table 2). The oligonucleotides were designed as previously described, ordered from IDT and grouped into pools up to 11 oligonucleotides per pool at 10-12µM total DNA per pool, regardless of number of oligos (N) 75 45741195.1 YU 8909 PCT Table 9. These pools were used for N+1 rounds of MAGE before cultures were plated. MAGE was performed as previously described. Forty-seven colonies were picked and screened using Multiplex allele-specific colony PCR (MASC-PCR), as previously described. Simultaneously, picked colonies were grown to confluence, diluted 1:15 into 150 µL of M9 minimal media, then 5 inoculated 1:100 into 150 µL LB and M9 to assess growth phenotypes (Max OD, doubling time, and lag time) (see Fitness Analysis section below). The colony containing the largest number of lowest frequency conversions and minimal deviations in growth fitness was chosen for subsequent rounds of MAGE. 10 Native RBS Predictions: predicted RBS translation rates of genes overlapped by TGA stop codons before and after recoding to TAA were calculated in alignment with previous protocols. MAGE and λ-Red-mediated recombination: Multiplex automated genomic engineering (MAGE), pORTMAGE, and λ-Red-mediated recombination were performed as previously 15 described. Cells were transferred to 0.1 cm cuvettes, electroporated (BioRad GenePulser™, 1.78 kV, 200 Ω, 25 µF), and immediately resuspended into 3 mL LB (MAGE) or 1 mL SOC medium (dsDNA), grown at 34 °C, 225 rpm. For tolC and galK negative selections, cultures were recovered for at least 7 hours to allow complete protein turnover before exposure to ColE1 and 2-deoxygalactose, respectively. Once all strains were conjugated into a final strain (see CAGE 20 Assembly method below), genomically-integrated λ-Red was displaced by tolC negative selection. To convert background mutations and remaining TGA codons a plasmid-based ssDNA recombineering approach was utilized. L-arabinose-inducible λ-Red-derived recombineering machinery was expressed on a temperature-curable plasmid. For dsDNA recombination, rhamnose-inducible episomal λ-Red-derived recombineering machinery was also expressed on a 25 temperature-curable plasmid containing L-arabinose-inducible ccdA antitoxin for negative selections as previous described for both marker removal and release factor placement / displacement. Both λ-Red plasmids contained tetA selectable markers. To cure plasmids, strains were incubated with λ-Red induction at 37°C overnight in 3-mL LB, plated onto solid media, incubated at 42 °C for 2-4 hours, then incubated at 37°C until colonies were 30 visible. Colonies were picked into 150 µL LB, incubated for 3 hours, then transferred 10 µL into LB + tetracycline to identify colonies lacking plasmids. Genotyping: Multiplex allele-specific colony PCR (MASC-PCR) was used to simultaneously detect up to 11 TGA-to-TAA conversions or background mutation reversions, as previously 76 45741195.1 YU 8909 PCT described with TAG conversions. All primers are designed with a 61°C annealing temperature, according to Harvard’s Kun’s Oligonucleotide Tm Calculator, while avoiding 3’ end binding in secondary structure, according to Benchling’s primer secondary structure prediction function. Each reaction consisted of KAPA 2G Fast Multiplex ReadyMix (Kapa Biosystems, # KK5802), 5 2 µl of template DNA, and 0.2 µM of each primer for each 10 µL reaction. MASC-PCR results were run on 2.2% agarose gels with ethidium bromide staining. After λ-Red-mediated recombination or conjugation, colony PCR was used to confirm the presence and absence of selectable markers at desired positions. Colony PCR (10 µL per reaction; annealing at 61°C – Kun’s Oligonucleotide Calculator) was performed using Kapa 2G Fast HotStart ReadyMix 10 following manufacturer’s protocols. Results were analyzed on a 1.5% agarose gel stained with ethidium bromide. Sanger sequencing was performed by Genewiz. CAGE Assembly: Conjugative^assembly genome engineering (CAGE) was performed using the protocol as previously described. Deviating from previous protocols, the donor strain had two 15 positive markers, e.g. a spectinomycin marker and a gentamycin marker, each flanking the recoded region, in addition to the kanamycin resistance-origin of transfer (kanR-oriT) cassette set ~3-5kb upstream (Figure 9). The donor strain also contained a modified RK24 plasmid in which all genes that end in a TAG codon were recoded to TAA. Cell spots were rinsed twice with 500 µL LB and collected to give a 1 mL 10-2dilution of conjugated cells, followed by an additional 20 10-fold dilution in a new tube.50 µL of 10-2and 10-3dilutions were each plated onto LB agar plate with appropriate antibiotics to select for both the recipient background selectable markers and the donor region selectable markers.47 candidate colonies were grown in a 96-well format and screened for desired genotypes via PCR (to confirm presence and absence of selectable markers) and MASC-PCR (to confirm the presence of interspersed desired codon replacements). 25 For large genomic transfers (>1Mb), final strains were sequence verified through WGS (see Genotyping method section below). The resulting strain contained the three selectable markers, all of which were subsequently deleted via dsDNA λ-Red tolC-mediated selection- counterselection (via sodium dodecyl sulfate or Colicin E1 selection, respectively), or maintained for the next conjugation. 30 Genomic Deletions: To reduce the overall number of genes requiring TGA recoding via MAGE, 16 multigenic regions containing a total of 229 genes (3 later categorized as pseudogenes) were identified for deletion based on previous work by the Blattner research lab (Figure 8). Each targeted deletion site (up to ~34kb in size) was displaced via tolC selectable marker 77 45741195.1 YU 8909 PCT displacement and counter-selection for subsequent marker removal in accordance with previous protocols. ColE1 selections on solid medium were performed as previously described. For pre- selection, 5 µL of recovered cultures were inoculated into 150 µL LB with either carbenicillin (control) or ColE1 with vancomycin (64µg / ml), in triplicate, within a 96-well plate, and 5 incubated at 34oC for 16 hrs to monitor growth along the progenitor strain with tolC. Strains exhibiting growth in both carbenicillin and ColE1 with vancomycin were considered positive for tolC deletion and subsequently plated onto LB agar plates with ColE1 for monoclonal colony selection and PCR screened to confirm the loss of tolC. Subsequent MASC analyses allowed for selection of strains with both deletion events and TGA conversions to reduce the total number of 10 MAGE cycles. After every tolC placement and gene displacement, strain growth curves were analyzed to assess gene deletion impact on strain phenotype (see Fitness Analysis methods section below). Major growth impairments were assessed. Additional major deletions (>50bp) resulted either from mutagenesis of highly repetitive noncoding genes, active transposable elements, or scarring from conjugations. 15 Whole Genome Sequencing (WGS) & Analysis: Whole genomes were isolated using the Qiagen DNeasy Blood and Tissue isolation kit. For high fidelity genome analysis performed after conjugations, raw reads were acquired via short-read 150bp (50x coverage) paired-end Illumina sequencing data were collected with Hiseq 4000 with libraries prepared by the Yale 20 Center for Genome Analysis (YCGA). Long read (<25kb – 50x coverage) data was prepared by Pacific Biosciences Single Molecule Real-Time (SMRT) Analysis. For rapid whole genome analysis, raw reads were acquired via Plasmidsaurus Oxford nanopore standard bacterial WGS (30x coverage) via bacterial pellet submissions suspended in DNA / RNA Shield. To perform analyses on raw reads, latest versions of breseq 0.38.1 computation pipeline were employed for 25 aligning sequence reads to rEc1.∆A (C321.∆A) reference genome, run using Ubuntu LTS on Windows Subsystem for Linux (WSL), in accordance to Barricklab.org. Summary.html outputs provided comprehensive lists of mismatches, indels, and missing or new junctions to monitor TGA conversion progress, deletions, and background mutagenesis. Mutations were then organized by type in Excel. 30 AlphaFold Structure of mRF2-oKP: 3D structure of mRF2-oKP in Fig.2D was acquired through Benchling AlphaFold online prediction from amino acid sequence submission (SEQ ID NO:4) and spatially oriented for view of primary reaction sites. 78 45741195.1 YU 8909 PCT Fitness Analysis - Kinetic Growth Curves: Kinetic growth (OD600) curves were obtained via monitoring strain growth within a Biotek Synergy HT plate reader. Each strain was grown in triplicate wells, 150 mL LB and 150 mL M9, within 96-well flat-bottom plates, incubated at 34 °C for intermediate λ-Red+ strains and 37 °C 5 for final strains, 225 rpm, for 16-24 hrs, and absorbance at 600 nm was read at 10-minute intervals. In preparation, strains were grown to confluence in LB, diluted 1:15 into 150 µL of M9 minimal media, and inoculated 1:100 into 150 µL LB and M9. Auxotrophic strains revealed no growth in M9. Quantification of growth phenotypes (lag time, growth rate and max OD) was computed using an in-house MATLAB script. To automatically determine boundaries between 10 different growth phases (lag, log, and stationery), the first derivative of growth curves was fit to a gaussian function. A temporal window of + / - 1*sigma around the time of max slope (MaxV) was defined as the mid-log range and timepoint 2*sigma - MaxV was defined as the transition from lag to log phase. To compute the doubling time, a baseline OD600 value of 0.01 was set as the log-base-2 transform of growth curve in the mid-log range was fit to a linear function. The 15 reciprocal of the slope of best fit was defined as the growth rate. Readthrough Fluorescence Assay: A dual fluorescence mCherry-YFP reporter was used to test stop codon readthrough as previously reported. This reporter was constitutively expressed from a low copy backbone 20 (p15A) to maximize dynamic range for detecting post transcriptional fluctuations. Each evaluated strain was transformed with four variants of the reporter (three stop codons and a sense codon) and plated on selective media for overnight growth. Colonies were picked in triplicate into 150 µl cultures of LB with appropriate selection in 96-well plates, grown with shaking at 225 rpm in 37 °C for 18 hours after which timepoints measurements were taken for mCherry 25 (Excitation: 585 nm, Emission: 635 nm, Gain: 100),YFP (Excitation: 500nm, Emission: 541 nm, Gain: 80), and OD600 (Absorbance: 600 nm) in a BioTek Synergy H1 plate reader (Agilent). Fluorescence values of all strain and plasmid conditions were OD600 normalized before dividing by internal positive controls described above to arrive at fractional mCherry and YFP signals reported. To assess cognate amber or opal suppression, a plasmid bearing supD with CUA or 30 UCA anticodon was co-transformed with the dual fluorescence reporter plasmid. Fluorescence values of all strain and plasmid conditions were OD600 normalized before dividing by internal positive controls described above to arrive at fractional mCherry and YFP signals reported. 79 45741195.1 YU 8909 PCT Bacteriophage Assays: For all phage experiments, growth was carried out in TK at 37°C; infection and propagation occurred in top (sloppy) TK poured onto solid TK incubated at 37 °C. Sloppy agar mixes were maintained at 42 °C before use. 5 Phage propagation: For propagation, Escherichia coli MG1655 was grown to mid-log phase in 3 mL of LB.200 µL of bacteria was added to 3 mL sloppy agar. Immediately following, 50 µL of phage (10 to 100-fold dilutions) was added directly from a frozen glycerol stock into the sloppy-bacterial culture.3 mL of sloppy-bacteria-phage culture was poured onto solid TK plates, 10 dispersed evenly, then left at room temperature to solidify. Plaques were then incubated at 37°C for ~16 hours to permit lysis to proceed to completion. The entire lysate was centrifuged to remove cell debris (12,000 rcf, 2 minutes), and 3 mL of lysate was filtered with 0.22µm filter column. 15 Phage titering: Bacteria strains were grown overnight at 37°C until OD reaches 2-3.20 µL of bacteria was added to 3 mL sloppy agar.3 mL of sloppy-bacteria culture was poured onto solid TK plates. After incubated at room temperature for 15 minutes, 3 µL of the phage dilutions (101to 108-fold dilutions) were dropped on the surface of the solidified sloppy agar. Once the drops have dried, plates were incubated overnight at 37 °C. Visible plaques were counted for the 20 individual drops. Titers (pfu / mL) were calculated with the following formula: PFU / ml where N is the number of plaques, DF is the dilution factor and V is the volume of phage dilution pipetted on the plate. nsAA Incorporation Assays: 25 Plasmid construction: Gene fragments for aaRS and tRNA were synthesized by Twist Bioscience and cloned into expression vectors by Golden Gate assembly. Plasmids were sequence verified by whole-plasmid sequencing (Plasmidsaurus or Quintarabio). All cloning was made in Mach1 (Thermofisher - Cat. #C862003). 30 Incorporation of BocK and pAcF into proteins: Recoded strains were transformed with OTS- reporter plasmids by standard electroporation protocols. Electroporated strains were recovered in 2 mL LB or SOC (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) for at least 2 hours before plating onto LB-agar plates with 80 45741195.1 YU 8909 PCT kanamycin (50 µg / mL) and incubated at 37°C overnight. Three single colonies from each plate were picked and grown in 800 µL LB supplemented with kanamycin (50 µg / mL) in a 96 deep- well plate sealed with a Breathe-Easy film (Sigma-Aldrich) and incubated at 37°C with shaking at 220 rpm for 20-24 hours. After overnight growth the cultures were back-diluted 1:50 onto a 5 clear-bottom black 96-well plate (Costar) in a total of 150 µL of LB supplemented with kanamycin (50 µg / mL), aTc (100 ng / mL), L-arabinose (0.05 % w / v), 1 mM pAcF and 10 mM BocK. Cell growth (absorbance at OD600) and GFP fluorescence (excitation 485 nm, emission 525, gain 70, bottom measurement) were measured in a BioTek Synergy H1 plate reader (Agilent) for 24 hours at 10 minutes intervals with linear shaking. Data was analyzed with a 10 custom Matlab script. GFP Fluorescence was normalized by OD600, and the maximum value after 10 hours was plotted as bar plots. Purification of ELP-6x(TGA / TAG)-GFP with nsAA: Single colonies of Ochre and Ochre.tW* (trpT-A37G) containing the dual-OTS dual-reporter plasmid were inoculated in 2 mL LB-kan in 15 a 14 mL falcon tube overnight at 37°C with shaking at 220 rpm. After overnight growth the cultures were diluted 1:100 in 25 mL LB-kan and grown until 0D600 at 0.6, where the cultures were supplemented with 1 mM pAcF and 10 mM BocK and induced with 100 ng / mL aTc and 0.05 % w / v L-arabinose. The cultures were grown at 37°C overnight after which the cells were harvested by centrifugation at 3,200 g for 20 minutes in a 50 mL centrifuge tube and stored at - 20 80 °C until protein purification. Protein Mass Spectrometry: ELP-GFP reporter protein purification: Frozen E. coli cell pellets were thawed on ice, and 25 pellets were lysed by sonication with lysis buffer consisting of 50 mM Tris-HCl (pH 7.4, 23°C), 500 mM NaCl, 0.5 mM EGTA, 1mM DTT, 10 % glycerol, 50 mM NaF, and 1 mM Na3O4V. The extract was clarified with two rounds of centrifugation performed for 20 minutes at 4°C and 14,000 x g. Cell-free extracts were applied to Ni-NTA metal affinity resin and purified according to the manufacturer's instructions. Wash buffers contained 50 mM Tris pH 7.5, 500 mM NaCl, 30 0.5 mM EGTA, 1mM DTT, 50 mM NaF, 1 mM Na3VO4, and 10 mM imidazole. Proteins were eluted with a wash buffer containing 250 mM imidazole. Eluted protein was subjected to 4 rounds of buffer exchange (20 mM Tris pH 8.0 and 100 mM NaCl) and concentrated using a 30 kDa molecular weight cutoff spin filter (Amicon). 81 45741195.1 YU 8909 PCT Protein digestion and mass spectrometry: Affinity purified, buffer exchanged ELP-GFP reporter protein, or whole cell lysates, were digested and analyzed by mass spectrometry as described previously with some modifications.5-10 μg ELP-GFP reporter protein was diluted with water and 20% SDS for a final volume of 115 μl and final concentration of 1% SDS. 5 Samples were denatured for 15 min at 55°C in a heat block. Reduction and alkylation of cysteines was performed with TCEP and 2-chloroacetamide (CAM) using a final TCEP and CAM concentration of 10 mM and 44 mM respectively. The reduction-alkylation reaction proceeded for 20 min at 55°C.6 μl of 50 mg / ml SP3 beads (Speed Bead, Cytiva) pre-washed and resuspended with water were added to samples for a final working volume of 134 μl. Binding of 10 protein to the beads was induced by adding 150 μl of 100% ethanol. The binding mixture was incubated in a ThermoMixer at 24°C for 10 min at 1,400 r.p.m. After binding, the beads were magnetized on a magnetic rack and supernatants were removed. This was followed by three rounds of bead washes with 500 μl of 80% ethanol per wash. All traces of 80% ethanol were removed after the last wash. Beads in each sample were resuspended with 50 μl of digestion 15 solution containing 0.4 μg of sequencing grade trypsin (Promega) in 50 mM Triethylammonium bicarbonate (TEAB) buffer (Sigma). Digests were incubated for 16 h at 37°C in a ThermoMixer at 1,400 r.p.m. Beads were magnetized and 50 μl supernatants were moved to fresh tubes. Beads were resuspended in 50 μl of 50 mM TEAB buffer and incubated for 5 min at 37°C in a ThermoMixer at 1,400 r.p.m to maximize peptide recovery. Beads were magnetized again and 20 the two 50 μl supernatants were combined. Peptides were dried in a vacuum centrifuge at room temperature. Dried peptides were reconstituted in 2 / 98 acetonitrile / water with 0.1% formic acid and analyzed by LC-MS / MS. LC-MS / MS was performed using a Vanquish Neo UHPLC system (Thermo) and an Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo). The analytical column employed was a 25 75 μm inner diameter, fused silica capillary tube (Molex) packed in-house to a length of 15 cm with 1.9 μm ReproSil-Pur 120 Å C18-AQ (Dr. Maisch) using methanol as the packing solvent. Column was attached to a PepSep Spray Adapter with a fused silica emitter (Bruker). Peptide separation was achieved using mixtures of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a 41-min gradient; 0 / 5, 30 / 30, 39 / 45, 40 / 55, 41 / 100 30 (min / %B, linear ramping between steps). The gradient was performed with a flowrate of 300 nl / min. At least one blank injection (5 μl 2% B) was performed between samples to eliminate peptide carryover on the analytical column.100 nmol of trypsin-digested BSA and 100 ng of trypsin-digested HeLa protein standard were run periodically between samples as quality control standards. The mass spectrometer was operated with the following parameters: (MS1) 60,000 82 45741195.1 YU 8909 PCT orbitrap resolution, 250% normalized AGC target, 50 ms maximum injection time, 300–1,400 m / z scan range; (data dependent-MS2) Ion Trap detector, 200% normalized AGC target, 13 ms maximum injection time, top 10 mode, 1.2 m / z isolation window, 30% normalized HCD collision energy, 40 s dynamic exclusion. Data were searched using MaxQuant version 1.6.10.43 5 with Deamidation (NQ), Oxidation (M), and Phospho (STY) as variable modifications and Carbamidomethyl (C) as a fixed modification with up to 3 missed cleavages, 5 AA minimum length, and 1% FDR against a modified Uniprot E. coli database containing custom MS-READ reporter proteins. MS-READ search results were analyzed using MaxQuant and Perseus version 1.6.2.2. 10 Statistical Analysis: Statistical significance for YFP:mCherry readthrough assays were calculated using unpaired t- tests in comparison to rEc∆1.∆A values, where * = p-value < 0.05, ** = p-value < 0.01, *** = p- value < 0.001. Error bars displayed standard errors of the mean, n = 3. Significance for phage15 infection assays were derived from unpaired t-tests in relation to E. coli MG1655, where * = p- value < 0.05, ** = p-value < 0.01, n.s. = not significant. Error bars display confidence interval 0.95 of n = 3. Significance for strain doubling times and maximum OD600were calculated using Mann-Whitney U tests comparing strain values with those of rEc∆1.∆A, where * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, n.s. = not significant. Error bars display 20 confidence interval 0.95 of n = 7-8 replicates (Fig.5) or n = 4 replicates (Figure 10). Statistical significance ELP-GFP fluorescence assays assessing nsAA incorporation efficiencies were derived from unpaired t-tests comparing to rEc∆1.∆A, where * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, **** = p-value < 0.0001. Error bars display confidence interval 0.95 of n =3. Calculations and graphs were generated using Graph Pad Prism (v10.1.0). Figures 25 were prepared using Adobe Illustrator 2023 (v28.2). Reverting Auxotrophic Phenotypes: When MAGE mutagenesis is performed within multiple strains to be conjugated together, off- target genomic mutations can arise that offer little phenotypic impairment alone, but when 30 combined with other mutations through conjugation can result in synthetic deleterious mutations with phenotypic impairments. Alongside TGA conversions (Figure 7), major deletions (Table 2), and a subset of intentional SNPs (e.g., SEQ ID NO:2), the rEcΔ2 strain contains 200 indels (Table 4), 130 ORF frameshifts, 526 single nucleotide polymorphisms (SNPs) including 169 synonymous SNPs and 301 nonsynonymous SNPs (Table 5), and 13 in-frame premature stop 83 45741195.1 YU 8909 PCT codons. When auxotrophies arose, particularly after conjugations where a negative phenotype affects all resultant offspring, two methods of prototrophy restoration were employed: (1) conjugative backcrossing with ancestral rEc∆1.∆A (C321), or (2) targeted mutation reversions via MAGE. 5 Conjugative Backcrossing with rEc∆2.∆A: Early in this research, when only an eighth or quarter of a genome is being recoded, there was high probability of mutations arising within the unrecoded background, representing a majority of the genomic regions. To address these, strains can be conjugatively backcrossed with 10 ancestral rEc∆2.∆A via CAGE, in accordance with previous protocols. This method was used routinely to “clean up” strains prior to major conjugations and ensured genomic background outside of recoded regions were genotypically parental and unmutated, thereby reducing probability of synthetic lethality leading to conjugation failure. 15 MAGE-Targeted Reversion of Off-Target Mutations: Early in this research, growth impairment mutations would arise within recoded regions, resulting in poor conjugated offspring phenotypes that could not be addressed with backcrossing. When multiple recoded regions were assembled, an accumulation of background mutations within recoded regions led to deleterious phenotypes in growth and antibiotic resistances, as well 20 as synthetic auxotrophies (i.e. an inability to grow in M9 minimal media supplemented with glucose). In one instance, partially recoded strains were not able to be conjugated. Literature searches indicateed that knockout mutations within genes fliK and kefB, responsible for flagellar assembly and potassium transporter, respectively, were implicated in disrupting conjugal ability3. MAGE-mediated reversions of these mutations restored strain conjugative capacities.25 Employing breseq WGS analysis of recoded strains along their lineages, various non- synonymous mutations within genomes that correlated with undesirable phenotypes were identified. The resulting list of mutations was examined through ecocyc.org database, uniprot.org mutation lists, and relevant literature to identify potentially phenotypically relevant mutations. Once a narrowed list of probable causative mutations was achieved, MAGE oligos 30 were used to revert targeted mutations. Growth curves or fluorescent assays were then used to assess changes in phenotypes resulting from mutation reversions. One demonstrative example of this method was the presence of an auxotrophy that arose post-conjugation between two partially recoded strains. Each strain was backcrossed with rEc∆2.∆A prior to conjugation, yet auxotrophy was not resolved. Via breseq analysis and ecocyc.org growth evidence, a mutation in 84 45741195.1 YU 8909 PCT purL suspected to lead to auxotrophy was identified. The gene purL is involved in purine biosynthesis, located within a partially TGA-recoded genomic region (Table 9. Once the purL mutation was reverted to WT via MAGE, the prototrophic phenotype was restored (Table 6). 5 RF2 Variant Sequences Nucleotide (gene: prfB) and amino acid (protein: release factor 2) sequences for wildtype B-wt and Ochre mB-oKP. Discrepancies between sequences are highlighted and in bold. Tripeptide sequences are underlined. 10 >RF2: B-wt nucleotide sequence atgtttgaaattaatccggtaaataatcgcattcaggacctcacggaacgctccgacgttctta gggggtatctttgactacgacgccaagaaagagcgtctggaagaagtaaacgccgagctggaac agccggatgtctggaacgaacccgaacgcgcacaggcgctgggtaaagagcgttcctccctcga agccgttgtcgacaccctcgaccaaatgaaacaggggctggaagatgtttctggtctgctggaa15 ctggctgtagaagctgacgacgaagaaacctttaacgaagccgttgctgaactcgacgccctgg aagaaaaactggcgcagcttgagttccgccgtatgttctctggcgaatatgacagcgccgactg ctacctcgatattcaggcggggtctggcggtacggaagcacaggactgggcgagcatgcttgag cgtatgtatctgcgctgggcagaatcgcgtggtttcaaaactgaaatcatcgaagagtcggaag gtgaagtggcgggtattaaatccgtgacgatcaaaatctccggcgattacgcttacggctggct20 gcgtacagaaaccggcgttcaccgcctggtgcgtaaaagcccgtttgactccggcggtcgtcgc cacacgtcgttcagctccgcgtttgtttatccggaagttgatgatgatattgatatcgaaatca acccggcggatctgcgcattgacgtttatcgcacgtccggcgcgggcggtcagcacgttaaccg taccgaatctgcggtgcgtattacccacatcccgaccgggatcgtgacccagtgccagaacgac cgttcccagcacaagaacaaagatcaggccatgaagcagatgaaagcgaagctttatgaactgg25 agatgcagaagaaaaatgccgagaaacaggcgatggaagataacaaatccgacatcggctgggg cagccagattcgttcttatgtccttgatgactcccgcattaaagatctgcgcaccggggtagaa acccgcaacacgcaggccgtgctggacggcagcctggatcaatttatcgaagcaagtttgaaag cagggttatga (SEQ ID NO:1) 30 >mRF2: mB-oKP nucleotide sequence atgtttgaaattaatccggtaaataatcgcattcaggacctcacggaacgctccgacgttctta gggggtatctttaactacgacgccaagaaagagcgtctggaagaagtaaacgccgagctggaac agccggatgtctggaacgaacccgaacgcgcacaggcgctgggtaaagagcgttcctccctcga agccgttgtcgacaccctcgaccaaatgaaacaggggctggaagatgtttctggtctgctggaa 85 45741195.1 YU 8909 PCT ctggctgtagaagctgacgacgaagaaacctttaacgaagccgttgctgaactcgacgccctgg aagaaaaactggcgcagcttgagttccgccgtatgttctctggcgaatatgacagcgccgactg ctacctcgatattcaggcggggtctggcggtacggaagcacaggactgggcgagcatgcttgag cgtatgtatctgcgctgggcagaatcgcgtggtttcaaaactgaaatcatcgaagagtcgaaag 5 gtgaagtggcgggtattaaatccgtgacgatcaaaatctccggcgattacgcttacggctggct gcgtacagaaaccggcgttcaccgcctggtgcgtaaacctccgtttgactccggcggtcgtcgc cacacgtcgttcagctccgcgtttgtttatccggaagttgatgatgatattgatatcgaaatca acccggcggatctgcgcattgacgtttatcgcgcgtccggcgcgggcggtcagcacgttaaccg taccgaatctgcggtgcgtattacccacatcccgaccgggatcgtgacccagtgccagaacgac10 cgttcccagcacaagaacaaagatcaggccatgaagcagatgaaagcgaagctttatgaactgg agatgcagaagaaaaatgccgagaaacaggcgatggaagataacaaatccgacatcggctgggg cagccagattcgttcttatgtccttgatgactcccgcattaaagatctgcgcaccggggtagaa acccgcaacacgcaggccgtgctggacggcagcctggatcaatttatcgaagcaagtttgaaag cagggttataatga (SEQ ID NO:2) 15 >RF2: B-wt peptide AA sequence MFEINPVNNRIQDLTERSDVLRGYLDYDAKKERLEEVNAELEQPDVWNEPERAQALGKERSSLE AVVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFRRMFSGEYDSADC YLDIQAGSGGTEAQDWASMLERMYLRWAESRGFKTEIIEESEGEVAGIKSVTIKISGDYAYGWL 20 RTETGVHRLVRKSPFDSGGRRHTSFSSAFVYPEVDDDIDIEINPADLRIDVYRTSGAGGQHVNR TESAVRITHIPTGIVTQCQNDRSQHKNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWG SQIRSYVLDDSRIKDLRTGVETRNTQAVLDGSLDQFIEASLKAGL* (SEQ ID NO:3) 25 >mRF2: mB-oKP peptide AA sequence MFEINPVNNRIQDLTERSDVLRGYLNYDAKKERLEEVNAELEQPDVWNEPERAQALGKERSSLE AVVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFRRMFSGEYDSADC YLDIQAGSGGTEAQDWASMLERMYLRWAESRGFKTEIIEESKGEVAGIKSVTIKISGDYAYGWL RTETGVHRLVRKPPFDSGGRRHTSFSSAFVYPEVDDDIDIEINPADLRIDVYRASGAGGQHVNR 30 TESAVRITHIPTGIVTQCQNDRSQHKNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWG SQIRSYVLDDSRIKDLRTGVETRNTQAVLDGSLDQFIEASLKAGL* (SEQ ID NO:4) 86 45741195.1 YU 8909 PCT Results Design and construction of ΔTAG / ΔTGA recoded strain The TGA codon is an attractive choice to engineer as a free codon as only 71 essential genes terminate in TGA among 1,216 total TGA codons that exist in the MG1655 genome 5 (Figure 7). Additionally, dual OTSs targeting UAG and UGA have been deployed within unrecoded E. coli for incorporating two distinct nsAAs concurrently, but are limited in efficacy by competition with native translation factors. Although recoding efforts can be accomplished by established DNA synthesis and genome engineering methods (e.g., multiplexed oligo-based mutagenesis - MAGE - and conjugation-based assembly - CAGE), determining essentiality of 10 TGA codons, engineering mutant RFs with UAA specificity, and addressing translational crosstalk from tRNA suppression were primary open questions for this recoding effort. Importantly, evidence of RFs and tRNAs with altered specificities indicate feasibility in engineering attenuated native UGA recognition to free TGA alongside TAG for reassignment with established OTSs. Completing these iterative steps would facilitate construction of a 15 bacterial strain lacking both native TAG and TGA codons that, when complemented by a noncompetitive mutant RF, are freed for reassignment as sense codons for dual nsAA incorporation. To construct a ΔTAG-ΔTGA recoded strain (rEcΔ2), the ΔTAG progenitor C321.ΔA, referred to as rEcΔ1.ΔA, was used. All TGA codons, with few exceptions, occur at the end of a 20 gene to terminate translation. Of the 1,213 annotated open reading frames (ORFs) terminating in TGA, 1,168 are annotated genes, while 45 are listed as pseudogenes according to GenBank. To reduce recoding efforts, 76 nonessential genes and 3 pseudogenes were removed within 16 targeted genomic deletions (Fig.1B). The remaining 1,134 terminal TGA codons (comprising 1,092 genes and 42 pseudogenes) were targeted for conversion to TAA via MAGE (Table 9. 25 Three formate dehydrogenase genes (fdhF, fdoG, and fdnG) contained internal TGA codons were not targeted for recoding due to their translational dependence on specialized selenocysteine insertion sequence (SECIS) elements for UGA decoding. Although most oligos were designed to introduce a single-nucleotide substitution to convert TGA to TAA, 380 terminal TGA codons overlap a neighboring gene and most cannot be 30 converted in this way without compromising expression of neighboring genes. This overlap can fall into one of three categories (Fig.1C). In the first category (26 instances), the A of TGA and ATG is shared, and thus mutating G to A in the TGA codon does not alter the ATG codon. In the second category (257 instances), both the T and G are shared between the ATG and TGA codons. Therefore, recoding TGA to TAA disrupts the start codon, which could preclude 87 45741195.1 YU 8909 PCT translation of the overlapped gene. To avoid this problem, the insertion of three bases – TAA – preserves both a new TAA codon and the ATG start codon, albeit with the ribosome binding site (RBS) shifted by three nucleotides from the start codon. Since the spacing of the RBS from the start codon can impact translation initiation rates, the RBS calculator was used to predict 5 translation initiation rates for both the wild type and recoded sequences ( and found little predicted deviation in translation initiation upon recoding (Fig.1D). The third category of overlap (97 instances) encompasses all instances where two genes overlap extensively so recoding inevitably introduces a mutation in the overlapped gene. In the latter two categories of TGA overlap, specialized oligos were designed to recode the TGA-containing gene to minimize 10 polar effects. To mitigate risk, the construction strategy for the rEcΔ2 strain constituted two major phases. First, construction of a preliminary ΔTGA-essentials strain rEcΔ2E.ΔA (recoding all 71 essential genes terminating in TGA to TAA) from progenitor strain rEcΔ1.ΔA. Second, construction of the final ΔTGA strain rEcΔ2.∆A by eliminating remaining TGA codons (Fig. 15 1E). Both phases employed iterative cycles of MAGE on distinct subdomains of the genome split amongst clonal progenitor strains, followed by CAGE to assemble recoded subdomains into combined-genomic strains within each phase. In Phase I, construction of rEcΔ2E.ΔA from rEcΔ1.ΔA involved the targeted conversion of 71 essential TGA-ending genes within two distinct genomic subdomains (A’ and B’) of rEcΔ1.ΔA clones. Additional oligos added to 20 MAGE pools converted 44 proximal genes. Three TGA-ending genes were deleted placing selectable markers. Recoded subdomains A’ and B’ of clones rEcΔ1.ΔA(A’) and rEcΔ1.ΔA(B’), respectively, were assembled via CAGE into rEcΔ2E.ΔA (Table 1). TGA conversions to TAA were confirmed via whole genome sequencing (WGS). In Phase II, construction of fully ΔTGA-recoded rEcΔ2.ΔA from ΔTGA-essentials 25 rEcΔ2E.ΔA involved the successful conversion of 1,012 remaining nonessential ORFs (980 genes and 35 pseudogenes) via MAGE (Table 9, alongside deletion of 229 nonessential ORFs (72 TGA genes and 3 TGA pseudogenes) using 15 additional targeted genomic deletions (Figure 8, Table 2). Deletions often coincided with placement of selectable markers employed in CAGE, with markers subsequently deleted via tolC placement-displacements. Conversions 30 and deletions were made concurrently across eight distinct genomic subdomains each within a rEcΔ2E.ΔA clone (Table 3). Notably, one terminal TGA within the nonessential glycerate kinase I gene, garK, was recalcitrant to conversion and ultimately deactivated by introducing a frameshift and premature nonsense mutation. Strains containing recoded subdomains were hierarchically assembled via CAGE into the final rEcΔ2.ΔA (Figure 9). TGA conversions and 88 45741195.1 YU 8909 PCT gene deletions were confirmed via WGS after each conjugative assembly. Out of 1,216 predicted TGA codons in E. coli, 1,195 were abolished, leaving 10 predicted pseudogenes, 8 transposable elements, and 3 internal selenocysteine codons unrecoded (Figure 7). WGS data and breseq analysis were used to assess background mutations, which arose 5 from ancestral inactivation of DNA mismatch repair protein MutS. Throughout the recoding and deletion process, background mutations were acquired across strain lineages toward the final rEcΔ2.∆A genome (Table 4 & Table 5). To account for emergent auxotrophies that arose during strain construction, cell fitness and growth were characterized after multiple successive rounds of MAGE and after each conjugative assembly, measuring maximum OD600 and doubling time 10 in lysogeny broth (LB), and minimal media (M9). When auxotrophy did arise, strains were initially backcrossed with ancestral rEcΔ1.∆A to overwrite background mutations arising outside of recoded subdomains. If backcrosses failed to restore prototrophy, implicated mutations within recoded regions were identified through WGS and reverted via MAGE (Table 6). Engineering and characterization of mutant release factor 2 15 Engineering UAA-specific translation termination is required to establish a functionally recoded ∆TAG-∆TGA GRO with TAA serving as the sole stop codon. As in most prokaryotes, E. coli release factor 1 (RF1), encoded by prfA, terminates translation at UAG and UAA, while release factor 2 (RF2), encoded by prfB, terminates translation at UGA and UAA. RF1 was previously deleted within ancestral rEcΔ1.∆A, confirming both the non-essential nature of RF1 20 in the absence of TAG codons and the ability of RF2 to serve as the sole cellular release factor. Upon removal of TGA in rEcΔ2.∆A, it was asked whether RF1, like RF2, could independently support cellular translation termination. Unlike RF1, E. coli RF2 serves central functions besides termination, including RF3-cooperative post-peptidyl transfer quality control and ArfA- cooperative codon-independent rescue of stalled ribosomal complexes from ‘non-stop’ mRNAs. 25 The inability to delete RF2 in the presence of RF1, despite an absence of genomic TGA codons, supports the essentiality of alternative RF2 functions aside from UGA termination, or indicates an insufficiency of RF1 to act as a solitary release factor in this genetic context. The unique capacity of RF2 to act as a sole release factor thus made it a target to engineer codon specificity limited to the UAA stop codon. Thus, it was sought to identify mutations that attenuate RF2- 30 mediated termination at UGA, while retaining essential functions in UAA termination and ribosomal rescue. In most bacterial RFs, stop codon specificity is conferred by the first and third amino acids of the RF tripeptide ‘anticodon’ motif (PXT in RF1, where X = A or V; SPF in RF2), which discriminate the second and third bases of the stop codon, respectively. However, 89 45741195.1 YU 8909 PCT alternative codon specificity is well precedented in nature. Most eukaryotic release factors (eRF1) terminate at all three stop codons, while rare cases terminate at one codon (UGA or UAA). Meanwhile, some mitochondrial RFs terminate at canonical sense codons (e.g. AGA / AGG). In some prokaryotes, single point mutations in RF2 were identified to alter codon 5 specificity, including some (e.g., E167K or F207T) known to expand codon recognition to all three stop codons, turning it into an “omnipotent” release factor. It was considered that one previously characterized E. coli RF2 mutation, S205P, might endow UAA-specificity. Occurring within the RF2 codon-recognition tripeptide motif, S205P converts wildtype tripeptide SPF to RF1-RF2 chimeric PPF. Previous studies conducted in vivo and in vitro characterizations of the 10 S205P mutation to interrogate E. coli RF codon recognition. Using a chimeric RF1-RF2 backbone, Ito et. al. recapitulated WT-RF2 termination with a SPF-tripeptide. Introduction of S205P (PPF-tripeptide) presented attenuated recognition of UGA in vitro, without compromising UAA termination. Meanwhile, expression in vivo failed to complement deletion of native RF2 in E. coli. A second study introduced S205P into WT-E. coli RF2 expressed from a plasmid. 15 Expression also failed to complement native RF2 knockout in E. coli. Given these results, experiments were designed to test if unlike WT-E. coli, which is dependent on UGA termination, the ∆TGA rEc∆2.∆A strain could uniquely tolerate RF2 S205P. Prior to introducing S205P to RF2, first introduced were three preliminary mutations to restore WT-RF2 termination efficiency and eliminate prfB reliance on TGA for RF2 translation 20 (Fig.2A). Derived from K-12 lab strains, rEc∆1.∆A contains an RF2 A246T mutation that hinders RF2 termination efficiency compared to that of WT-E coli. The first mutation, T246A, re-establishes RF2 methylation by PrmC and improves strain fitness. To make RF2 translation compatible with attenuated UGA termination, both the internal autoregulatory TGA and the terminal TGA were converted to TAA. The prfB mRNA contains an internal RBS upstream of a 25 “slippery” CUU-UGA that facilitates RF2-mediated autoregulation, resulting in premature prfB translation termination when RF2 concentrations are high, and full-length protein translation via a +1 frameshift when RF2 concentrations are low. While most analyzed bacterial prfB mRNA contain this internal CUU-UGA, Chlorobium tepidum contains CUU-UAA. The conversion of internal TGA to TAA resulted in a D26N mutation which conferred minor rEc∆2.∆A growth 30 defects relative to WT-RF2 ( Figure 10). Meanwhile, the terminal TGA of prfB overlaps the RBS for lysS encoding lysine tRNA synthetase. To preserve the RBS and minimally impact lysS translation, TAA was inserted directly upstream of the terminal TGA (Fig.2A). The resulting mutant release factor 2 was dubbed mRF2-o (“o” signifying RF2 reliance on the “ochre” codon – UAA – for translation) (Table 1). 90 45741195.1 YU 8909 PCT Once a TGA-independent prfB was established, S205P was introduced into rEcΔ2.∆A mRF2-o to generate mRF2-oP, yet failed to recover viable clones. However, once RF1 was introduced, generating mRF2-oP became permissible (Table 1). These results reveal that despite whole-genome ∆TGA recoding, S205P was not tolerated in mRF2-o, ∆RF1, indicating mRF2-oP 5 function was not sufficient to sustain viability. AlphaFold structural prediction (Fig.2B) indicates wild-type S205 presents up to 4 hydrogen bonds within the codon recognition loop of RF2, while P205 presents one, likely altering codon recognition loop stability and function. In further support, once mRF2-oP was established, RF1 deletion was only achieved when accompanied by a spontaneous P205 reversion to S, or via acquisition of a spontaneous 10 compensatory mutation in RF3, which monitors peptide quality control alongside accelerating RF1 / RF2-mediated termination and release from the ribosome. The RF3 mutation was only revertible when RF2 P205 reverted to S or when RF1 was reintroduced. To facilitate a viable ΔRF1 RF2-S205P strain, MAGE with oligos designed to introduce mutations at 9 unique sites within mRF2-o alongside and including S205P was employed 15 (Figure 11). It was considered that RF2 residues found uniquely within bacterial genomes containing low frequencies of TGA codons may possess poorer UGA recognition. Targeted mutations were identified through protein sequence alignments of E. coli RF2, RF1, and RF2 sequences from low-TGA-frequency bacterial genomes, with residues cross-referenced with literature to avoid conferring UAG recognition. While various mutations were achieved, S205P 20 arose only in co-occurrence with E170K within a viable rEcΔ2.ΔA strain. RF2 E170K is a charge-flip mutation characterized in previous RF mutagenesis studies, and arose spontaneously within rEcΔ1.ΔA optimization-oriented adaptive evolution. Previous RF complementation assays revealed that adjacent residue charge-flips, like E167K, altered or induced omnipotent stop codon recognition in RF2, while E170K presented no identifiable impact to RF function. 25 Introduction of S205P and E170K into mRF2-o generated mRF2-oKP (Fig.2A-2B). To examine the importance of E170K in the mRF2-oKP strain, MAGE was employed to revert K170 back to E, but failed to yield viable clones in absence of RF1, confirming the essentiality of E170K to support viability of an RF2 S205P mutant. To assess whether mRF2-oKP abolishes UGA recognition, an in vivo assay was 30 conducted that reports on release activity of mutant and wildtype RF2 variants at all three stop codons and GCG sense codon as a control. Derived from established methods, this assay determines an RF’s ability to terminate translation of an mCherry gene ending in a target codon upstream of a YFP gene (Fig.2C, Figure 12). If no termination occurs at the target codon, translation reads through mCherry to YFP, expressing both proteins. However, if successful 91 45741195.1 YU 8909 PCT termination occurs, only mCherry is expressed, yielding low YFP fluorescence. Figures 2D-2E present fluorescence of mCherry and YFP expression as a function of termination at a target codon, normalized to fluorescent expression from GCG (+ control). Results revealed low YFP expression at all stop codons, regardless of RF status (Fig.2D). In rEcΔ1.ΔA strains lacking 5 RF1, genes ending in TAG are targeted for degradation via transfer messenger RNA (tmRNA) tagging, reducing UAG codon readthrough and thus GFP expression. This is consistent with the low expression levels observed for UAG-ending mCherry and downstream YFP. To provide more robust readouts, modified UAG- and UGA-suppressor serine tRNAs (supD) that precisely base-pair with their respective codons to examine direct codon competition between RF variants 10 and suppressor tRNAs were employed, effective for assessing codon reassignment (Fig.2E). YFP expression results for mRF2-oKP indicate termination at UAA alongside a ~42-fold enhanced readthrough at UAG and ~45-fold enhanced readthrough at UGA relative to no suppressors, resulting in 11-fold and 8-fold increases in UGA YFP fluorescence from rEc∆2.∆A mRF2-oKP compared to WT-RF2 strains rEc∆1.∆A and rEc∆2.∆A, respectively. These results15 demonstrate that mRF2-oKP recognition of UGA is significantly attenuated compared to WT- RF2, as mRF2-oKP is outcompeted by the UGA-specific supD tRNA. As a result, RF2 mutations E170K and S205P functionally reassign UGA from a stop codon to an open codon amenable to amino acid reassignment. In addition, these cumulative RF2 mutations render S205P viable in a ∆TGA genomic background, strongly indicateing that UAA-specificity was 20 achieved concurrent with attenuated UGA recognition. The new mRF2-oKP ∆TAG-∆TGA recoded strain was designated as “Ochre”, referencing its use of the ochre codon, TAA, as its sole cellular stop codon. To assess whether S205P (not E170K) is responsible for attenuation of UGA recognition, P205 was reverted back to S within mRF2-oKP to generate mRF2-oK. The same in vivo 25 YFP:mCherry readthrough assays was then repeated, without suppressors, for previously derived RF1+mRF2-oP (S205P) alongside mRF2-oK (E170K) (Figure 13). The high mCherry and low YFP fluorescence for UAG from the RF1+mRF2-oP strain is indicative of the presence of RF1 terminating at UAG. For UGA, results reveal low YFP fluorescence from both WT-RF2 and mRF2-oK variants, indicating little impact from E170K on WT RF2 termination. However, 30 S205P in absence of E170K (RF1+mRF2-oP) presents high YFP fluorescence, indicating significant native suppression at UGA and attenuation of RF2 recognition. This result stands in contrast to mRF2-oKP (S205P with E170K) without supD suppressors (Fig.2C), revealing S205P to be uniquely responsible for attenuation of UGA termination. These data indicate E170K restores mRF2-oP termination function at UAA sufficient for cell viability in absence of 92 45741195.1 YU 8909 PCT RF1 and partially restores termination function at UGA with significant attenuation relative to WT-RF2. Characterization of mutant release factor 2 by phage infection To further examine impacts of RF2 engineering on protein translation, plate-based phage 5 infection assays were conducted that have been previously used to assess the impact of genomic recoding and translation factor deletions in establishing genetic code orthogonality. Hosts that lacked machinery for decoding essential phage codons mistranslated viral proteins, halting propagation and conferring resistance to phage. Lambda (λ) phage genes containing TAG codons are targeted for degradation via tmRNA tagging when expressed in a strain lacking RF1 10 recognition of the UAG codon, preventing phage infection. Experiments were designed to test if abolition of UAG and UGA termination will confer strains with resistance to phage that employ essential genes ending in TAG or TGA. RF-variant strains and predecessors were challenged with two genotypically distinct bacteriophages: λ (containing TAG, TGA, and TAA) and Mu (µ) (containing TGA and TAA only). Strains incubated with serially diluted phage were used to 15 quantify plaque-forming units per milliliter (PFU / ml), presenting no visible plaques in resistant strains and reduced plaque sizes in partially resistant strains (Figure 14). Results revealed that all strain variants lacking RF1 were resistant to λ (Fig.3A), retaining the genetic orthogonality of rEc∆1.∆A conferred by deletion of RF1. To specifically examine the impact of UGA decoding alone, strains were challenged with µ (lacking TAG). If RF2 decoding of UGA is 20 abolished, a strain should be resistant to µ infection by degrading the products of TGA-ending genes. Indeed, strains presenting mRF2-oP (S205P alone), with RF1 to compensate for inviability, demonstrate robust resistance to µ (Fig.3B), indicating abolition of UGA termination. However, deleting RF1 and introducing E170K to generate mRF2-oKP (i.e. Ochre) renders the strains susceptible to infection, indicating minor restoration of mRF2 UGA decoding 25 for phage translation, consistent with readthrough data (Fig.2D). Taken together, these phage assays indicate (i) robust RF2 decoding of UGA is not necessary for cell viability in rEc∆2, as demonstrated by mRF2-oP resistance to µ, and (ii) S205P functionally abolishes UGA termination, which is partially restored in the presence of E170K, consistent with readthrough results. 30 Eliminating UGA suppression by tRNATrpA proteomics assay and reporter protein were established to examine how UGA is decoded within Ochre. Mass spectrometry revealed striking levels of Trp suppression comprising nearly 80% of residues at UGA, indicating significant suppression by tRNATrp(CCA) (Fig.4) as the native E. coli tRNATrp(Ec-tRNATrp) anticodon loop wobble pairs with UGA. To 93 45741195.1 YU 8909 PCT reduce native Trp suppression and create an open coding channel for reassignment for nsAA incorporation, trpT was modified, encoding tRNATrp(CCA), to abolish UGA recognition. An A37G mutation within S. cerevisiae tRNATrp(Sc-tRNATrp), had been shown to abolish Sc- tRNATrpUGA suppression. The sequence surrounding the anticodon region of Ec-tRNATrpto 5 Sc-tRNATrpwas compared and a common A36-A37-A38 motif in the anticodon loop of both tRNAs was identified. This motif is required for the modification of A37 to N6-(isopentenyl) adenosine (i6A37) in both prokaryotes and eukaryotes, yet additional modification of A37 (ms2i6A37) occurs in prokaryotes and is not present in Sc-tRNATrp. Based on these observations, experiments were designed to test if the UGA suppression activity was linked to 10 A37 modification in E. coli and that the A37G mutation could abolish UGA suppression in E. coli. A37 was mutated in native Ec-tRNATrpto G37 (tW*) to disrupt the A36-A37-A38 recognition motif and prevent base modification (Fig.4A). YFP:mCherry fluorescent assays were used with a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase tRNATyr(UCA) pair 15 (MjTyrRS-OTS) designed to encode Tyr at UGA to evaluate both OTS activity and native tRNATrpsuppression at UGA. A significant reduction in fluorescent YFP / mCherry expression in Ochre.tW* was observed compared to WT tRNATrp, indicating a reduction of native tRNATrpsuppression (Fig.4B). To confirm decreased Trp suppression in Ochre.tW*, the established Mass Spectrometry Reporter for Exact Amino Acid Decoding (MS-READ) to assay amino acid 20 incorporation at UGA in WT and A37G tRNATrpstrains containing the MjTyr-OTS. Mass spectrometry analysis confirmed OTS-mediated Tyr incorporation at UGA codons, and a small amount (<1%) of Cys incorporation. However, while Trp was the most abundant amino acid at UGA in Ochre cells with WT tRNATrp, it was completely absent in Ochre.tW* (Fig.4C). These results confirm that a single base substitution in Ec-tRNATrppreserves native Trp decoding while 25 eliminating UGA suppression, removing the wobble effect and establishing an open UGA codon amenable for reassignment. With the deletion of RF1 and attenuation of UGA recognition by mRF2-oKP and tW*, four codons (UAG, UGA, UAA, & UGG) that naturally display significant translational crosstalk can be rendered translationally isolated and functionally exclusive: open, open, stop, Trp, respectively (Fig.1A). 30 Phenotypic characterization of recoded strains: To assess the impacts of TGA recoding and RF2 and tRNATrpmutations on cellular fitness, doubling times (DT) and maximum cellular density at OD600 (MaxOD) of each strain were measured in nutrient-rich (LB) and minimal (M9) media (Fig.5). Assessing the cumulative impacts of whole-genome TGA replacement and translation engineering, results revealed greater 94 45741195.1 YU 8909 PCT increases in DTs for Ochre.tW* (~32% M9, ~37% LB) than for Ochre (~18% M9, ~28% LB) when compared to ancestral rEcΔ1.∆A, with greater change in MaxOD in Ochre.tW* (increase ~12% M9, decrease ~14% LB) than in Ochre (increase 6% M9, no change LB). To distinguish the impacts of TGA recoding from translation engineering, TGA-recoded rEcΔ2.∆A was 5 compared with ancestral rEcΔ2.∆A, and translationally-engineered Ochre / Ochre.tW* strains with rEcΔ2.∆A. Assessing the impacts of genome-scale TGA replacements, results reveal no reduction in MaxOD (no change M9, increase ~5% LB) as DTs elongated (~9% M9, ~22% LB) from ancestral rEcΔ1.∆A to rEcΔ2.∆A, demonstrating growth rate impairments after ∆TGA recoding. Assessing the impacts of translation engineering, results present a greater elongation in 10 DTs for Ochre.tW* (~21% M9, ~12% LB) than Ochre (~9% M9, ~6% LB), as well as greater increases in MaxOD for Ochre.tW* (~7% M9, ~12% LB) than for Ochre (~1% M9, ~1% LB). Taken together, these results reveal that growth rates are impaired by both TGA recoding and cumulative translation engineering of RF2 exacerbated by tW*. To dissect the impacts of individual RF mutations on growth, RF-variant strains were 15 assessed in isogenic rEc∆2 backgrounds (Figure 10) and compared to WT-RF2-containing rEc∆2.∆A. D26N conversion of RF2 autoregulatory TGA to TAA extended LB DT from WT- RF2 (rEc∆2.∆A) by ~8%, decreasing MaxOD by ~2%. Adding E170K atop D26N largely restored LB DT and MaxOD back to WT-RF2 levels. Meanwhile, introducing S205P atop D26N and E170K (generating mRF2-oKP) slowed LB DT by ~12% from WT-RF2, increasing MaxOD 20 by ~2%. To further assess the impact of S205P and E170K decoupled, RF2 variants were assessed with RF1 reintroduced. mRF2-oKP / RF1+presented ~21% longer LB DT and ~7% reduction in MaxOD compared to WT-RF2 / RF1+. Meanwhile, reverting K170 to E (mRF2- oP / RF1+) presented a similar ~20% longer LB DT and ~5% reduction in MaxOD relative to WT- RF2 / RF1+. These data indicat fitness impairments in Ochre result from cumulative effects of 25 RF2 mutations, with S205P as a key contributor. When comparing DT and MaxOD of rEcΔ1.∆A and rEcΔ2.∆A independent of RF mutations (Fig.5, Fig.15), the accumulated effects of deliberate genomic deletions (Table 2) and overlapping gene mutations likely played at least a minor role in impairing fitness. Most phenotypic disparities likely arose from synthetic effects of accumulated unintended background 30 mutations acquired through selections, recoding, and conjugations (Table 4, Table 5, Figure 8). This factor establishes opportunity for targeted and evolved fitness improvements, as well as analysis into the impacts of uncharacterized mutations on individual protein expression, function, and associated pathways. 95 45741195.1 YU 8909 PCT Dual incorporation of nsAAs into proteins: To assess the practical implications of isolating four codons from translational crosstalk, opening two for reassignment, the functional capacity of Ochre and Ochre.tW* to incorporate two distinct nsAAs at UAG and UGA in recombinant reporter proteins was assessed. Previous 5 recoding of TAG and deletion of RF1 in rEcΔ1.ΔA demonstrated multi-site incorporation of 30 nsAAs within elastin-like polypeptides (ELPs) with 95% accuracy, compared to upwards of 50- 88% efficiencies at single sites within WT-E. coli. To reassign both UAG and UGA codons, a dual-OTS fluorescent reporter plasmid was constructed for the incorporation of para-acetyl-L- phenylalanine (pAcF) at UGA and Nε-Boc-L-lysine (BocK) at UAG (Fig.6, Fig.16). This 10 plasmid contains a bicistronic cassette with two aaRSs, pAzFRS2.t1 and chPylRS, controlled by L-arabinose-inducible promoter pBAD. The Mj-tRNA(CUA), paired with pAzFRS2.t1, was assigned to UGA by changing its anticodon to UCA while an optimized PylT, paired with chPylRS, was assigned to UAG. Both tRNAs were expressed from a single proK promoter with a valX linker for dual expression. For a reporter, a TetR-anhydrotetracycline (aTc)-inducible 15 elastin-like polypeptide fused to a green-fluorescent protein (ELP-GFP) with the ELP sequence modified for MS-READ was expressed. Three ELP constructs were tested to assess the efficiency of nsAA encoding at either UAG, UGA or both UAG and UGA: (1) 3 TAG sites or (2) 3 TGA sites for single nsAA incorporations, or (3) 6 sites with alternating TAG and TGA combinations for dual incorporation (Fig.6A). Four strains were tested for single and dual nsAA 20 incorporation: rEcΔ1.ΔA, rEcΔ2.ΔA, Ochre, & Ochre.tW* (Fig.6B-6D). For nsAA incorporation at UAG and / or UGA, 10mM BocK and / or 1mM pAcF were added to growth media, respectively, along with L-arabinose 0.05% w / v and 100 ng / mL aTc. Given the absence of RF1 in all four strains, it was believed there would be minimal differences between strains in efficiencies of incorporation of BocK at three UAG. In contrast, it was considered that efficient 25 incorporation of pAcF at three UGA sites might significantly improve in Ochre variants where genomic TGA is removed and native competition for UGA is attenuated with mRF2-oKP and tW*. First analyzed was single nsAA incorporation at three codon sites within the ELP-GFP reporters. Upon OTS expression with ELP-3xTAG-GFP in the presence of BocK, rEc∆1.∆A and 30 rEc∆2.∆A present roughly similar increases in GFP fluorescence (4- and 6-fold, respectively), compared with an absence of nsAA (Fig.6B). Similarly, Ochre and Ochre.tW* strains presented 11- and 12-fold increases in fluorescence compared to no nsAA, reveal no negative impact on nsAA incorporation efficiencies at UAG resulting from ∆TGA recoding and RF2 engineering. 96 45741195.1 YU 8909 PCT Upon OTS expression with ELP-3xTGA-GFP in the presence of pAcF, both rEc∆1.∆A and rEc∆2.∆A present similar absolute fluorescence, revealing that UGA reassignment was not achieved by ∆TGA recoding alone (Fig.6C). In contrast, Ochre and Ochre.tW* present ~5- and 7-fold increased fluorescence with pAcF, respectively, compared to both rEc∆1.∆A and 5 rEc∆2.∆A, resulting from ~6-fold increases in fluorescence for both Ochre strains compared to no nsAA. These data reveal the significance of RF2 and tRNATrptranslation engineering in establishing UGA codon isolation, orthogonality, and reassignment. Finally, it was desired to investigate strain capacities for efficient dual-incorporation of BocK and pAcF at three UAG and three UGA sites concurrently within ELP-6xTAG / TGA-GFP 10 (Fig.6D). Upon OTS expression with BocK and pAcF, rEc∆1.∆A and rEc∆2.∆A present 2- and 4-fold increases in fluorescence relative to no nsAAs added, while the difference in GFP fluorescence between rEc∆2.∆A and rEc∆1.∆A were marginal. In contrast, Ochre and Ochre.tW* both presented an ~4-fold increase in GFP fluorescence compared to rEc∆2.∆A and 7- and 8-fold respective increases relative to rEc∆1.∆A, resulting from 18- and 14-fold 15 respective increases relative to no nsAAs. These data indicate enhanced incorporation of two nsAAs at six alternating sites. These results demonstrate the importance of engineering RF2 and Ec-tRNATrpto reduce UGA recognition, while maintaining UAG orthogonality. The attenuation of native codon competition from wildtype translation factors that cleave and suppress at UGA demonstrably supported stop codon reassignment for multi-site encoding of two distinct nsAAs 20 at UAG and UGA. To assess the accuracy of nsAA incorporation, mass spectrometry was used to directly assay BocK and pAcF incorporation at the six UAG / UGA sites in a modified MS-READ protein. Importantly, BocK and pAcF combinations were designed to allow for unambiguous identification of each MS-READ reporter segment (Fig.6E). MS-MS spectra for each MS- 25 READ peptide segment confirmed 3x BocK and 3x pAcF incorporation at the six positions. Product ion spectra were more clearly differentiated by a strong 100Da neutral loss signature at BocK sites (^ in Fig.6F). Peptide intensities for the two reporter segments revealed Trp incorporation in Ochre with WT tRNATrp, which competed with pAcF incorporation at UGA, in addition to Lys, Gln, and Tyr incorporation at UAG (Fig.6G-6H). Dual incorporation of BocK 30 and pAcF was completely free of Trp contamination in Ochre.tW* and accompanied by a striking reduction of Lys, Gln, and Tyr incorporation at UAG, yielding greater than 99% average on-target incorporation at all sites (Fig.6G-6H). This result compared to 93% on-site incorporation for Ochre, without tRNATrp engineering, reveals the impact of the A37G mutation to enhance incorporation efficiency and mitigate competition from native UGA 97 45741195.1 YU 8909 PCT suppressors. Taken together, these results reveal that recoding alone does not result in a general enhancement in nsAA encoding efficiency at UGA, whereas recoding combined with RF2 and tRNATrpengineering show a striking increase in ELP-GFP expression, highlighting the importance of combining recoding and compatible translation factor engineering to achieve 5 robust and multi-site incorporation of nsAAs in protein at high accuracy. Discussion This study describes an integrated genome, biomolecular, and protein engineering effort that eliminates all redundant stop codons, resulting in TAA as the sole genomic stop codon. Specifically, translational impact of whole-genome recoding of TGA stop codons to TAA in E. 10 coli was computationally projected, then mutagenic oligos were designed to recode 1,195 instances of TGA stop codons to TAA in rEcΔ1.∆A while minimizing unintended polar effects. This effort generated rEcΔ2.∆A: a bacterial GRO lacking all instances of TAG and TGA stop codons. Translational compatibility was established by engineering native RF2 and tRNATrpto attenuate native cognate recognition of UGA, while retaining tRNATrpspecificity for UGG, RF2 15 specificity for UAA, and orthogonality of UAG. Through expression of compatible OTSs coupled with translation factor engineering for single-codon specificity, GRO Ochre contains a single functional stop codon UAA, with UAG and UGA codons reassigned for efficient multi- site incorporation of two distinct nsAAs at six UAG and UGA codons into proteins with recombinant peptide purity >99%, surpassing previous in vivo efforts. The establishment of this 20 GRO with four translationally isolated codons, one non-degenerate stop codon and two open for reassignment, offers a high purity, functional chassis for production of multi-functional designer proteins with expanded chemistries. An important focus of this work was structurally guided engineering of multiple important translation factors responsible for decoding the three stop codons and tryptophan UGG 25 to mitigate translational crosstalk in decoding, translationally isolating four codons for exclusive functions. These translation engineering efforts and the universal conservation of broad translational structures offer a new lens of RF codon reassignment to be applied to future GRO efforts across domains of life and toward both fully non-degenerate genetic codes, as well as fully orthogonal codes that employ non-canonical stop codons for robust genetic isolation. These 30 efforts affirm that release factor decoding of stop codons is a delicate balance involving multiple translation factors. Although the introduction of S205P into RF2 attenuates UGA termination (Fig.2e), in vivo assays indicat the mutation may compromise UAA termination as well (Fig. 2d, Fig.3b), leading to cell inviability without compensatory mutations (e.g., E170K) to re- establish UAA function. The surface charge exchange from E170K (Fig.2B) may influence 98 45741195.1 YU 8909 PCT interactions rates of mRF2 with the negatively charged ribosome, or even positioning of mRF2 within the ribosomal pockets, impacting codon recognition and permitting some degree of UGA termination by mRF2-oKP outcompeting native tRNATrp(CCA) wobble suppression (Fig.2C – “No Suppressor”), but not UGA-specific tRNAs (supD and TyrOTS) (Fig.2C – “UGA 5 Suppressor”; Fig.4B). Further refinements to RF structures may yield RF variants with absolute single-codon specificity. Given the limited number of mutations tested to attenuate UGA termination, along with the abolition of UGA termination found in rEc∆2.A mRF2-oP (Figure 13), there exists opportunity for broader mutational sampling and engineering of RFs to further alter codon specificity. In addition, spontaneous mutations to RF3 arising from RF2 10 engineering may offer mechanistic insights into interactions and functions of RF3 in translation termination, as well as open yet another door to tune translation. Finally, engineering ribosomal components may present alternative means for attenuating native UGA recognition. Mutations within 16S and 23S ribosomal RNA (rRNA) have been found to exclusively increase UGA suppression without compromising UAG or UAA. Similarly, mutagenesis or deletion of 15 ribosomal protein L11 has been demonstrated to increase both UAG and UGA stop-codon readthrough by altering the interactions between RFs and the ribosome, reducing their catalytic efficiency. Any of these methods may offer promising avenues toward achieving absolute orthogonality of the UGA codon. Building off previous GROs, the freeing of an additional stop codon can facilitate stricter 20 and more robust biocontainment strategies, engineered with dependencies on two distinct nsAAs rather than one. With additional engineering to abolish UGA decoding by RFs, the alternative genetic code could further obstruct horizontal transfer of genetic elements (e.g., resistance against multiple phage). As the understanding of RF structural engineering and decoding deepens, furthering RF engineering inspired by non-canonical mitochondrial stop codons (e.g. 25 AGG) could permit reassignment of canonical sense codons as stop codons within recoded strains. These orthogonal stop codons could serve to terminate recoded genes within host cells, offer robust bidirectional genetic isolation by prematurely terminating invading transcripts (e.g., phage), and prevent host genes from escaping into the environment (i.e., transgene escape) by being functionally illegible to natural translations systems. 30 This study has allowed us to make significant and fundamental breakthroughs in the science of the genetic code and translation by employing a synthetic biology approach that recodes genomes and repurposes the function of translation components in an integrated framework. This approach offers a unique ability to probe the genome and biomolecular factors, which could reveal new principles to explain how genetic information is conserved, encoded, 99 45741195.1 YU 8909 PCT and exchanged. More broadly, this study helps to establish design rules to probe and engineer genomes alongside translation factors to achieve a deeper and predictive understanding of the universal translation machinery and the canonical genetic code, setting the stage for engineering genomes with non-degenerate genetic codes. This research also offers valuable applications in 5 safeguarding genetically modified organisms with enhanced genetic isolation, biocontainment, and expanded capacity for producing entirely new classes of synthetic proteins, biomaterials, or therapeutics comprising diverse chemistries. Tables Table 1 - Translation Factor Variant Nomenclature: Table includes symbols for key 10 translation factor genotypes and protein variants used in this study. Symbols A, B, & tW refer to genes prfA, prfB, & trpT, respectively. Symbol "o" stands for ochre (TAA-only prfB variant). Symbols K & P refer to amino acid substitutions E170K & S205P, respectively. Symbol “*” refers to A37G nucleotide substitution attenuating UGA suppression in tRNATrp. Symbol ORF Nt Status Product AA Status A prfA WT RF1 WT * 15 Table 2 - Major Intentional Deletions. Large genomic deletions intentionally created via tolC- mediated displacement (selection & counterselection). Most deleted regions pertained to crytpic prophages. All sites were listed as unessential and deleted by the Blattner research lab (doi:10.1101 / gr.217202; doi:10.1126 / science.1126439). ORF names listed in blue are TGA- ending genes. ORF names listed in green are TGA-ending pseudogenes. Brackets '[]' indicate 20 partial ORF deletions - position relative to rEc∆1.∆A reference genome. position mutation Genomic ORF Range 100 45741195.1 YU 8909 PCT 262,362 Δ33,787 bp A ykfI–[intF] 571311 Δ8538 b B l H– l I a e - e o c u v s o s o juga ve e ediates: The genome of rEc∆1.∆A is subdivided into two subdomains (A’ & B’). A’ contains 35 essential genes, while B’ contains 36 essential genes. Two clones of rEc∆1.∆A were categorized as rEc∆1.∆A(A’) and rEc∆1.∆A(B’). 5 Essential TGA-terminating genes within each region were targeted for recoding within each respectively named strain. During MAGE, additional oligos targeting adjacent TGA-terminating genes were included, resulting in 40 conversions within region A’ in rEc∆1.∆A(A’) and 68 conversions in B’ within rEc∆1.∆A(B’). Resulting partially recoded genomes were combined via CAGE, resulting in rEc∆E.∆A. Eight copies of rEc∆E.∆A were each assigned a particular 10 genomic subdomain (A through H). All TGA-terminating genes within each subdomain was similarly targeted within each respective strain. Once nearly all genes are converted, genomes are hierarchically assembled via CAGE until a fully recoded strain rEc∆2.∆A is achieved. Actual numbers of TGA ORFs recoded after conjugations vary as recoded codons are lost via genomic crossovers during conjugations and subsequently recoded again throughout the CAGE assembly 15 process. NOTE: number of ORFs includes pseudogenes converted, but not transposable elements or pseudogenes unconverted. 101 45741195.1 YU 8909 PCT Genomic Start End Locus # UGA ORFs Recoded within Strain Regions: Locus Region ^ ) ) ) ) ) ) ) ) ) B) C) F) F) H) C) (2014)) alignment of Ochre whole genome sequencing raw reads with a rEc∆1.∆A annotated 5 reference. Tables exclude intentional indels, whole gene deletions, & heavy mutagenic events. ORFs impacted by Indels (Ochre) P l N I i D l i T l Table 5 – Background SNP Summary: SNPs and genes are categorized by breseq (Deatherage, et al., Methods Mol Biol 1151, 165-188, doi:10.1007 / 978-1-4939-0554-6_12 (2014)) alignment 10 of Ochre whole genome sequencing raw reads with a rEc∆1.∆A annotated reference. Tables exclude intentional SNPs and those within proximity of heavy mutagenic events. 102 45741195.1 YU 8909 PCT Nonsynonymous, synonymous, and nonsense categorizations of SNPs were not provided for pseudogenes. SNPs (Ochre) 2bp A→T A→C A→G T→A T→C T→G C→A C→T C→G G→A G→T G→C Total ORFs 527 2 3 4 1 Coding Sense SNPs Coding Nonsense SNPs All Nonsense Codons Nonsyn Syn Total G→A C→T Total TAA TGA TAG Total3 5 Table 6: Reverting Mutations Implicated in Auxotrophy (Growth Data) – Growth data (doubling time – DT; maximum cellular density at OD600 – MaxOD) in nutrient rich media (LB) and minimal media (M9) before and after reversion of acquired mutations back to wildtype sequences. Mutation reversions in rEc∆2E.∆A(AB) & (C) did not alleviate auxotrophic phenotypes. Identified mutations in purL, cysU, & hisI each individually conveyed auxotrophy. 10 Only when all three genes were reverted to wildtype sequences was prototrophy (growth in M9) restored. Reverting the mutation in argG alone revealed variable growth effects in a strain dependent manner (data not shown). Ancestral rEc∆1.∆A strain and optimization mutations serve as a control. LB M9 LB M9i DT M D DT M D R DT M D DT M D N =82162215 YU 8909 PCT Table 9: TGA Conversions. List of all instances of predicted TGA opal-codon-containing genes, along with pseudogenes with TGAs abolished. Table includes gene names, relative loci within rEc∆1.∆A reference genome, overlapping genes, mutations made to convert TGA to TAA, gene orientation, and breseq reference database annotations.- 0 signifies absence, 1 signifies presence. 5 '→' = + sense strand, '←' = - sense strand. NOTE: some mismatching gene names are synonyms. 104 45741195.1 YU 8909 PCT 105 45741195.1 YU 8909 PCT 106 45741195.1 YU 8909 PCT 107 45741195.1 YU 8909 PCT 108 45741195.1 YU 8909 PCT 109 45741195.1 YU 8909 PCT 110 45741195.1 YU 8909 PCT 111 45741195.1 YU 8909 PCT 112 45741195.1 YU 8909 PCT 113 45741195.1 YU 8909 PCT 114 45741195.1 YU 8909 PCT 115 45741195.1 YU 8909 PCT 116 45741195.1 YU 8909 PCT 117 45741195.1 YU 8909 PCT 118 45741195.1 YU 8909 PCT 119 45741195.1 YU 8909 PCT 120 45741195.1 YU 8909 PCT 121 45741195.1 YU 8909 PCT 122 45741195.1 YU 8909 PCT 123 45741195.1 YU 8909 PCT 124 45741195.1 YU 8909 PCT 125 45741195.1 YU 8909 PCT 126 45741195.1 YU 8909 PCT 127 YU 8909 PCT Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. 5 Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific forms of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 128 45741195.1

Claims

YU 8909 PCT We claim:

1. A genomically recoded organism comprising no more than 25 genomic instances of the TGA codon.

2. A genomically recoded organism comprising two or more of single nucleotide substitutions, TAA and / or TAG insertions, and deletions to eliminate most or all of the TGA codons relative to a progenitor cell.

3. A genomically recoded organism comprising no more than 25 genomic instances of the TAA codon.

4. A genomically recoded organism comprising two or more of single nucleotide substitutions, TAG and / or TGA insertions, and deletions to eliminate most or all of the TAA codons relative to a progenitor cell.

5. The genomically recoded organism of any one of claims 1-4 further comprising one or both of (i) a mutant release factor 2 (RF2) with reduced ability to terminate translation at UGA and / or a nucleic acid encoding the same and (ii) a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same, relative to a progenitor cell.

6. A genomically recoded organism comprising one or both of (i) a mutant release factor 2 (RF2) with reduced ability to terminate translation at UGA and / or a nucleic acid encoding the same and (ii) a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same, relative to a progenitor cell.

7. The genomically recoded organism of any of claims 2-6, wherein greater 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% of TGA codons eliminated relative to the progenitor cell.

8. The genomically recoded organism of claim 7, wherein less than 100% of the TGA codons are eliminated.

9. The genomically recoded organism of one of claims 1-8, wherein TGA codon(s), if present, are only present in pseudogenes, ins, and / or selenocystine-encoding codons.

10. The genomically recoded organism of one of claims 1-9, wherein TAG is absent from the genome.

11. The genomically recoded organism of any one of claims 1-10, wherein release factor 1 (RF1) has reduced activity or is absent.

12. The genomically recoded organisms of any one of claims 1-2 or 5-11, wherein TAA is the only functional stop codon, or any one of claims 3-11, wherein TGA is the only functional stop codon. 129 45741195.1YU 8909 PCT 13. The genomically recoded organism of any one of claims 1-2 or 5-12, wherein the TGA codon is a functionally open codon that can be reassigned, or any one of claims 3-12, wherein the TAA codon is a functionally open codon that can be reassigned.

14. The genomically recoded organism of any one of claims 1-11, wherein the TAG codon is a functionally open codon that can be reassigned.

15. The genomically recoded organism of any one of claims 1-14, wherein crosstalk between four codons TAG, TGA, TAA, and TGG is attenuated rendering each codon a unique translational function: open, open, stop, and standard amino acid encoding optionally wherein the standard amino acid is Trp; or open, stop, open, and standard amino acid encoding optionally wherein the standard amino acid is Trp.

16. The genomically recoded organism of any one of claims 5-15 comprising a mutant RF2 and / or nucleic acid encoding the same.

17. The genomically recoded organism of claim 16, wherein the mutant RF2 comprises (i) one or more of N26, K170, P205, and A246 relative to SEQ ID NO:3 and optionally has an amino acid sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% to the amino acid sequence of SEQ ID NO:3, or (ii) the corresponding amino acids in a homolog thereof, optionally wherein the homolog comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity thereto.

18. The genomically recoded organism of claim 16, wherein the mutant RF2 comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, preferably comprising all of N26, K170, P205, and A246 relative to SEQ ID NO:

3.

19. The genomically recoded organism of any one of claims 5-18 comprising a mutant prfB gene having one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to SEQ ID NO:

1. 130 45741195.1YU 8909 PCT 20. The genomically recoded organism of claim 19, where the mutant prfB gene comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

2.

21. The genomically recoded organism of any one of claims 5-20, comprising a mutant tRNATrpwith reduced ability to decode UGA and / or a nucleic acid encoding the same.

22. The genomically recoded organism of claim 21, wherein the mutant tRNATrpis encoded by nucleic acid sequence comprising a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:

5.

23. The genomically recoded organism of claim 22, wherein the mutant tRNATrpis encoded by a nucleic acid sequence comprising at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

6.

24. The genomically recoded organism of any one of claims 5-20 comprising a mutant trpT gene comprising a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:

5.

26. The genomically recoded organism of claim 24, wherein the mutant trpT gene comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

6.

26. The genomically recoded organism of any one of claims 1-2 or 5-25 further comprising a first heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate tRNA capable of decoding TGA; or any one of claims 3-25 further comprising a first heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate tRNA capable of decoding TAA.

27. The genomically recoded organism of any one of claims 10-26 further comprising a second heterologous orthogonal aminoacyl tRNA synthetase (AARS) and cognate tRNA capable of decoding TGA where TGA has been reduced or removed as stop codon and TAA where TAA has been reduced or removed as a stop codon.

28. The genomically recoded organism of any one of claims 1-27 further comprising a heterologous mRNA or nucleic acid encoding the same comprising one or more TGA codons where TGA has been reduced or removed as stop codon and TAA codons where TAA has been reduced or removed as a stop codon.

29. The genomically recoded organism of any one of claims 1-27 further comprising a heterologous mRNA or nucleic acid encoding the same comprising one or more TAG codons. 131 45741195.1YU 8909 PCT 30. The genomically recoded organism of any one of claims 1-27 further comprising a heterologous mRNA or nucleic acid encoding the same comprising one or more TGA codons and one or more TAG codons.

31. The genomically recoded organism of any one of claims 1-30, wherein the organism is a bacterium, yeast, fungi, insect, plant, or animal cell(s).

32. The genomically recoded organism of claim 31, wherein the bacterium is E. coli.

33. A culture comprising a plurality of genomically recoded organisms of any one of claims 1-32.

34. A method of making a polypeptide comprising one or more instances of a non- standard amino acid comprising expressing a first messenger RNA (mRNA) encoding the target protein in a system comprising: a first orthogonal translation system (OTS) comprising a nucleic acid sequence encoding a first orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the genomically recoded organism (GRO) of any one of claims 1-33, and a plurality of a first non-standard amino acid, wherein the first mRNA comprises a nucleic acid sequence comprising at least one instance of a first codon reduce or absent in the GRO, wherein the first orthogonal AARS can charge its cognate tRNA with the first non- standard amino acid, and wherein the first cognate tRNA comprises an anticodon that can bind to the codon reduced or absent from the GRO.

35. The method of claim 34, further comprising a second orthogonal translation system (OTS) comprising a nucleic acid sequence encoding a second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the GRO, and a plurality of as second non-standard amino acid, wherein the first mRNA or a second heterologous mRNA comprises a nucleic acid sequence comprising at least one instance of a second codon reduced or absent in the GRO, wherein the second orthogonal AARS can charge its cognate tRNA with the second non- standard amino acid, and wherein the second cognate tRNA comprises an anticodon that can bind to a second codon reduced or absent from the GRO. 132 45741195.1YU 8909 PCT 36. A method of making a polypeptide comprising one or more instances of a non- standard amino acid comprising expressing a first messenger RNA (mRNA) encoding the target protein in a system comprising: a first orthogonal translation system (OTS) comprising a nucleic acid sequence encoding a first orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the genomically recoded organism (GRO), a second orthogonal translation system (OTS) comprising a nucleic acid sequence encoding a second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the GRO and a plurality of first and second non-standard amino acids, wherein the first mRNA comprises a nucleic acid sequence comprising one or more instances of a first codon and / or one or more instances of a second codon of a second codon reduced or absent in the GRO, wherein the first and second orthogonal AARS’s can charge their cognate tRNA with first and second non-standard amino acids, respectively, and wherein the first and second cognate tRNA comprises anticodons that can bind to different codons reduced or absent from the GRO.

37. The method of claims 34, 35 or 36 wherein the first and / or second mRNA comprise between 1-100 instances includes, or any specific integer or subrange in between, of the first codon that is reduced or eliminated from the GRO, the second codon that is reduced or eliminated from the GRO, or a combination thereof.

38. The method of any one of claims 34-37, wherein the target polypeptide is made in greater yield and / or purity compared to making the polypeptide using the same system in the GRO’s progenitor cell.

39. A mutant RF2 comprising a reduced ability to terminate translation at UGA relative to wildtype RF2.

40. The mutant RF2 of claim 39 comprising one or more of N26, K170, P205, and A246, preferably all of N26, K170, P205, and A246, relative to SEQ ID NO:

3.

41. The mutant RF2 of claims 39 or 40 comprising at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, preferably comprising all of N26, K170, P205, and A246 relative to SEQ ID NO:

3.

42. A nucleic acid encoding the mutant RF2 of any one of claims 39-41. 133 45741195.1YU 8909 PCT 43. The nucleic acid of claim 42 comprising an expression control sequence operably linked thereto.

44. A mutant prfB gene comprising one or more of conversion of one or both the internal autoregulatory TGA of the native sequence to TAA; conversion of the terminal TGA to TAA and / or insertion TAA directly upstream of the terminal TGA to preserve the RBS and minimize impact on translation of the adjacent lysS gene; coding or recoding of the corresponding codons to ensure the presence of amino acids N26, K170, P205, and / or A246, or a combination thereof relative to SEQ ID NO:

1.

45. The mutant prfB of claim 44, wherein the mutant prfB gene comprises at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

2.

46. A nucleic acid comprising the mutant prfB of claims 44 or 45 further comprising an expression control sequence operably linked thereto.

47. A mutant tRNATrpwith reduced ability to decode UGA.

48. The mutant tRNATrpcomprising a sequence encoded by a nucleic acid sequence comprising a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:

5.

49. The mutant tRNATrpof claims 47 or 48 comprising a nucleic acid sequence encoded by a nucleic acid sequence comprising at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

6.

50. A nucleic acid encoding the mutant tRNATrpof any one of claims 47-49 51. The nucleic acid of claim 50 further comprising an expression control sequence.

52. A mutant trpT gene comprising a A37 mutation, preferably an A37G substitution, relative to SEQ ID NO:

5.

53. The mutant trpT gene of claims 52 comprising at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

6.

54. A nucleic acid comprising the mutant trpT gene of claims 52 or 53 further comprising an expression control sequence operably linked thereto.

55. A GRO comprising one or more, optionally all, of the features of Table 9.

56. The GRO referred to herein as rEc∆2.∆A.mB-oKP.tW* (Ochre.tW*).

57. A GRO comprising a genome comprising stop codons, wherein the stop codons consist of a single stop codon sequence.

58. The GRO of claim 57, wherein one or more release factors provide translation termination only at the single stop codon sequence. 134 45741195.1YU 8909 PCT 59. The GRO of claims 57 or 58, wherein the GRO further comprise a heterologous translation system comprising one or two orthogonal translation systems (OTS) comprising a nucleic acid sequence encoding a first and optionally second orthogonal AARS and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integrated into the GRO, and a mRNA encoding a polypeptide, wherein the mRNA sequences includes one or both of reassigned stop codons as sense codons encoding nsAAs. wherein the first and second orthogonal AARS can charge their cognate tRNA with different non-standard amino acid, and wherein the first and second cognate tRNA comprises different anticodons that can bind to the reassigned stop codons.

60. A method of making a GRO comprising a genome consisting of a single stop codon sequence, the method comprising recoding other stop codon sequences to the stop codon sequence, optionally further comprising altering one or more release factors to provide translation termination only at the single stop codon sequence.

61. A nucleic acid encoding a polypeptide, wherein the nucleic acid sequence comprises one or more UAG / TAG codons and one or more UGA / TGA codons, wherein the polypeptide comprises nsAAs and the UAG / TAG codons and UGA / TGA codons encode for the nsAAs.

62. A GRO as described herein in the description, figures, tables, or combination thereof.

63. A method, composition, device, or process as described herein in the description, figures, tables, or combination thereof. 135 45741195.1