genetic code

Abstract

Mobile genetic elements or cells that are resistant to horizontal gene transfer, and methods for obtaining the cells, are provided. Also provided are methods for suppressing horizontal transfer of genetic information between a mobile genetic element and a first cell, cells utilizing novel gene codon schemes and related subject matter, and kits containing cells and mobile genetic elements that are orthogonal to one another. Also provided are methods for altering the susceptibility of a gene to mutations that alter the encoded amino acid sequence, methods for evolving or improving proteins, and methods for making a target gene more resistant to mutations. Additionally, the use of cells to make polymers and methods comprising using cells to make polymers are provided.

Description

[Technical Field]

[0001] Cells that are resistant to mobile genetic elements or horizontal gene transfer, and methods for obtaining such cells, are provided. Also provided are methods for suppressing horizontal transfer of genetic information between mobile genetic elements and cells, cells utilizing novel gene codon schemes and related subject matter, and kits containing cells and mobile genetic elements that are orthogonal to one another. Also provided are methods for altering the susceptibility of genes to mutations that alter the encoded amino acid sequence, methods for evolving or improving proteins, and methods for making target genes more resistant to mutations. Additionally, the use of cells to make polymers and methods comprising using cells to make polymers are also provided. [Background technology]

[0002] The nearly universal genetic code defines the correspondence between codons in genes and amino acids in proteins (FH Crick, L. Barnett, S. Brenner, RJ Watts-Tobin, General nature of the genetic code for proteins. Nature 192, 1227-1232 (1961); MW Nirenberg, JH Matthaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci USA 47, 1588-1602 (1961)). Because all forms of life use essentially the same genetic code, evolutionary innovations can be shared between organisms using horizontal gene transfer (HGT) (RJ Hall, FJ Whelan, JO McInerney, Y. Ou, MR Domingo-Sananes, Horizontal Gene Transfer as a Source of Conflict and Cooperation in Prokaryotes. Front Microbiol 11, 1569 (2020); K. Vetsigian, C. Woese, N. Goldenfeld, Collective evolution and the genetic code. Proc Natl Acad Sci USA 103, 10696-10701 (2006)). The sharing of genetic information between organisms is a major driver of evolution in prokaryotes and some eukaryotes (D. de la Torre, JW Chin, Reprogramming the genetic code. Nat Rev Genet 22, 169-184 (2021)).

[0003] However, a near-universal genetic code also has its disadvantages; mobile genetic elements (or selfish genetic elements)—including transposons, viruses, and plasmids—can exploit the code's universality, exploiting host cell machinery to read their genes and propagating themselves at the host organism's expense. There is a clear tension between maintaining a common genetic code to enable the acquisition of beneficial innovations through HGT and utilizing the common code for their own purposes and eliminating selfish genetic elements (RJ Hall, FJ Whelan, JO McInerney, Y. Ou, MR Domingo-Sananes, Horizontal Gene Transfer as a Source of Conflict and Cooperation in Prokaryotes. Front Microbiol 11, 1569 (2020); EV Koonin, AS Novozhilov, Origin and evolution of the genetic code: the universal enigma. IUBMB Life 61, 99–111 (2009)).

[0004] Several deviations from the standard genetic code have been documented in mitochondria and chloroplasts, and the most characterized reassignments involve stop codons (M. Kollmar, S. Muhlhausen, Nuclear codon reassignments in the genomics era and mechanisms behind their evolution. Bioessays 39, (2017); J. Ling et al., Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria. Nucleic Acids Res 42, 499-508 (2014); A.L. Borges et al., Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes. Nat Microbiol 7, 918-927 (2022)). Known sense codon reassignments in nuclear genomes are rare. "CTG yeast" decodes the CUG codon (which encodes leucine in the standard code) primarily as serine (97% of the time, with the remaining 3% still assigned to leucine) (MA Santos, AC Gomes, MC Santos, LC Carreto, GR Moura, The genetic code of the fungal CTG clade. CR Biol 334, 607-611 (2011)). Viruses against CTG yeast are essentially unknown, suggesting that sense codon reassignment could protect against viruses (DJ Taylor, MJ Ballinger, SM Bowman, JA Bruenn, Virus-host co-evolution under a modified nuclear genetic code. PeerJ 1, e50 (2013)).Although recent studies have provided computational evidence for arginine codon reassignment in bacilli (Y. Shulgina, SR Eddy, A computational screen for alternative genetic codes in over 250,000 genomes. Elife 10, (2021)), there are no experimentally verified examples of sense codon reassignment in bacteria.

[0005] 、DG Gibson et al., Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215-1220 (2008)、DG Gibson et al., One-step assembly of DNA fragments in complete form synthetic Mycoplasma genitalium genome Proc Natl Acad Sci USA 105, 20404-20409 (2008)、J Fredens et al., Total synthesis of Escherichia coli with a recoded genome. (2019)) The phylogenetic relationships of the genome are recognized with a recoded nature (J. Fredens et al., Total genome synthesis of E. Nature). 569, 514-+ (2019)、FJ Isaacs et al., Precise manipulation of chromosomes in vivo enables genome-wide codon replacement Science 333, 348-353 (2011)、MJ Lajoie et al., Genomically recoded organisms functions, Science 342. 357-360 (2013))。We synthesized a 4 Mb Escherichia coli genome in which we compressed the genetic code by removing all annotated occurrences of the serine-encoding TCG and TCA sense codons and the TAG stop codon; this generated a new strain, Syn61 (J. Fredens et al., Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514-+ (2019)). We then further evolved the strain to encode tRNAs (serU, tRNA) that decode TCG and TCA codons. CGA Ser and serT, tRNA UGA Ser The gene for Sense codon reassignment (Syn61Δ3) and the gene for RF-1 (prfA), which terminates protein synthesis at a TAG stop codon, were deleted. The resulting organism, Syn61Δ3, is unable to read all codons in the near-universal genetic code and is therefore unable to read horizontally transmitted genes containing the deleted codons from its genome, as exemplified by its resistance to various bacteriophages (W. E. Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)).

[0006] It is widely hypothesized that refactoring the structure of the genetic code through the reassignment of sense codons to different canonical amino acids can generate organisms with novel properties and create genetic firewalls that limit the leakage of genetic information from synthetic to natural organisms (K. Vetsigian, C. Woese, N. Goldenfeld, Collective evolution and the genetic code. Proc Natl Acad Sci USA 103, 10696-10701 (2006); EV Koonin, AS Novozhilov, Origin and evolution of the genetic code: the universal enigma. IUBMB Life 61, 99-111 (2009); G. Pines, JD Winkler, A. Pines, RT Gill, Refactoring the Genetic Code for Increased Evolvability. mBio 8, (2017); J. Calles, I. Justice, D. Brinkley, A. Garcia, D. Endy, Fail-safe genetic codes designed to intrinsically contain engineered organisms. Nucleic Acids Res 47, 10439-10451 (2019)). However, these hypotheses remain to be tested. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] FH Crick, L. Barnett, S. Brenner, RJ Watts-Tobin, General nature of the genetic code for proteins. Nature 192, 1227-1232 (1961) [Non-patent document 2] MW Nirenberg, JH Matthaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci USA 47, 1588–1602 (1961).

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[0008] In the experiments disclosed herein, the genetic code of a synthetic E. coli strain was refactored to exhibit semantic and functional orthogonality with respect to the universal genetic code, allowing for the creation of an orthogonal horizontal gene transfer system. [Means for solving the problem]

[0009] In one aspect, a cell is provided that comprises a genome in which at least a first type of sense codon has been recoded such that a first endogenous tRNA is unnecessary; the cell does not express the first endogenous tRNA; the cell expresses a first modified tRNA that can decode the first type of sense codon, wherein the first modified tRNA is charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon; the cell comprises a gene required for viability, the gene comprising at least one occurrence of the first type of sense codon, and the cell is viable when the first type of sense codon in the gene is decoded as the first amino acid.

[0010] In another aspect, a cell comprises a genome in which a first type of sense codon and a second type of sense codon are recoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; the cell expresses a first anticodon-swapped tRNA derived from a naturally occurring first parent tRNA, the first anticodon-swapped tRNA being charged with a first amino acid, the first parent tRNA being an isoacceptor for the first amino acid, and the first amino acid being a and not the naturally cognate amino acid of the first type of sense codon; wherein the cell expresses a second anticodon-swapped tRNA derived from a naturally occurring second parent tRNA, the second anticodon-swapped tRNA being charged with a second amino acid, the second parent tRNA being an isoacceptor for the second amino acid, and the second amino acid is not the naturally cognate amino acid of the second type of sense codon; and wherein the first and / or second modified tRNA are incapable of decoding any type of codon other than the first type of sense codon and / or the second type of sense codon.

[0011] In another aspect, a cell comprises a genome in which a first type of sense codon and a second type of sense codon have been recoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; the cell expresses a first modified tRNA that can decode the first type of sense codon, the first modified tRNA being charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon; and the cell expresses a second modified tRNA that can decode the first type of sense codon, the first modified tRNA being charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon. and expressing a second modified tRNA capable of decoding a second type of sense codon, wherein the second modified tRNA is charged with a second amino acid that is not the naturally cognate amino acid of the second type of sense codon: i) the first amino acid is alanine and the second amino acid is alanine; ii) the first amino acid is alanine and the second amino acid is histidine; iii) the first amino acid is alanine and the second amino acid is leucine; or iv) the first amino acid is alanine and the second amino acid is alanine. v) the first amino acid is histidine and the second amino acid is alanine; vi) the first amino acid is histidine and the second amino acid is histidine; vii) the first amino acid is histidine and the second amino acid is leucine; viii) the first amino acid is histidine and the second amino acid is proline; ix) the first amino acid is leucine and the second amino acid is alanine; x) the first amino acid is leucine and and the second amino acid is histidine; xi) the first amino acid is leucine and the second amino acid is proline; xii) the first amino acid is proline and the second amino acid is alanine; xiii) the first amino acid is proline and the second amino acid is histidine; xiv) the first amino acid is proline and the second amino acid is leucine; or xv) the first amino acid is proline and the second amino acid is proline.

[0012] In another aspect, provided is a cell having increased resistance to horizontal gene transfer or mobile genetic elements, the cell having been modified to reassign at least one type of sense codon to an amino acid that is not associated with a sense codon in the normal genetic code, and comprising a gene necessary for survival that is functional when decoded according to the reassigned genetic code and is non-functional when decoded according to the normal genetic code.

[0013] In another aspect, there is provided a method of increasing a cell's resistance to mobile genetic elements or horizontal gene transfer, wherein the cell has been modified to reassign at least one type of sense codon to an amino acid that is not associated with the sense codon in the canonical genetic code; the method comprising modifying a gene required for survival to contain at least one occurrence of the reassigned sense codon, wherein the cell is viable when the reassigned sense codon in said gene is decoded as the reassigned amino acid, the cell is non-viable when the reassigned sense codon in said gene is decoded according to the canonical genetic code, or the reassigned sense codon in said gene when decoded according to the canonical genetic code contributes, at least in part, to reduced survival.

[0014] In another aspect, a kit is provided that includes a first cell recoded according to a first orthogonal coding scheme and a second cell recoded according to a second orthogonal coding scheme, wherein the first and second coding schemes are orthogonal to each other.

[0015] In another aspect, mobile genetic elements are provided that are recoded according to an orthogonal coding scheme.

[0016] In another aspect, provided is a method of suppressing horizontal transfer of genetic information between a mobile genetic element and a first cell, comprising incubating a mobile genetic element and a first cell, wherein the mobile genetic element is a mobile genetic element disclosed herein, and the first cell comprises tRNAs that decode codons according to the canonical genetic code or according to a coding scheme that is orthogonal to that of the mobile genetic element.

[0017] In another aspect, a method is provided for altering the susceptibility of a gene to mutations that alter the encoded amino acid sequence, comprising: i) identifying a target gene; and ii) incubating cells containing the target gene, wherein the cells contain a tRNA capable of decoding at least one sense codon for the reassigned amino acid.

[0018] In an additional aspect, there is provided a use of the cells disclosed herein for the production of a polymer. In one embodiment, there is provided a method for making a polymer, the method comprising culturing the cells disclosed herein, providing the cells with a nucleic acid sequence encoding the polymer, and obtaining the polymer. [Brief explanation of the drawings]

[0019] [Figure 1-1] ~ [Figure 1-4]Figure 1 shows that the compressed genetic code is non-orthogonal. (A) Relationship between the TCG and TCA codons in a gene, the decoders for these codons in cells using wild-type (WT) decoding and Syn61Δ3 decoding (Δ3), and the corresponding protein sequence synthesized. The anticodon of the tRNA that reads the TCG or TCA codon is shown (decoder). The amino acid (aa, amino acid) used by the tRNA is shown. Gray for the decoder indicates that the tRNA is loaded with serine. Gray for a codon indicates that the codon is in a non-codon-compressed gene and its decoding as serine creates the correct protein sequence. Pink for the decoder / amino acid pair indicates that the tRNA is deleted. Pink for a codon indicates that the codon is absent from the gene because the gene was designed using codon compression. (B) Functionality assessment of wild-type (SpecR WT) and codon-compressed spectinomycin resistance (recSpecR(ΔTCG,TCA)) genes in cells that use the full complement of tRNAs to decode all codons in the reading frame (Syn61 WT, left panel) and cells lacking tRNAs that decode TCG and TCA codons (Syn61Δ3, right panel). Cells were spotted onto agar plates in the presence or absence of spectinomycin and incubated overnight. Growth of cells in the presence of spectinomycin indicates that the indicated SpecR gene is functional in the indicated strain (C, D). Protein synthesis and horizontal gene transfer outcomes predicted from recipient cells harboring mobile genetic elements and the indicated decoders and codons in essential genes. (C) A mobile genetic element encoding that gene according to the canonical genetic code, where TCG and TCA encode serine, cannot be horizontally transferred to Syn61Δ3 cells that lack decoders for the TCG and TCA codons. Translation stops at the TCG and TCA codons, and full-length proteins are not synthesized from essential genes within mobile genetic elements that contain TCG and TCA codons.(D) Mobile genetic elements encoding genes according to the canonical genetic code, which also carry genes for tRNAs that decode TCG and TCA codons, can be horizontally transferred to Syn61Δ3 cells. The tRNAs encoded by the mobile genetic elements can rescue the decoding of TCG and TCA codons within essential genes in the mobile genetic elements to produce the correct proteins. (E) Transfer of tRNA-encoding mobile genetic elements through conjugation; colony counts show successful transconjugants received from approximately 10 cells. WT mobile genetic elements (F WT) can be transferred to cells with the WT translation machinery (Syn61 WT) but not to cells lacking tRNAs that decode TCG and TCA (Syn61Δ3). WT mobile genetic elements (F(WT+serT)) encoding tRNAs that decode TCG and TCA codons as serine can be transferred to both Syn61 WT and Syn61Δ3. [Figure 2-1] ~ [Figure 2-4]This figure shows that sense codon reassignment generates a novel genetic code. (A) Total synthesis of a codon-compressed genome followed by tRNA and terminator deletion resulted in Syn61Δ3. Discovery of tRNAs directing the incorporation of distinct natural amino acid codons. (B) Isoacceptor tRNAs for the indicated amino acids, with anticodons modified to the Watson-Crick complement of the TCG or TCA codon, were introduced into cells in the indicated pairwise combinations. We used a GFP gene bearing a TCG or TCA codon at position 3 and electrospray ionization mass spectrometry (ESIMS) to determine the identity of the amino acid incorporated at each codon. When pairs of isoacceptors for distinct amino acids were used, each codon resulted in the specific incorporation of the amino acid bound to the Watson-Crick paired isoacceptor. The second peak measured in proline incorporation results from incomplete methionine cleavage at the N-terminus. A complete list of found and predicted masses is provided in Data File S1. (C) 16 novel genetic codes in which TCG and TCA codons are reassigned to Ala, His, Leu, and Pro. [Figure 3-1] ~ [Figure 3-2]

[0033] Figure 1 shows semantic orthogonality in genetic systems. (A) Relationship between TCG and TCA codons in genes, decoders for these codons in cells using wild-type (WT) decoding and decoding by tRNAAla CGA and tRNAHis UGA in Syn61Δ3, and the corresponding synthesized protein sequences. The anticodon of the tRNA that reads the TCG or TCA codon is shown (decoder). The amino acid (aa) used by the tRNA is shown. Grey for a codon indicates that its decoding as serine will create the correct protein sequence. Yellow for a codon indicates that its decoding as alanine will create the correct protein sequence. Green for a codon indicates that its decoding as histidine will create the correct protein sequence. (B) Functionality assessment of SpecR WT (using the native genetic code) and O-SpecR (TCG-Ala, TCA-His), codon-compressed according to the Syn61 recoding scheme, where Ala codons are replaced with TCG and His codons are replaced with TCA. The gene is read in cells using the WT translation machinery (Syn61 WT) and in cells where TCG is decoded as Ala and TCA is decoded as His (Syn61Δ3(tRNACGA Ala, tRNAUGA His)). Cells were spotted onto agar plates in the presence or absence of spectinomycin and incubated overnight. Growth of cells in the presence of spectinomycin indicates that the indicated SpecR gene is functional in the indicated strain. [Figure 4-1] ~ [Figure 4-3]Diagram of orthogonal and mutually orthogonal horizontal gene transfer systems. (A) Horizontal gene transfer between two bacterial strains is prohibited (dashed gray arrows), while horizontal gene transfer between cells sharing a common genetic code is possible (solid arrows). (B) Orthogonal horizontal gene transfer of mobile genetic elements. Colony counts indicate the number of exconjugants received from approximately 10 donor cells harboring the indicated mobile genetic elements. A WT mobile genetic element (F WT) is transferred to cells with the WT translation machinery (Syn61 WT) but not to cells in which TCG has been reassigned to alanine and TCA to histidine (Syn61Δ3(tRNACGA Ala, tRNAUGA His)). An orthogonal mobile genetic element (OF1) is transferred to Syn61Δ3(tRNACGA Ala, tRNAUGA His) but not to Syn61 WT. (C) Mutually orthogonal horizontal gene transfer systems. Colony counts indicate successful transconjugants received from approximately 10 donor cells carrying the indicated mobile genetic elements. The orthogonal mobile genetic element (O-F2) was exclusively transferred to cells in which TCG was reassigned to histidine and TCA was reassigned to alanine (Syn61Δ3(tRNACGA His, tRNAUGA Ala)); O-F2 was not transferred to Syn61Δ3(tRNACGA Ala, tRNAUGA His) or Syn61 WT. Neither F WT nor O-F1 could be transferred to Syn61Δ3(tRNACGA His, tRNAUGA Ala). (D) extends the experiment illustrated in (C) and demonstrates similar specificity for O-F3 and O-F4. [Figure 5-1] ~ [Figure 5-3]Orthogonal code locking blocks the invasion code. (A, B) Expected protein synthesis and horizontal gene transfer results from recipient cells harboring mobile genetic elements and the indicated decoders and codons in essential genes. (A) Transfer of a WT mobile genetic element encoding a tRNA that decodes TCG and TCA codons as Ser to a cell in which TCG has been reassigned to Ala and TCA has been reassigned to His. Essential genes in WT mobile genetic elements containing TCG and TCA codons are missynthesized, with each TCG and TCA codon in the gene stochastically decoded as Ser or His / Ala. This is expected to impair horizontal gene transfer. (B) Transfer of a WT mobile genetic element encoding a tRNA that decodes TCG and TCA codons as serine to a cell in which TCG has been reassigned to Ala and TCA has been reassigned to His. Essential genes in WT mobile genetic elements containing TCG and TCA codons are erroneously synthesized, with each TCG and TCA codon in the gene stochastically decoded as Ser or His / Ala. In addition, essential genes in host cells—where TCG is used to encode Ala and TCA is used to encode His—are erroneously synthesized. This is expected to eliminate horizontal gene transfer. (C) Horizontal gene transfer is eliminated in recipient cells and cells using orthogonal genetic coding in the essential genes of the mobile genetic element. F(WT+serT) was used as the mobile genetic element in all experiments. Colony counts indicate successful transconjugants received from approximately 10 cells. Recipient cells and the spectinomycin resistance gene variant (SpecR gene) in the recipient cells are shown. Correct reading of the indicated SpecR gene in the recipient cells becomes essential upon addition of spectinomycin. (D) T4-like phages encoding seryl-tRNAUGA infect Syn61Δ3 but not cells carrying the orthogonal genetic code. Plaque counts indicate the number of successfully replicating phages obtained from infection with 1.1 × 10 PFU / mL (phage 12) and 7.5 × 10 PFU / mL (phage 6).The cells, as in C, contain the cognate spectinomycin resistance gene; all experiments were performed in the presence of spectinomycin. [Figure 6] (Figure S1) This figure shows that the compressed genetic code is non-orthogonal. Functionality assessment of wild-type (HygR WT) and codon-compressed hygromycin resistance (recHygR(ΔTCG,TCA)) genes in cells that use the full complement of tRNAs to decode all codons in the reading frame (Syn61 WT, left panel) and cells in which the genes for tRNAs that decode TCG and TCA codons have been deleted (Syn61Δ3, right panel). Cells were spotted onto agar plates in the presence or absence of hygromycin and incubated overnight. Growth of cells in the presence of hygromycin indicates that a given HygR gene is functional in a given strain. [Figure 7-1] ~ [Figure 7-4](Figure S2) tRNA transfer disrupts genetic isolation. A wild-type mobile genetic element (F WT) encoding the chloramphenicol resistance gene and the lux operon, encoding enzymes for luciferin synthesis, was conjugated from donor cells (Syn61 WT) to recipient cells (Syn61Δ2, a strain derived from Syn61Δ3 in which prfA had been reintroduced via lambda Red recombination) containing the pSC101-Hyg plasmid encoding hygromycin resistance and the pKW20 plasmid (K. Wang et al., Defining synonymous codon compression schemes by genome recoding. Nature 539, 59-64 (2016)). After conjugation, cells were selected on agar plates containing hygromycin and chloramphenicol, ensuring that only cells containing the pSC101 vector and F WT survive. (A) Chemiluminescence image of the selection plate from the conjugation assay. Three colonies surviving conjugation on the selection plate lit up as a result of luciferin production, indicating they received FWT. (B) Genotyping of two colonies (the third colony did not grow in liquid medium) picked from the selection plate. Controls included Syn61 WT (containing prfA, serT, and serU encoded in the genome; these cells did not contain the pSC101-Hyg plasmid) and Syn61Δ3 (prfA, serT, and serU were deleted from the genome; these cells contained the pSC101-Hyg plasmid). Genotyping for the pSC101-Hyg plasmid confirmed that the clones were recipient cells. Genotyping of the prfA locus revealed that both clones that survived selection contained the prfA gene at the endogenous genomic locus. Genotyping of the serT locus revealed that both clones possessed serT at the endogenous genomic locus. Genotyping of the serU locus demonstrated that the clone did not harbor serU at the endogenous genomic locus.(C) Next-generation sequencing (NGS) sequence alignments to the reference genome for the recipient and donor strains. We observed that at the serT locus, the clone's sequence matched the donor reference (no gaps or insertions were observed when aligned to the Syn61 WT sequence; an insertion was observed when aligned to the Syn61Δ3 sequence). However, at the serU locus, the clone's sequence matched the Syn61Δ3 sequence (insertions were observed when aligned to the Syn61 WT sequence; no gaps or insertions were observed when aligned to the Syn61Δ3 sequence). Vertical colored lines indicate mismatches between sequencing reads and the reference. Gray indicates paired-end reads where the pairs are correctly oriented and positioned relative to each other. Reads shown in green / red indicate anomalous paired-end reads where the pairs are misoriented or mispositioned relative to each other. (D) Sequencing allowed us to determine the origin of the genomic sequence for each clone; Syn61Δ3 (and thereby Syn61Δ2) contains mutations resulting from its evolution from Syn61 WT, and these mutations act as watermarks that allow us to decipher whether the DNA sequence is derived from the donor (Syn61 WT) or recipient (Syn61Δ2) genome. We find that the majority of the genome is that of the recipient. However, multiple segments spanning a ∼400 kb stretch of donor DNA are integrated into the recipient genome. The pattern of genomic DNA integration was unique to each sequenced clone, but both clones contained the serT locus. [Figure 8-1] ~ [Figure 8-2](Figure S3) Isoacceptor tRNAs with altered anticodons are active and specific. (A) Production of sfGFP-His6 from the sfGFP3TCG or sfGFP3TCA gene in Syn61Δ3 cells harboring the indicated isoacceptor tRNA chimeras with CGA or UGA anticodons. While serT and serU decode both TCG and TCA codons, the chimeric isoacceptors show specificity for codons with Watson-Crick complementarity at all three bases of the codon-anticodon interaction. For all but proM, which has a UGA anticodon, the level of GFP produced by all chimeric tRNAs with Watson-Crick complementarity codons was at least comparable to that produced from the WT GFP gene without the TCG or TCA codons, suggesting that the chimeric isoacceptor tRNAs are efficient and specific. The protein yield for WT sfGFP is 17 mg / L. sfGFP-3-TCG / TCA expression is comparable to WT sfGFP with respect to the cognate decoder. (B) The chimeric isoacceptor retains specificity for aminoacylation with the amino acid specified by the parent isoacceptor. In cells containing a chimeric tRNA with an anticodon that is the Watson-Crick complement of the codon at position 3 of sfGFP, the identity of the amino acid incorporated in response to TCG or TCA at position 3 of sfGFP was confirmed by electrospray ionization mass spectrometry (ESI-MS). Only the mass corresponding to the correct amino acid was detected. The second peak measured for proline incorporation results from incomplete methionine cleavage at the N-terminus. Predicted and actual masses for expression of sfGFP3TCG; predicted mass for sfGFP-3-Ser: 27755.13 Da, actual mass: 27756.00 Da. sfGFP-3-Ala predicted mass: 27739.13 Da, actual mass: 27740.60 Da. sfGFP-3-His predicted mass: 27805.19 Da, actual mass: 27806.20 Da. sfGFP-3-Leu predicted mass: 27781.21 Da, actual mass: 27782.00 Da.sfGFP-3-Pro predicted mass: 27765.17 Da, actual mass: 27765.40 Da. Predicted and actual masses for expression of sfGFP3TCA: sfGFP-3-Ser predicted mass: 27755.13 Da, actual mass: 27756.00 Da. sfGFP-3-Ala predicted mass: 27739.13 Da, actual mass: 27741.20 Da. sfGFP-3-His predicted mass: 27805.19 Da, actual mass: 27805.60 Da. sfGFP-3-Leu predicted mass: 27781.21 Da, actual mass: 27782.00 Da. Predicted mass of sfGFP-3-Pro: 27765.17 Da, actual mass: 27765.20 Da. [Figure 9-1] ~ [Figure 9-3](Figure S4) Illustrates semantic orthogonality in the gene system. (A) Cells using canonical decoding (Syn61 WT) and cells in which TCG and TCA codons were reassigned according to the appropriate code (Syn61Δ3(tRNACGAHis, tRNAUGAAla), Syn61Δ3(tRNACGAAla, tRNAUGALeu), Syn61Δ3(tRNACGALeu, tRNAUGALeu), Syn61Δ3(tRNACGAPro, tRNAUGALeu), Syn61Δ3(tRNACGAAla, tRNAUGAAla), Syn61Δ3(tRNACGAAla, tRNAUGALeu). Functionality assessment of the gene (SpecR WT) written in the normal genetic code and codon-reassigned for spectinomycin resistance (O-SpecR(His-TCG, Ala-TCA), O-SpecR(Ala-TCG, Leu-TCA), O-SpecR(Leu-TCG, Leu-TCA), O-SpecR(Pro-TCG, Leu-TCA), O-SpecR(Ala-TCG, Ala-TCA), O-SpecR(Ala-TCG, Pro-TCA)) in the tRNAUGAPro (Pro-tRNAUGAPro). Cells were spotted onto agar plates in the presence or absence of spectinomycin and incubated overnight. Growth of cells in the presence of spectinomycin indicates that the indicated SpecR gene is functional in the indicated strain. (B) Functional evaluation of HygR WT (using the native genetic code) and O-HygR (TCG-Ala, TCA-His), which has been codon-compressed according to the Syn61 recoding scheme, replacing Ala codons with TCG and His codons with TCA. The gene is read in cells using the WT translation machinery (Syn61 WT) and in cells in which TCG is decoded as Ala and TCA is decoded as His (Syn61Δ3(tRNACGA Ala, tRNAUGA His)). [Figure 10-1] ~ [Figure 10-3](Figure S5) Mutational landscape of WT and codon-compressed genetic codes. Depiction of the mutational landscape for various amino acids in the WT and codon-compressed genetic codes. Amino acids that exhibited a mutational landscape are highlighted (serine in gray, alanine in yellow, histidine in green, leucine in orange, and proline in blue). Amino acids that were the only point mutations removed from the highlighted amino acids were connected and marked with a line. [Figure 11-1] ~ [Figure 11-4] (Figure S6) Refactored genetic codes alter the mutational landscape. A description of the mutational landscape for the refactored genetic codes created in this study. TCG reassignments are shown on the left. TCA reassignments are shown above. For each code, the serine and reassigned amino acids are highlighted. Amino acids with only one point mutation removed from the highlighted amino acids are connected and marked. [Figure 12-1] ~ [Figure 12-4] Figures 2A and 2B show raw spectra. (A) The completed mass spectrum (before deconvolution) for the sfGFP-3-TCG measurement (as shown in Figure 2B) is shown. The intensity at each mass is shown as the total ion count. (B) The completed mass spectrum (before deconvolution) for the sfGFP-3-TCA measurement (as shown in Figure 2B) is shown. The intensity at each mass is shown as the total ion count. [Figure 13-1] ~ [Figure 13-2]Figure 1 shows the fidelity of TCG / TCA decoding by isoacceptor tRNAs at position 11 of ubiquitin. The indicated amino acid isoacceptor tRNAs with anticodons modified to the Watson-Crick complement of the TCG or TCA codon were introduced into Syn61Δ3 in the indicated pairwise combinations. We used ubiquitin genes with either the TCG or TCA codon at position 11 and electrospray ionization mass spectrometry (ESI-MS) to determine the identity of the amino acid incorporated at each codon. When pairs of isoacceptors for distinct amino acids were used, each codon resulted in the specific incorporation of the amino acid bound to the Watson-Crick paired isoacceptor. We calculated the minimum specificity for decoding the TCG or TCA codon in the presence of both tRNACGAXXX and tRNAUGAYYY, where XXX and YYY are distinct amino acids (Methods). We observed clear incorporation of Ala at TCA in cells containing tRNACGA Ala and tRNAUGA Leu; we estimate ≥78.2% Leu incorporation at this codon; this may result in part from misacylation of tRNAUGA Leu and / or from competitive decoding of the TCA codon by tRNACGA Ala. For all other spectra, the specificity of decoding the codon using the correct versus incorrect anticodon ranged from ≥96% to ≥99.8%. [Figure 14-1] ~ [Figure 14-4]This figure shows that sense codon reassignment does not result in detectable off-target incorporation at the TCT codon. (A) Isoacceptor tRNAs with anticodons modified to the Watson-Crick complement of the TCG or TCA codon for the indicated amino acids were introduced into cells in pairwise combinations as indicated. We used electrospray ionization mass spectrometry (ESI-MS) to determine the identity of the amino acid incorporated into the TCT codon at position 3 of GFP. The masses detected in the presence of all isoacceptor pairs correspond to the incorporation of serine at the TCT codon. Expected masses: serine: 27755.13 Da; alanine: 27739.13 Da; histidine: 27805.19 Da; leucine: 27781.21 Da; proline: 27765.17 Da. All measured masses (27754.5 ± 0.5 Da) correspond to serine incorporation in TCT. The limits of fidelity measurement (method) for these spectra ranged from 97.4% to 99.6%, and we observed no peaks for incorporation of amino acids other than serine. (B) The corresponding mass spectrum (before deconvolution) for the sfGFP-3-TCT measurement (as shown in A) is shown. The intensity at each mass is shown as the total ion count. [Figure 15-1] ~ [Figure 15-4]This figure shows that sense codon reassignment does not result in detectable off-target incorporation at the TCC codon. (A) Isoacceptor tRNAs with anticodons modified to the Watson-Crick complement of the TCG or TCA codon for the indicated amino acids were introduced into cells in the pairwise combinations shown. We used electrospray ionization mass spectrometry (ESI-MS) to determine the identity of the amino acid incorporated at the TCC codon in position 3 of GFP. The masses detected in the presence of all isoacceptor pairs correspond to the incorporation of serine at the TCC codon. Expected masses: 27755.13 Da for serine; 27739.13 Da for alanine; 27805.19 Da for histidine; 27781.21 Da for leucine; and 27765.17 Da for proline. All measured masses (27754.5 ± 0.5 Da) correspond to serine incorporation in TCC. The fidelity measurement limits (method) for these spectra ranged from 98.0% to 99.5%, and we observed no peaks for incorporation of amino acids other than serine. (B) The corresponding mass spectrum (before deconvolution) for the sfGFP-3-TCC measurement (as shown in A) is shown. The intensity at each mass is shown as the total ion count. [Figure 16-1] ~ [Figure 16-4] Figure 13 shows raw ubiquitin spectra. (A) Shown is the corresponding mass spectrum (before deconvolution) for Ub-11-TCG measurement (as shown in Figure 13). The intensity at each mass is shown as the total ion count. (B) Shown is the corresponding mass spectrum (before deconvolution) for Ub-11-TCA measurement (as shown in Figure 13). The intensity at each mass is shown as the total ion count. [Figure 17-1] ~ [Figure 17-2]Figure 1 shows MS-MS of the amino acid incorporated at position 11 of ubiquitin. (A) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCG gene in the presence of tRNAUGA Ser. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The serine at position 5 of the peptide (marked with a green asterisk) confirms correct decoding of the TCG codon in Ub-11-TCG by tRNAUGA Ser. (B) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCA gene in the presence of tRNAUGA Ser. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The serine at position 5 of the peptide (marked with a green asterisk) confirms the correct decoding of the TCA codon in Ub-11-TCA by tRNAUGA Ser. [Figure 18-1] ~ [Figure 18-2] Figure 1 shows MS-MS of the amino acid incorporated at position 11 of ubiquitin. (A) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCG gene in the presence of tRNACGA Ala. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The alanine at position 5 of the peptide (marked with a green asterisk) confirms correct decoding of the TCG codon in Ub-11-TCG by tRNACGA Ala. (B) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCA gene in the presence of tRNAUGA Ala. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The alanine at position 5 of the peptide (marked with a green asterisk) confirms the correct decoding of the TCA codon in Ub-11-TCA by tRNAUGA Ala. [Figure 19-1] ~ [Figure 19-2] Figure 1 shows MS-MS analysis of the amino acid incorporated at position 11 of ubiquitin. (A) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCG gene in the presence of tRNACGA His. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The histidine at position 5 of the peptide (marked with a green asterisk) confirms correct decoding of the TCG codon in Ub-11-TCG by tRNACGA His. (B) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCA gene in the presence of tRNAUGA His. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The histidine at position 5 of the peptide (marked with a green asterisk) confirms the correct decoding of the TCA codon in Ub-11-TCA by tRNAUGA His. [Figure 20-1] ~ [Figure 20-2]Figure 1 shows MS-MS of the amino acid incorporated at position 11 of ubiquitin. (A) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCG gene in the presence of tRNACGA Leu. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The leucine at position 5 of the peptide (marked with a green asterisk) confirms correct decoding of the TCG codon in Ub-11-TCG by tRNACGA Leu. (B) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCA gene in the presence of tRNAUGA Leu. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The leucine at position 5 of the peptide (marked with a green asterisk) confirms the correct decoding of the TCA codon in Ub-11-TCA by tRNAUGALeu. [Figure 21-1] ~ [Figure 21-2] Figure 1 shows MS-MS analysis of the amino acid incorporated at position 11 of ubiquitin. (A) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCG gene in the presence of tRNACGA Pro. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The proline at position 5 of the peptide (marked with a green asterisk) confirms correct decoding of the TCG codon in Ub-11-TCG by tRNACGA Pro. (B) Tandem mass spectrometry spectrum of a peptide containing position 11 of the ubiquitin protein after digestion of the ubiquitin protein expressed from the Ub-11-TCA gene in the presence of tRNAUGA Pro. y-ions are displayed in red; b-ions are displayed in blue. The peptide sequence is shown at the bottom of each spectrum. The proline at position 5 of the peptide (marked with a green asterisk) confirms the correct decoding of the TCA codon in Ub-11-TCA by tRNAUGA Pro. [Figure 22-1] ~ [Figure 22-6] Screening of anticodon-modified tRNAs. (A) We expressed and purified ubiquitin-His6 with a TCG codon at position 11 (Ub-11-TCG-His6) in the presence of the indicated tRNAs with a CGA anticodon; expression was performed in Syn61Δ3 and purified by Ni-NTA chromatography. To assess the amino acid specificity of the anticodon-modified tRNAs, we performed mass spectrometry of the intact protein. The predicted and measured masses are shown for each tRNA. The predicted mass is for the amino acid of the parent isoacceptor; if the mass differs from this, the closest canonical amino acid incorporation is also shown as an additional predicted mass. The following tRNAs incorporated amino acids other than those predicted by the parent isoacceptor (or produced results that could not be unambiguously assigned to the parent isoacceptor amino acid) and were not further considered: AsnT, CysT, GlnV, LysQ, MetV, MetY, PheU, ValV, and ValW. The following tRNAs incorporated the parent isoacceptor amino acid: ArgU, ArgX, ArgQ, ArgW, GltU, GlyU, HisR, ProK, ProL, ProM, ThrT, TrpT, and TyrV. (B) For the subset of tRNAs that incorporated the parent isoacceptor amino acid, we expressed Ub-11-TCG-His6 in the presence of the indicated tRNA with a CGA anticodon; expression was performed in Syn61Δ3, and lysates were probed with anti-His6 after SDS-PAGE (we replaced ThrT with ThrU). From these experiments, we selected ProM and HisR as good candidates for further characterization. In control experiments without tRNA expression, we did not detect ubiquitin from Ub-11-TCG. SerU is a natural TCG decoder and serves as a positive control. [Figure 23]Figure 1 shows doubling time. Doubling time for Syn61Δ3 in the presence of various pairwise combinations of anticodon-modified tRNAs was measured in 2xYT. Strains contained no tRNA(-), or serT (tRNAUGA Ser) was present as a control. Most anticodon-modified tRNAs caused no or moderate changes in the doubling time of Syn61Δ3. To facilitate parallel measurements of doubling time, experiments were performed in a 96-well format, 200 μL volume (Methods). For comparison, Syn61Δ3 (tRNA(-)-free control) has a doubling time of 49.77 ± 0.8 min when grown in shake flasks. [Figure 24A-1] ~ [Figure 24A-3] Figure 1. Mutually orthogonal horizontal gene transfer systems. Colony numbers indicate successful exconjugants received from approximately 10 donor cells carrying the indicated mobile genetic elements. Mobile genetic elements (F(WT(TCG-Ser, TCA-Ser)), O-F1(TCG-Ala, TCA-His), O-F2(TCG-His, TCA-Ala), O-F3(TCG-Ala, TCA-Ala), O-F4(TCG-Ala, TCA-Pro)) were exclusively propagated into cells with cognate reassignments of TCG and TCA codons (Syn61 WT(tRNACGA Ser, tRNAUGA Ser), Syn61Δ3(tRNACGA Ala, tRNAUGA His), Syn61Δ3(tRNACGA His, tRNAUGA Ala), Syn61Δ3(tRNACGA Ala, tRNAUGA Ala), Syn61Δ3(tRNACGA Ala, tRNAUGA Pro)). Transconjugation was not observed for noncognate code / decoder systems. [Figure 25]Figure 1 shows that orthogonal code locking blocks the invasion code. Horizontal gene transfer of the WT mobile genetic element (F(WT+serT)) is eliminated in recipient cells and cells using the refactored genetic code in the essential gene of the mobile genetic element. Colony counts show successful transconjugants received from approximately 10 cells. Recipient cells and hygromycin resistance gene variants (HygR gene) in recipient cells are shown. Correct reading of the expressed HygR gene in recipient cells becomes essential upon addition of hygromycin. [Figure 26-1] ~ [Figure 26-3] Figure 1 shows that orthogonal code locking blocks the invasion code. Horizontal gene transfer of the WT mobile genetic element (F(WT+serT)) is eliminated in recipient cells and cells using the refactored genetic code in the essential gene of the mobile genetic element. Colony counts show successful transconjugants received from approximately 10 cells. Recipient cells and the spectinomycin resistance gene variant (SpecR gene) in recipient cells are shown. Correct reading of the expressed SpecR gene in recipient cells becomes essential upon addition of spectinomycin. [Figure 27-1] ~ [Figure 27-3]Whole-genome sequencing of purified phages reveals the seryl-tRNA gene. (A) We evaluated 31 phage enrichments from environmental samples for their ability to form plaques in the presence of spectinomycin in a code-compacted strain (Syn61Δ3) and a refactored, code-locked strain (Syn61Δ3(tRNACGA Ala, tRNAUGA His)O-SpecR(Ala-TCG, His-TCA)). Thirteen environmental samples formed plaques in Syn61Δ3, whereas none formed plaques in the strain with the refactored genetic code (Methods). Two individual phages (designated 6 and 12) were sequentially purified from a single plaque in Syn61WT (as described in Methods). Broad-coverage NGS enabled de novo genome assembly for these phages. Their genome sizes were 164,924 and 167,593, respectively. Both phages belong to the Tequatrovirus genus (T4-like phages) and show >97% identity to Citrobacter phage ZZ23 (NC_054901) and E. coli phage U115 (MZ753803), respectively. Both genomes contain a seryl-tRNA gene encoding φtRNAUGA Ser. (B) Predicted secondary structure of the phage encoding seryl-tRNA (φtRNAUGA Ser) and serT (the endogenous E. coli tRNAUGA Ser gene). All E. coli seryl-aaRS identity elements are present in φtRNAUGA Ser (bases shown in red), consistent with the viral tRNA being a substrate for E. coli seryl-tRNA synthetase. The sequences shown are SEQ ID NO:69 (endogenous E. coli tRNAUGA Ser gene) and SEQ ID NO:70 (φtRNAUGA Ser). (C) Electron microscopy images of isolated phages 6 and 12. Both phages exhibit characteristic Myoviridae morphology, consistent with the phage genome sequence. [Figure 28]This figure shows that providing Syn61Δ3 with tRNAUGA Ser from phages 6 and 12 allows infection by T4 phage. We evaluated the ability of T4 phage to form plaques in various strains. While T4 formed plaques in Syn61 WT, Syn61Δ3 is resistant to T4 plaque formation due to the absence of tRNAs that decode the TCG and TCA codons. T4 plaque formation is rescued in Syn61Δ3 by expression of either serT (encoding E. coli tRNAUGA Ser) or φtRNAUGA Ser, both encoded in the genomes of phages 12 and 06. Cells were infected with approximately 10 to 10 PFU / mL. [Figure 29-1] ~ [Figure 29-4] Genetic code refactoring and code locking block viral replication. (A) Pools of phage 12 and 06 clones encoding φtRNAUGASer on their genomes form plaques in both Syn61 WT and Syn61Δ3. Various strains with distinct refactored and locked genetic codes show a complete loss of plaque formation. (B) Titration of phages 6 and 12 (approximately 10 to 10 PFU / mL) in strains with the indicated genetic codes. No plaque formation was observed in strains with refactored and locked genetic codes. [Figure 30-1] ~ [Figure 30-4]This figure shows that phage replication is a more complex biological function than conjugation. Plaque formation from T-4-like phage infection is a more complex biological process than colony formation after successful horizontal gene transfer from conjugation. (A) Comparative genomic analysis of T-4-like phages (phage 06 and phage 12) and the RK2 F plasmid. Size is represented in kilobases on the x-axis. Positions where TCA codons occur are marked with vertical yellow lines (top). Positions where TCG codons occur are marked with vertical red lines (bottom). (B) Codon usage in mobile genetic elements. The combined frequency of target codons (TCA and TCG) is shown on the x-axis. TCA codon frequency is shown in yellow, and TCG codon frequency is shown in red. (C) For plaque formation, phage particles must first attach to bacterial cells and inject their DNA into the cytosol. Subsequently, genes from the phage genome are transcribed and translated by the host machinery to produce viral proteins. Furthermore, the phage genome is replicated inside the cell. Finally, these components must mature into fully functional phage particles that escape the cell and infect neighboring cells. This complex process requires numerous structural and regulatory proteins to be correctly expressed from the phage genome. (D) For colony formation from recipient cells harboring horizontally transmitted conjugative plasmids, donor cells must first attach to the recipient. Subsequently, the F plasmid is transmitted through the conjugative channel and recircularized inside the recipient cell. The attachment, transmission, and recircularization processes depend exclusively on proteins expressed in the donor cell. Proteins are then expressed from the conjugative elements that allow their replication and segregation during cell division. Division of recipient cells harboring the transmitted plasmid leads to colony formation without further conjugation events. [Figure 31-1] ~ [Figure 31-3]Code locking ensures the stability of the refactored genetic code. (A) Cells were passaged every 12 hours and assessed for the presence of the refactored code. Cells with code locking retained their genetic code in all cases, demonstrating the stability of the code refactoring, while in cells without code locking, the refactored code was not stable. Cells contained either the codon-compressed SpecR resistance gene (unlocked: -) or the cognate SpecR gene (SpecR TCG-Ala, TCA-His / SpecR TCG-Leu, TCA-Leu) (locked: +); all experiments were performed in the presence of spectinomycin. (B) A phage encoding seryl-tRNAUGA infects cells with an unstable genetic code but not cells with a stably refactored code. Cells from the end of the time course (A), with or without code locking, were subjected to infection with a T-4-like phage (phage 06 / 12). Plaque counts indicate the number of successfully replicating phage obtained from infection with approximately 5 x 10 PFU (phage 12) and approximately 1 x 10 PFU (phage 6). Cells contained either the codon-compressed SpecR resistance or the cognate SpecR gene (as in (A)); all experiments were performed in the presence of spectinomycin. [Figure 32] Figure 1 shows the structure of the major capsid protein gp23. (A) Protein structure of the major capsid protein gp23 from T4 phage. Three serine residues are present and are encoded using TCA (highlighted in orange), thereby subjecting it to ambiguous decoding in cells with a refactored genetic code. (B) Binding interface of the major capsid protein gp23; shown are the three subunits of gp23 that are part of a hexameric capsid subunit. The serine residue encoded by TCA in the middle subunit (gray) is shown in orange. [Figure 33]Figure 1 shows a phage growth assay. A T-4-like phage encoding seryl-tRNA (tRNAUGA Ser) successfully infects Syn61Δ3 but not cells with a refactored, locked genetic code. Plaque counts indicate the number of phage particles in a 7.5 uL volume after 24 hours of growth in cultures containing the indicated cells. [Figure 34-1] ~ [Figure 34-2] Figure 1 shows the effect of code refactoring and code locking on the pattern of horizontal gene transfer. A T-4-like phage encoding seryl-tRNA (tRNAUGA Ser) successfully infects Syn61Δ3 but not cells with a refactored genetic code. Plaque counts indicate the number of successfully replicating phage obtained from infection with 1.1 x 10 plaque-forming units (PFU) / ml (phage 12) and 7.5 x 10 PFU / ml (phage 6). DETAILED DESCRIPTION OF THE INVENTION

[0020] Code Locking There is a need to prevent mobile genetic elements, such as viruses, from contaminating cells. For example, industrial-scale fermentation of bacteria for the production of commercial products can be contaminated with mobile genetic elements, such as viruses. This can result in economic losses and disrupt critical supply demands. Although there are existing methods to protect cells from such contamination (see WO 2020 / 229592 A1 or (WE Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)), both of which are incorporated herein by reference), the present inventors demonstrate herein that risks from mobile genetic elements, including tRNAs, remain. Although attempts have been made to reduce the risks from such mobile genetic elements (see Nyerges et al. “Swapped genetic code blocks viral infections and gene transfer”, https: / / doi.org / 10.1101 / 2022.07.08.499367), there remains a need for technologies that can make cells resistant to mobile genetic elements, including tRNAs.

[0021] Provided herein are "code-locked" cells. The genomes of these cells are recoded to reduce or eliminate instances of at least one type of sense codon, which in turn allows for the removal of the endogenous cognate tRNA since it is no longer needed by the cell (see W. E. Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)). The inventors discovered that including a tRNA specific to the removed sense codon but charged with an amino acid with a non-naturally associated sense codon reduces the risk of, but does not eliminate, contaminating mobile genetic elements containing the relevant tRNA (see Figure 5 of this disclosure). This has been described elsewhere (see Nyerges et al.). The inventors overcame this challenge by generating cells containing genes necessary for cell viability, where the genes have been recoded so that sense codons are reassigned to amino acids different from those in the canonical genetic code. The inventors have surprisingly found that including such genes reduces or eliminates horizontal gene transfer from mobile genetic elements that enable the use of genetic code that does not match the normal genetic code or that of the target cell. The inventors have also found that this approach results in the maintenance of the exogenous tRNAs introduced for code refactoring, thereby maintaining resistance to horizontal gene transfer or mobile genetic elements.

[0022] Thus, in a first aspect, there is provided a cell comprising a genome in which at least a first type of sense codon has been recoded such that a first endogenous tRNA is unnecessary, wherein the cell does not express the first endogenous tRNA; the cell expresses a first modified tRNA that can decode the first type of sense codon, wherein the first modified tRNA is charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon; the cell comprises a gene required for viability, the gene comprising at least one occurrence of the first type of sense codon, and the cell is viable when the first type of sense codon in the gene is decoded as the first amino acid.

[0023] The cells may have increased resistance to horizontal gene transfer or mobile genetic elements, as discussed in the following section. Accordingly, in a fourth aspect, provided herein is a cell having increased resistance to horizontal gene transfer or mobile genetic elements, the cell having been modified to reassign at least one type of sense codon that is not associated with a sense codon in the normal genetic code to an amino acid, and the cell comprises a gene necessary for survival that is functional when decoded according to the reassigned genetic code and is non-functional when decoded according to the normal genetic code. The gene may be necessary for survival alone or in combination with other genes.

[0024] As discussed in Example 7, cells modified by the above-described means exhibit improved maintenance of resistance to horizontal gene transfer or mobile genetic elements, whereby the increased resistance can be maintained over long periods of time compared to cell cultures that do not contain the codelocked bacteria.

[0025] The gene required for survival can be an exogenous gene. For example, the gene can be a gene commonly used as a positive selectable marker. In some examples, the gene is an antibiotic resistance gene. Exemplary embodiments include a spectinomycin resistance gene or a hygromycin resistance gene.

[0026] In another example, a gene required for survival may be an essential gene in the genome of a cell.As used herein, a gene is "essential" if its product is necessary for cell survival.For example, if suppression of the expression of the functional form of the protein encoded by the gene results in cell non-survival, the gene is considered to be essential.

[0027] A gene required for viability may contain at least one reassigned codon in which mutation of the corresponding residue in the translated product results in a loss of function. In particular, the reassigned codon may be positioned so that decoding the codon according to the normal genetic code results in a loss of function for the product. For example, if a cell contains a tRNA that normally associates with serine but can decode a codon that charges alanine, and the gene contains the codon in which alanine is present in the natural product, the product of the gene may have a serine at that position and be non-functional. The foregoing examples of specific amino acids are merely illustrative, and any may be used. In particular, any of the reassignment schemes shown in Figure 2C may be used. Thus, a cell may contain a gene required for viability, the gene containing at least one occurrence of a first type of sense codon, and the cell is non-viable when the first type of sense codon is decoded according to the normal genetic code.

[0028] A gene required for survival may contain multiple reassigned codons. The multiple reassigned codons may be individually or cumulatively positioned such that a product containing an unreassigned amino acid (as discussed in the previous paragraph) is nonfunctional. Thus, a cell may contain a gene required for survival, the gene containing multiple occurrences of a first type of sense codon, which, when decoded according to the canonical genetic code, renders the cell nonviable. At least one occurrence of a first type of sense codon in a gene required for survival may contribute, at least in part, to reduced survival when decoded according to the canonical genetic code, and may contribute, in combination with other characteristics, such as other reassigned codons or other types of reassigned codons, to a complete loss of survival. In some cases, multiple reassigned codons, potentially of multiple types, may be present in a gene required for survival, or multiple genes required for survival may exist. Any individual instance of a reassigned codon may contribute, at least in part, to reduced survival when decoded according to the canonical genetic code, and a complete loss of survival may be due to the effects of translation of the multiple reassigned codons according to the canonical genetic code.

[0029] The cells of the present disclosure may contain more than one gene necessary for survival that contains at least one reassigned codon.

[0030] The cells of the present disclosure can comprise a genome recoded with respect to sense codons of a second type.

[0031] In some embodiments, the genome of the cell is recoded so that the first endogenous tRNA is unnecessary and the second endogenous tRNA is unnecessary. The cell may not express or contain the first or second endogenous tRNA. In examples, the cell expresses or contains a second modified tRNA that can decode a second type of sense codon. The second modified tRNA is charged with a second amino acid, and the second amino acid is not the naturally cognate amino acid of the second type of sense codon.

[0032] The gene required for viability may contain at least one occurrence of a second kind of sense codon, and the cell is viable when the second kind of sense codon in the gene is decoded as the second amino acid. This gene may be the same gene required for viability that contains the first kind of sense codon, or it may be a different gene.

[0033] In some cases, a cell is not viable when the second type of sense codon in a gene required for survival is decoded according to the normal genetic code. The gene may contain multiple occurrences of the second type of sense codon, and the cell may be not viable when the occurrences are decoded according to the normal genetic code. At least one occurrence of the second type of sense codon in a gene required for survival may at least partially contribute to reduced survival when decoded according to the normal genetic code, and may contribute to the complete loss of survival in combination with other characteristics, such as other reassigned codons or other types of reassigned codons. The complete loss of survival may be due to the translational effects of multiple reassigned codons according to the normal genetic code.

[0034] The cell may contain at least one gene necessary for survival that contains a first type of sense codon and at least one different gene necessary for survival that contains a second type of sense codon. The cell may contain genes necessary for survival that contain both the first and second types of sense codons. It is also possible to contain any combination of genes necessary for survival and any combination of reassigned codons.

[0035] Cells of the present disclosure may be viable if their genes are decoded according to the rearranged genetic code, and may be non-viable if their genes are decoded at least in part according to the canonical genetic code.

[0036] Modified tRNA can be derived from naturally occurring tRNA, which is modified so that the codon can decode into an amino acid that the codon is not associated with in the normal genetic code.For example, the residue of the anticodon of tRNA can be replaced so that the tRNA has a different codon specificity, and such a tRNA can be called anticodon-swapped tRNA.Alternatively, tRNA can be charged with an amino acid that is not naturally associated, thereby providing the tRNA with the ability to decode the codon into an amino acid that the codon is not associated with in the normal genetic code.Modified tRNA can also be modified in other ways, for example, additional sequences can be added.Modified tRNA can be charged with the natural amino acid that the parent tRNA is naturally associated with.

[0037] The modified tRNA may be derived from a naturally occurring tRNA (which may be referred to as a parent tRNA). For example, the modified tRNA may be derived from a tRNA that is endogenous to the cell. The modified tRNA may be derived from an isoacceptor tRNA for a particular amino acid in the cell. For example, if the cell is E. coli, the modified tRNA may be derived from an E. coli tRNA that is an isoacceptor for the first or second amino acid. The modified tRNA may be derived from a naturally occurring tRNA found in a mobile genetic element, such as a viral tRNA. The modified tRNA may contain an identity element that is recognized by an aminoacyl-tRNA synthetase endogenous to the cell. The modified tRNA may retain the identity element of the parent tRNA.

[0038] The present inventors have demonstrated herein that episomes encoding the components of the translational machinery that are necessary for the translation of genes required for survival according to the reassigned genetic code can be essential for cells.Therefore, these episomes are stably maintained by the cells of the first aspect.Thus, in one embodiment, the first modified tRNA can be encoded by an episome, for example, a plasmid, in cells.Episomes may further comprise other genes that are desired to be stably maintained.

[0039] In a particular example, the cell is E. coli and comprises a genome recoded for a first and a second type of sense codon (e.g., TCA and TCG). The first modified tRNA can be an E. coli isoacceptor tRNA for a first amino acid (e.g., alanine) modified to include an anticodon complementary to the first type of sense codon (e.g., TCA). The second modified tRNA can be an E. coli isoacceptor tRNA for a second amino acid (e.g., histidine) modified to include an anticodon complementary to the first type of sense codon (e.g., TCG).

[0040] In some examples, the first modified tRNA cannot decode the second type of sense codons and / or the second modified tRNA cannot decode the first type of sense codons. In further examples, the first modified tRNA cannot decode any type of codons other than the first type of sense codons and / or the second modified tRNA cannot decode any type of codons other than the second type of sense codons.

[0041] The first and second amino acids can be the same amino acid, e.g., they can both be alanine, histidine, leucine, or proline. In other examples, the first and second amino acids can be different, e.g., one can be alanine while the other is leucine. Some exemplary reassignment schemes are shown in Figure 2C.

[0042] Cells produce tRNA XXX Xaa where XXX is the sense codon type and Xaa is the charged amino acid. Thus, cells capable of using a particular reassignment scheme can be defined as containing the relevant tRNAs. For example, in certain embodiments, cells capable of reassignment of TCG to alanine and TCA to histidine can be described as containing the tRNAs CGA Ala and tRNA UGA Hiscells that can reassign TCG to histidine and TCA to alanine contain tRNA CGA His and tRNA UGA Ala Includes.

[0043] The first and / or second amino acid may be a naturally occurring amino acid. The naturally occurring amino acid may be any naturally occurring proteinogenic amino acid. A "naturally occurring proteinogenic amino acid" is any one of L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, glycine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-arginine, L-serine, L-threonine, L-valine, L-tryptophan, and L-tyrosine, L-pyrrolidine, and L-selenocysteine. The naturally occurring amino acid may be a regular amino acid. A "canonical amino acid" is any one of L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, glycine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-arginine, L-serine, L-threonine, L-valine, L-tryptophan, and L-tyrosine.

[0044] The cell of the first aspect may be of any species or type disclosed herein. For example, the cell may have a genome recoded for the codons TCA and TCG, and may encode tRNAs Ser UGA and tRNA Ser CGA The bacterial cell may be a bacterial cell lacking TCA and TCG, and the TCA and TCG have been reassigned. The genome of the cell may be recoded by any means discussed herein. The reassignment scheme for the cell of the first embodiment may be any of those disclosed herein, for example, one of the schemes illustrated in Figure 2C.

[0045] The cells may be Syn61, a line derived from Syn61, or recoded in the same manner as Syn61. The cells may be Syn61Δ3, a line derived from Syn61Δ3, or modified in the same manner as Syn61Δ3.

[0046] Features of the first aspect related to "code locking" may be applied to the recoding scheme of the second or third aspect, whereby any features of the first, second and third aspects may be combined or are not mutually exclusive. Features of the second and third aspects, such as tRNAs and coding schemes, may be applied to the first aspect.

[0047] The cell of the first aspect may have increased resistance to mobile genetic elements. The cell of the first aspect may have improved maintenance of resistance to mobile genetic elements; for example, resistance may be maintained in long-term cell culture compared to control cultures that do not contain code-locked cells.

[0048] Orthogonal Coding Schemes There are an ever-increasing number of applications involving genetically modified organisms, and there is a need to limit the transfer of genetic information from these organisms to naturally occurring organisms. The inventors provide herein orthogonal coding schemes that inhibit the transfer of genetic information to naturally occurring organisms or to organisms that utilize alternative orthogonal coding schemes. For example, mobile genetic elements that utilize one of the orthogonal coding schemes cannot be transferred to or expressed by naturally occurring organisms.

[0049] Others have attempted to generate synthetic genetic information to suppress horizontal gene transfer (Nyerges et al. "Swapped genetic code blocks viral infections and gene transfer," https: / / doi.org / 10.1101 / 2022.07.08.499367). However, the present inventors provide herein a screening method that allows for the development of active and specific tRNAs. (See Figure 8 (Figure S3)). As one of skill in the art will appreciate, the screens disclosed in the Examples can be adapted for use with codons other than TCA and TCG, allowing for the development of active and specific tRNAs that do not decode off-target codons.

[0050] Thus, in a second aspect, there is provided a cell comprising a genome in which a first type of sense codon and a second type of sense codon have been re-encoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; the cell expresses a first anticodon-swapped tRNA derived from a naturally occurring first parent tRNA, the first anticodon-swapped tRNA being charged with a first amino acid, the first parent tRNA being an isoacceptor for the first amino acid, and the first amino acid being a first species of and not the naturally cognate amino acid to the sense codon of the second type; wherein the cell expresses a second anticodon-swapped tRNA derived from a naturally occurring second parent tRNA, the second anticodon-swapped tRNA being charged with a second amino acid, the second parent tRNA being an isoacceptor for the second amino acid, and the second amino acid is not the naturally cognate amino acid to the sense codon of the second type; and wherein the first and / or second modified tRNA are incapable of decoding any type of codon other than the first type of sense codon and / or the second type of sense codon.

[0051] In certain embodiments, the tRNA does not decode a particular codon if the rate of misincorporation is undetectable by the screening methods disclosed herein or too low to affect cellular fitness. Thus, the tRNA of the second aspect may not have a detectable rate of misincorporation of non-target codons, or may not have a rate of misincorporation that would be appropriate given the size of the host genome. In one embodiment, the tRNA of the second aspect is the tRNA exemplified in Example 3, i.e., tRNA CGA Ala , tRNA UGA Ala , tRNA CGA His , tRNA UGA His , tRNA CGA Leu , tRNA UGA Leu and tRNA CGA Leu , tRNA UGA Leu It is as active and specific as either one of

[0052] An anticodon-swapped tRNA is one in which the residue of the anticodon has been replaced so that the tRNA has a different codon specificity. Anticodon-swapped tRNAs may also be modified in other ways, for example, additional sequences may be added.

[0053] The present inventors have surprisingly discovered that sense codons that normally encode the same amino acid and are normally decoded by the same tRNA or overlapping tRNAs due to wobble base pairing can be used to encode multiple alternative amino acids. Such sense codons are predicted to be only capable of single reassignment. The present inventors provide herein a screening method that allows the development of tRNAs with desired activity and specificity. This discovery is advantageous because it allows the inventors to generate a large number of different orthogonal codes, for example, in an exemplary organism containing two reassigned serine codons. As illustrated, the inventors generated 16 refactored codes from only two reassigned sense codons and four amino acids.

[0054] Thus, in some cases, the first and second types of sense codons are normally decoded by the same tRNA or overlapping tRNAs due to wobble base pairing. The first anticodon-swapped tRNA may be unable to decode any type of codon other than the first type of sense codon, and the second anticodon-swapped tRNA may be unable to decode any type of codon other than the second type of sense codon. This allows the first and second types of sense codons to be used to encode two different amino acids without misincorporation.

[0055] The first type of sense codon and the second type of sense codon may have the formula XXN. This means that the first and second bases are the same, while the third base is different. In an example, the first anticodon-swapped tRNA cannot decode the second type of sense codon, and the second anticodon-swapped tRNA cannot decode the first type of sense codon.

[0056] In some examples, the first anticodon-swapped tRNA cannot decode any type of codon other than the first type of sense codon, and / or the second anticodon-swapped tRNA cannot decode any type of codon other than the second type of sense codon.

[0057] In certain embodiments, the first anticodon-swapped tRNA does not decode a TCC or TCT codon, and the second anticodon-swapped tRNA does not decode a TCC or TCT codon. As an example, this can be advantageous when the first or second type of recoded sense codon is TCA or TCG, because misincorporation at a TCC or TCT codon can reduce cellular fitness. For example, some E. coli genomes contain 9,999 TCC codons and 9,566 TCT codons, and thus misincorporation can affect fitness. In certain embodiments, the tRNA does not decode a particular codon if the rate of misincorporation is undetectable in the screening methods disclosed herein or too low to affect cellular fitness. Thus, the tRNA may not have a detectable rate of misincorporation at TCC or TCT, or may not have a rate of misincorporation that would be appropriate given the size of the host genome. In one embodiment, the tRNA is a tRNA exemplified in Example 3. CGA Ala , tRNA UGA Ala , tRNA CGA His , tRNA UGA His , tRNA CGA Leu , tRNA UGA Leu and tRNA CGA Leu , tRNA UGA Leu have a rate of misintegration in TCC or TCT not higher than any one of

[0058] In the second embodiment, the anticodon-swapped tRNA is charged with a natural amino acid with which it is not naturally associated. This allows the anticodon-swapped tRNA to be charged with the same amino acid as the parent tRNA from which it is derived. The anticodon-swapped tRNA may contain an identity element that is recognized by an aminoacyl-tRNA synthetase endogenous to the cell.

[0059] The anticodon-swapped tRNA can be derived from a tRNA that is endogenous to the cell. The anticodon-swapped tRNA can be derived from an isoacceptor tRNA for a particular amino acid in the cell. For example, if the cell is E. coli, the anticodon-swapped tRNA can be derived from an E. coli tRNA that is an isoacceptor for the first or second amino acid. The anticodon-swapped tRNA can be derived from a naturally occurring tRNA found in a mobile genetic element, such as a viral tRNA. The anticodon-swapped tRNA can retain the identity elements of the parent tRNA.

[0060] In examples, the first and second types of sense codons may both canonically encode serine, may both canonically encode alanine, or may both canonically encode leucine.

[0061] In certain embodiments, the first type of sense codon is TCA and the second type of sense codon is TCG.

[0062] The first and / or second type of sense codons may be reassigned to any naturally occurring proteinogenic or canonical amino acid. In exemplary embodiments, a first type of sense codon, e.g., a canonical serine codon, may be reassigned to one of alanine, histidine, leucine, and proline, and a second type of sense codon, e.g., a canonical serine codon, may be reassigned to one of alanine, histidine, leucine, and proline.

[0063] In certain examples, TCA may be reassigned to any non-serine naturally occurring proteinogenic or canonical amino acid, and / or TCG may be reassigned to any non-serine naturally occurring proteinogenic or canonical amino acid. In further exemplary embodiments, TCA may be reassigned to one of alanine, histidine, leucine, and proline, and TCG may be reassigned to one of alanine, histidine, leucine, and proline. In some examples, the reassignment scheme is disclosed in Figure 2C.

[0064] In other examples, the first amino acid and the second amino acid are different types of amino acids. The following is an exemplary reassignment scheme: Reassignment of TCG to alanine and TCA to histidine Reassignment of TCG to alanine and TCA to leucine Reassignment of TCG to alanine and TCA to proline Reassignment of TCG to histidine and TCA to alanine Reassignment of TCG to histidine and TCA to leucine Reassignment of TCG to histidine and TCA to proline Reassignment of TCG to leucine and TCA to alanine Reassignment of TCG to leucine and TCA to histidine Reassignment of TCG to leucine and TCA to proline Reassignment of TCG to proline and TCA to alanine Reassignment of TCG to proline and TCA to histidine Reassignment of TCG to proline and TCA to leucine.

[0065] The first or second anticodon-swapped tRNA can be derived from a parent tRNA encoded by ArgQ, ArgU, GltU, HisR, ProK, ProL, ProM, TrpT, ThrU, ThrT, TyrU, TyrV, AlaT, or LeuQ. Thus, the first or second anticodon-swapped tRNA can be encoded by any one of the genes, and the tRNA has an anticodon modified to recognize a sense codon that is not normally associated with the amino acid that the parent tRNA charges. In a specific example, the first or second anticodon-swapped tRNA can be derived from a parent tRNA encoded by HisR, ProM, AlaT, or LeuQ. The gene encoding the tRNA can be derived from E. coli.

[0066] In some examples, the first and second anticodon-swapped tRNAs are derived from a parent tRNA encoded by the group consisting of: ArgQ, ArgU, GltU, HisR, ProK, ProL, ProM, TrpT, ThrU, ThrT, TyrU, TyrV, AlaT, and LeuQ. In some examples, the first and second anticodon-swapped tRNAs are derived from a parent tRNA encoded by the group consisting of: HisR, ProM, AlaT, and LeuQ.

[0067] In some examples, the ArgQ, ArgU, GltU, HisR, ProK, ProL, ProM, TrpT, ThrU, ThrT, TyrU, TyrV, AlaT, or LeuQ gene is unmodified except for the anticodon. In other examples, the gene may contain additional sequences or may be truncated. In certain examples, the encoded tRNA may contain identity elements of the parent tRNA. In other examples, the gene may contain one or more modifications, and the encoded tRNA remains functional. The gene may contain no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution, addition, or replacement.

[0068] In some examples, the anticodon is swapped to UGA or CGA. In some examples, the first anticodon-swapped tRNA is swapped to UGA and the second anticodon-swapped tRNA is swapped to CGA.

[0069] In particular examples, the ArgQ-derived tRNA is according to SEQ ID NO: 43 or 44, the GltU-derived tRNA is according to SEQ ID NO: 19 or 20, the HisR-derived tRNA is according to SEQ ID NO: 49 or 50, the ProK-derived tRNA is according to SEQ ID NO: 55 or 56, the ProL-derived tRNA is according to SEQ ID NO: 57 or 58, the ProM-derived tRNA is according to SEQ ID NO: 59 or 60, the TrpT-derived tRNA is according to SEQ ID NO: 61 or 62, the ThrU-derived tRNA is according to SEQ ID NO: 25 or 26, the ThrT-derived tRNA is according to SEQ ID NO: 23 or 24, the TyrV-derived tRNA is according to SEQ ID NO: 63 or 64, the AlaT-derived tRNA is according to SEQ ID NO: 65 or 66, and the LeuQ-derived tRNA is according to SEQ ID NO: 67 or 68. Any of these sequences may contain one or more modifications, provided that the encoded tRNA remains functional. Any of these sequences may or may not contain 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 substitution, addition, or replacement. The modifications may be in regions that do not encode identity elements. Any of these sequences may be modified to encode a different anticodon. The alternative anticodon may not be the naturally associated anticodon.

[0070] The cell of the second aspect may be of any species or type disclosed herein. For example, the cell may have a genome recoded for the codons TCA and TCG, and may encode tRNAs Ser UGA and tRNA Ser CGAThe cell may be a bacterial cell lacking Syn61. The genome of the cell may be recoded by any of the means discussed herein. The cell may be Syn61, a Syn61-derived strain, or a strain recoded by the same means as Syn61. The cell may be Syn61Δ3, a Syn61Δ3-derived strain, or modified by the same means as Syn61Δ3.

[0071] Reassignment schemes can vary in their efficiency. For example, Figure 4C compares two different gene coding schemes and notes the difference in colony numbers. Reassignment schemes can affect cellular fitness. The contribution of a validated reassignment scheme is therefore a valuable contribution.

[0072] Thus, in a third aspect, there is provided a cell comprising a genome in which a first type of sense codon and a second type of sense codon have been re-encoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; the cell expresses a first modified tRNA that can decode the first type of sense codon, the first modified tRNA being charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon; The cell expresses a second modified tRNA capable of decoding a second type of sense codon, the second modified tRNA being charged with a second amino acid that is not the naturally cognate amino acid of the second type of sense codon: i) the first amino acid is alanine and the second amino acid is alanine; ii) the first amino acid is alanine and the second amino acid is histidine; iii) the first amino acid is alanine and the second amino acid is leucine; or iv) the first amino acid is alanine, v) the first amino acid is histidine and the second amino acid is alanine; vi) the first amino acid is histidine and the second amino acid is histidine; vii) the first amino acid is histidine and the second amino acid is leucine; viii) the first amino acid is histidine and the second amino acid is proline; ix) the first amino acid is leucine and the second amino acid is alanine; x) the first amino acid is leucine. xii) the first amino acid is proline and the second amino acid is alanine; xiii) the first amino acid is proline and the second amino acid is histidine; xiv) the first amino acid is proline and the second amino acid is leucine; or xv) the first amino acid is proline and the second amino acid is proline.

[0073] The modified tRNA can be as described in the first or second embodiment. In particular, the first modified tRNA may be unable to decode the second type of sense codon, and / or the second modified tRNA may be unable to decode the first type of sense codon. In some cases, the first modified tRNA cannot decode any type of codon other than the first type of sense codon, and / or the second modified tRNA cannot decode any type of codon other than the second type of sense codon. This high specificity is only possible by the screening method disclosed herein.

[0074] Modified tRNAs are derived from tRNAs, and may be naturally occurring tRNAs that have been altered to enable decoding of codons to amino acids not associated with the codon in the normal genetic code. For example, the anticodon residue of a tRNA may be replaced so that the tRNA has a different codon specificity; such a tRNA may be referred to as an anticodon-swapped tRNA. Modified tRNAs may also be modified in other ways, for example, additional sequences may be added. Modified tRNAs may be charged with the natural amino acid with which the parent tRNA is naturally associated. Modified tRNAs may be derived from naturally occurring tRNAs (which may be referred to as parent tRNAs). For example, modified tRNAs may be derived from tRNAs endogenous to the cell. Modified tRNAs may be derived from isoacceptor tRNAs for a particular amino acid in the cell. For example, if the cell is E. coli, the modified tRNA may be derived from an E. coli tRNA that is an isoacceptor for the first or second amino acid. Modified tRNAs may also be derived from naturally occurring tRNAs found in mobile genetic elements, such as viral tRNAs. The modified tRNA can contain an identity element that is recognized by an aminoacyl-tRNA synthetase endogenous to the cell. The modified tRNA can retain the identity element of the parent tRNA.

[0075] The recoding scheme can be any of those discussed herein. In certain embodiments, the first type of sense codon is TCA and the second type of sense codon is TCG.

[0076] The cell of the third aspect may be of any species or type disclosed herein. For example, the cell may have a genome recoded for the codons TCA and TCG, and may encode tRNA Ser UGA and tRNA Ser CGA The cell may be a bacterial cell lacking Syn61, a Syn61-derived strain, or a strain recoded in the same manner as Syn61. The cell may be Syn61Δ3, a Syn61Δ3-derived strain, or modified in the same manner as Syn61Δ3.

[0077] Kits containing cells utilizing mutually orthogonal coding schemes The inventors have discovered that cells utilizing a first orthogonal code can be mutually orthogonal to cells utilizing a second orthogonal code (see FIG. 4C). Thus, such cells may coexist with each other and with cells utilizing the canonical genetic code, and horizontal gene transfer does not occur between cells that do not have the same coding scheme.

[0078] Accordingly, in a sixth aspect of the present invention there is provided a kit comprising a first cell recoded according to a first orthogonal coding scheme and a second cell recoded according to a second orthogonal coding scheme, wherein the first and second coding schemes are orthogonal to each other.

[0079] In one example, the kit comprises a first cell of the first, second or third aspect and a second cell of the first, second or third aspect, wherein the first and second cells utilize coding schemes that are orthogonal to each other.

[0080] In some examples, the first and / or second orthogonal genetic coding scheme is any disclosed herein, for example, any of the orthogonal genetic codes of FIG. 2C.

[0081] In one example, the kit can include a first cell, which can be a bacterial cell such as E. coli, that utilizes the reallocation scheme illustrated in Figure 2C. A second cell of the kit, which can be a bacterial cell such as E. coli, can utilize a different reallocation scheme illustrated in Figure 2C. In some examples, the cell is based on or derived from Syn61.

[0082] In certain instances, the first cell CGA Ala and tRNA UGA His or tRNA CGA His and tRNA UGA Ala Includes.

[0083] The kit may further include cells that utilize the authentic genetic code.

[0084] The kit may further comprise a first mobile genetic element recoded according to a first orthogonal coding scheme. The kit may further comprise a second mobile genetic element recoded according to a second orthogonal coding scheme. The first and / or second mobile genetic element may be a mobile genetic element disclosed herein.

[0085] In one example, a kit may include a mobile genetic element in which at least one, multiple, or all instances of a codon that normally encodes alanine (i.e., a GCN codon) in at least one gene required for horizontal transfer of genetic information have been replaced with TCG or TCA, and the kit may include cells expressing a modified tRNA capable of decoding TCG or TCA to alanine. The tRNA may be as disclosed for the first, second, or third aspect.

[0086] In one example, the kit may include a mobile genetic element in which at least one, multiple, or all instances of a codon that normally encodes histidine (i.e., a CAT / C codon) in at least one gene required for horizontal transfer of genetic information has been replaced with TCG or TCA, and may include cells expressing a modified tRNA that can decode TCG or TCA to histidine. The tRNA may be as disclosed for the first, second, or third aspect.

[0087] In particular examples, the kit may include a mobile genetic element in which at least one, multiple, or all instances of the GCN codon in at least one gene required for horizontal transfer of genetic information have been replaced with a TCG codon; and at least one, multiple, or all instances of the CAT / C codon in at least one gene required for horizontal transfer of genetic information have been replaced with a TCA codon.

[0088] The kit may further include a third, fourth, fifth, or additional cells, each of which utilizes a coding scheme that is orthogonal to all other cells.

[0089] Mobile genetic elements We demonstrate that mobile genetic elements that utilize orthogonal genetic codes cannot be transferred to cells that utilize regular genetic codes or to cells that utilize genetic codes that are orthogonal to each other. Horizontal gene transfer is shown to be suppressed in mobile genetic elements, such as F plasmids, which are transferred via conjugation. This is an improvement over orthogonal genetic elements that must be electroporated, such as elements that are not truly mobile.

[0090] Thereby, in a seventh aspect, there is provided a mobile genetic element recoded according to an orthogonal coding scheme.

[0091] The orthogonal coding schemes may be those discussed herein, including any in FIG. 2C.

[0092] In one embodiment, a mobile genetic element is provided in which at least one, multiple, or all instances of a particular type of sense codon in at least one gene required for horizontal transfer of genetic information have been replaced with a sense codon that normally encodes a different amino acid.

[0093] In one embodiment, a mobile genetic element is provided in which at least one, multiple, or all instances of a codon that normally encodes alanine, leucine, histidine, proline, or any combination thereof in at least one gene required for horizontal transfer of genetic information has been replaced with a sense codon that does not encode the respective amino acid. In some instances, the new sense codon may normally encode serine, e.g., TCA or TCG.

[0094] In one embodiment, a mobile genetic element is provided in which at least one, several, or all instances of a codon that normally encodes alanine, leucine, histidine, proline, or any combination thereof in at least one gene required for horizontal transfer of genetic information has been replaced with TCG or TCA.

[0095] For example, at least one, multiple, or all occurrences of a codon that normally encodes alanine (the GCN codon) in at least one gene required for horizontal transfer of genetic information can be replaced in the mobile genetic element with a codon that has been reassigned to alanine. In one embodiment, a mobile genetic element is provided in which at least one, multiple, or all instances of a codon that normally encodes alanine (i.e., the GCN codon) in at least one gene required for horizontal transfer of genetic information have been replaced with TCG or TCA.

[0096] Alternatively or additionally, at least one, multiple, or all instances of a codon that normally encodes histidine (the CAT / C codon) in at least one gene required for horizontal transfer of genetic information may be replaced with a codon that has been reassigned to histidine. In one embodiment, a mobile genetic element is provided in which at least one, multiple, or all instances of a codon that normally encodes histidine (i.e., the CAT / C codon) in at least one gene required for horizontal transfer of genetic information has been replaced with TCG or TCA.

[0097] In one embodiment, a mobile genetic element is provided in which at least one, multiple, or all instances of a codon that normally encodes alanine (GCN codon) in at least one gene required for horizontal transfer of genetic information have been replaced with TCG, and at least one, multiple, or all instances of a codon that normally encodes histidine (CAT / C codon) in at least one gene required for horizontal transfer of genetic information have been replaced with TCA.

[0098] A mobile genetic element comprises at least one gene necessary for horizontal transfer of genetic information recoded according to a reassignment scheme. The mobile genetic element may comprise two, three, four, or more such genes. Genes within the mobile genetic element that are not necessary for horizontal transfer of genetic information may be recoded to have a condensed coding scheme, e.g., one or more types of sense codons may be absent from the gene. Genes within the mobile genetic element that are not necessary for horizontal transfer of genetic information may also comprise one or more codons that have been reassigned (e.g., replaced with alternative codons according to a reassignment scheme). Thus, in some embodiments, all genes within the mobile genetic element are recoded to be absent from one or more types of sense codons, and the one or more genes and the mobile genetic element comprise at least one gene necessary for horizontal transfer of genetic information recoded according to a reassignment scheme.

[0099] In some examples, the mobile genetic element may be a plasmid or a virus. The mobile genetic element may be a phage. The mobile genetic element may be an F plasmid.

[0100] In one aspect of the invention there is provided a kit comprising a first mobile genetic element as disclosed herein and a first cell of the first, second or third aspect disclosed herein, wherein the first mobile genetic element and the first cell utilize the same genetic coding scheme.

[0101] In one example, the kit can include a mobile genetic element in which at least one, multiple, or all instances of a codon that normally encodes alanine (GCN codon) in at least one gene required for horizontal transfer of genetic information have been replaced with TCG or TCA, and can include cells expressing a modified tRNA capable of decoding TCG or TCA to alanine. The tRNA can be as disclosed for the first, second, or third aspect. Alternatively or additionally, the kit can include a mobile genetic element in which at least one, multiple, or all instances of a codon that normally encodes histidine (CAT / C codon) in at least one gene required for horizontal transfer of genetic information have been replaced with TCG or TCA, and can include cells expressing a modified tRNA capable of decoding TCG or TCA to histidine.

[0102] In other examples, the kit can include mobile genetic elements and cells utilizing any of the orthogonal genetic codes disclosed herein, for example, any of the orthogonal genetic codes of Figure 2C.

[0103] The kit may include a second mobile genetic element and a second cell of the first, second, or third aspect disclosed herein, wherein the second mobile genetic element and the second cell utilize the same genetic coding scheme and the first mobile genetic element and the second mobile genetic element utilize different genetic coding schemes. In some examples, the genetic coding schemes of the second mobile genetic element and the second cell are any disclosed herein, such as any of the orthogonal genetic codes of Figure 2C.

[0104] The kit may further comprise a third, fourth, fifth or further mobile genetic element and cell, each pair of mobile genetic element and cell being compatible and orthogonal to all other pairs.

[0105] Methods for increasing cell resistance to mobile genetic elements or horizontal gene transfer In a fifth aspect of the present invention, there is provided a method of increasing a cell's resistance to mobile genetic elements or horizontal gene transfer, wherein the cell is modified to reassign at least one type of sense codon to an amino acid that is not associated with the sense codon in the canonical genetic code, the method comprising modifying a gene required for survival to contain at least one occurrence of the reassigned sense codon, wherein the cell is viable when the reassigned sense codon in said gene is decoded as the reassigned amino acid, the cell is non-viable when the reassigned sense codon in said gene is decoded according to the canonical genetic code, or the reassigned sense codon in said gene, when decoded according to the canonical genetic code, contributes at least in part to reduced survival.

[0106] The increased resistance can be resistance that is maintained for a long period of time, for example, resistance that is not lost during long-term cell culture (see Example 7). Thus, the cells of the fifth aspect can exhibit resistance to horizontal gene transfer or mobile genetic elements that is maintained for a long period of time compared to cells that are not code-locked.

[0107] The genome of the cell can be recoded to remove at least one example of a sense codon. The recoding can be any of those disclosed herein, e.g., TCA or TCG. The cell can also be recoded to remove at least one endogenous tRNA, e.g., tRNA Ser UGA or tRNA Ser CGA The assignment may be due to the insertion of one or more modified tRNAs. The modified tRNA may be any of those disclosed herein, e.g., an anticodon-swapped isoacceptor tRNA for alanine, leucine, histidine, or proline.

[0108] The gene required for survival can be any, including any disclosed herein. For example, the gene can be an essential gene or a positive selectable marker.

[0109] The cells obtained may be cells of the first, second or third aspect of the invention and the method may be modified accordingly.

[0110] Resistance to horizontal gene transfer Cells of the present disclosure, including those of the first, second, and third aspects, can be resistant to horizontal gene transfer. For example, the cells can be resistant to the transfer of genetic information from mobile genetic elements, including plasmids (such as F plasmids), viruses (including phages), and the like.

[0111] In particular, cells of the present disclosure can be resistant to the propagation of genetic information from mobile genetic elements that contain associated tRNAs, which can be capable of decoding one or more reassigned codons according to the canonical genetic code.

[0112] In some embodiments, cells may reduce or completely eliminate propagation of the F plasmid containing the relevant tRNA, a property that can be tested using the method of Figure 5C of the present application.

[0113] The cell may be a bacterium and may be resistant to the bacteriophage disclosed in Nyerges et al. "Swapped genetic code blocks viral infections and gene transfer", https: / / doi.org / 10.1101 / 2022.07.08.499367. The cell may be Escherichia coli and may be resistant to the bacteriophage.

[0114] The cells are also resistant to horizontal gene transfer from the cells to other types of cells. For example, cells of the first, second, or third aspects, or cells produced by the method of the fifth aspect, may not be able to transfer synthetic genes to or from wild-type bacteria. Cells of the present disclosure may not be able to transfer synthetic genes to wild-type bacteria of the same species. The synthetic genes may follow any of the reassigned coding schemes disclosed herein.

[0115] Additionally, the cells of the present disclosure are resistant to horizontal gene transfer from the cells to other cells that do not use the same reassigned coding scheme. Thus, the cells of the present disclosure cannot transfer synthetic genes to bacteria that cannot decode the synthetic genes according to a particular reassigned coding scheme. While other bacteria can also utilize the reassigned coding scheme, if the scheme is orthogonal to the cells of the present disclosure, horizontal gene transfer is thereby inhibited.

[0116] Methods for modifying the susceptibility of a gene to mutations that alter the encoded amino acid sequence - Patents.com The cells, codes, and techniques disclosed herein enable methods to alter the susceptibility of genes to mutations that alter the encoded amino acid sequence. Thus, the refactored codes disclosed herein can be used to speed up or slow down the rate of protein evolution.

[0117] The canonical genetic code is somewhat conservative, in that a point mutation may not change the encoded amino acid. Additionally, a point mutation may change the encoded amino acid to another amino acid with similar properties (e.g., a conservative substitution) or dissimilar properties (e.g., a non-conservative substitution). The number of differences between codon types may vary, which may affect the chance that a point mutation will result in: no change, a conservative change, or a non-conservative change to the encoded amino acid. We provide code and techniques for implementing such a code to alter the mutational landscape (see, e.g., Figure 10 (Figure S5) and Figure 11 (Figure S6)).

[0118] Thus, in one aspect, there is provided a method for altering the susceptibility of a gene to mutations that alter its encoded amino acid sequence, comprising: i) identifying a target gene; and ii) incubating cells containing the target gene, wherein the cells contain a tRNA capable of decoding at least one sense codon into a reassigned amino acid; A method is provided, comprising:

[0119] The target gene may be one or more target genes. The target gene may be a synthetic or natural gene. Suitably, the synthetic gene may have altered codon usage to support the evolutionary trajectory. In some aspects, the target gene may follow a condensed genetic code.

[0120] The cell may be any of the cells disclosed herein, e.g., any of the first, second, or third aspects. The cell may be a bacterial cell, e.g., E. coli, recoded for the first and second types of sense codons. The cell may be Syn61, Syn61-derived, or recoded by the same means as Syn61. The cell may be Syn61Δ3, Syn61Δ3-derived, or recoded by the same means as Syn61Δ3.

[0121] In one example, the reassignment scheme can be any of those illustrated in Figure 2C. These schemes can be used to alter the mutational landscape shown in Figure 11 (Figure S6).

[0122] The cells may be incubated under conditions that favor or allow mutations to occur. The method may be for the purpose of evolving or improving a protein. The method may be for the purpose of making the target gene more resistant to mutations, e.g., to protect cells from deleterious mutations.

[0123] Sense codon recoding This section further describes an exemplary embodiment of recoding, which is applicable to all aspects disclosed herein.

[0124] Endogenous tRNA is not considered to be expressed if endogenous tRNA does not exist in a form that can decode its cognate codon.Therefore, endogenous tRNA can be removed using any means that suppresses the production of functional form of endogenous tRNA in cells.For example, endogenous gene can be deleted, or part of the gene can be deleted to suppress expression.Regulatory sequence can be deleted or changed to suppress expression.Alternatively, nonsense, frameshift or missense mutation can suppress the expression of tRNA in functional form.

[0125] As used herein, " recoding " refers to the replacement of a certain type of codon with a different codon, so that the occurrence of the codon is removed from genome.The sense codon of recoding can be replaced with synonymous codon, so as to produce different codon usage without changing the encoded polypeptide.Alternatively, sense codon can be replaced with non-synonymous codon, for example, if the change in the sequence of the encoded polypeptide does not affect survival.The endogenous tRNA that is deleted is unnecessary from the viewpoint of recoding.As used herein, " unnecessary " means that it is not necessary for cell survival.

[0126] A viable cell is one that can be metabolically active. In certain embodiments, a viable cell can grow when cultured in an appropriate medium and under appropriate conditions for a particular species or strain. Such cells can be said to be capable of being cultured. As an example, if the cell is a bacterial cell, e.g., E. coli, assessment of viability can be performed by culturing the bacterium in a medium containing LB medium or on agar containing LB agar at 37°C. The medium or agar can be supplemented with 2% glucose. Bacterial growth can be monitored using standard approaches, e.g., OD 600 Alternative approaches or approaches adapted to particular cells, bacterial strains, bacterial species or in terms of the inclusion of marker genes are well known to those skilled in the art.

[0127] The endogenous tRNA that decodes the replaced (or deleted) sense codon(s) may be deleted, such that the tRNA decodes only the replaced (or deleted) sense codon(s); or, alternatively, if the tRNA decodes the replaced (or deleted) sense codon(s) and the unreplaced (or deleted) sense codon(s), the cell remains viable if the tRNA is dispensable for the unreplaced (or deleted) sense codon(s) (i.e., one or more of the sense codons decoded by the tRNA are decoded by one or more alternative tRNAs). For example, if the genome of a prokaryotic cell lacks a TCA sense codon, the tRNA Ser UGA serT, which encodes tRNA, can be deleted, and / or if the genome lacks a TCG sense codon, Ser CGA serU, which encodes tRNA, can be deleted. Ser UGA and tRNA Ser CGA does not express.

[0128] The number of occurrences of the first and / or second type of sense codons to be recoded is adapted to allow for the removal of the cognate tRNAs corresponding to the sense codons while maintaining cell viability. For example, this can be achieved by removing all natural occurrences of the first and second type of sense codons from an essential gene. In particular, a gene is considered essential if a "blank" codon within the gene (i.e., a codon for which the cell does not contain the corresponding tRNA or release factor) results in reduced cell viability. Thus, in one embodiment, all genes in a cell that cannot tolerate a blank codon without reduced viability are recoded, but genes that can tolerate a blank codon may not be recoded. This allows one skilled in the art to assess whether all essential genes have been recoded by assessing whether the cognate tRNAs are dispensable. Notably, some embodiments require at least one essential gene to contain the first and / or second type of sense codon; however, in such embodiments, the codons are reassigned and not in their naturally occurring positions.

[0129] The cells of the present disclosure, including the cells of the first, second and third aspects, can be recoded for the first, second, third, fourth, fifth or further types of sense codons. The recoding of the first and second types of sense codons is exemplified herein, and those skilled in the art will understand that the principles for recoding can be extended to reduce the occurrence of further types of sense codons in the genome of a cell. For example, further types of sense codons can be replaced with synonymous codons to eliminate specific occurrences without changing the encoded sequence, and an appropriate number of specific types of sense codons can be removed so that at least one additional endogenous tRNA is not required and does not need to be expressed by the cell.

[0130] In certain embodiments, the genome comprises 100 or more, 200 or more, or 300 or more essential genes that do not have natural occurrences of sense codons of the first and / or second types. For example, all, or substantially all, of the essential genes in the genome may not have natural occurrences of sense codons of the first and / or second types. In some embodiments, the essential genes are: ribF, lspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, tsf, pyrH, frr, dxr, ispU, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lp xB, dnaE, accA, tilS, proS, yafF, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, folD, entD, mrdB, mrdA, nadD, holA, rlpB, leuS, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asn S, fabA, mviN, rne, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, minE, minD, pth, prsA, ispE, lolB, hemA, prfA, prmC, kd sA, topA, ribA, fabI, tyrS, ribC, ydiL, pheT, pheS, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM , gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, lepB, rpoE, pssA , yfiO, rplS, trmD, rpsP, ffh, grpE, csrA, ispF, ispD, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, parE,It can be selected from one or more of the list consisting of ribB, cca, ygjD, tdcF, yraL, yhbV, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsI, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, yhhQ, bcsB, glyQ, gpsA, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spoT, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, yihA, ftsN, murI, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ and dnaC.,

[0131] RibF, lspA, ispH, dapB, folA, imp, yabQ, lpxC, secM, secA, can 、folK、hemL、yadR、dapD、map、rpsB、tsf、pyrH、frr、dxr、ispU、cdsA、yaeL、 yaeT、lpxD、fabZ、lpxA、lpxB、dnaE、accA、tilS、proS、yafF、hemB、secD、se cF、ribD、ribE、thiL、dxs、ispA、dnaX、adk、hemH、lpxH、cysS、folD、entD、mr dB、mrdA、nadD、holA、rlpB、leuS、lnt、glnS、fldA、cydA、infA、cydC、ftsK、 lolA、serS、rpsA、msbA、lpxK、kdsB、mukF、mukE、mukB、asnS、fabA、mviN、rne 、fabD、fabG、acpP、tmk、holB、lolC、lolD、lolE、purB、minE、minD、pth、prs A、ispE、lolB、hemA、prfA、prmC、kdsA、topA、ribA、fabI、tyrS、ribC、ydiL、p heT、pheS、rplT、infC、thrS、nadE、gapA、yeaZ、aspS、argS、pgsA、yefM、met G、folE、yejM、gyrA、nrdA、nrdB、folC、accD、fabB、gltX、ligA、zipA、dapE、 dapA、der、hisS、ispG、suhB、tadA、acpS、era、rnc、lepB、rpoE、pssA、yfiO、 rplS、trmD、rpsP、ffh、grpE、csrA、ispF、ispD、ftsB、eno、pyrG、chpR、lgt、f baA、pgk、yqgD、metK、yqgF、plsC、ygiT、parE、ribB、cca、ygjD、tdcF、yraL、 yhbV、infB、nusA、ftsH、obgE、rpmA、rplU、ispB、murA、yrbB、yrbK、yhbN、rps I、rplM、degS、mreD、mreC、mreB、accB、accC、yrdC、def、fmt、rplQ、rpoA、rp sD、rpsK、rpsM、secY、rplO、rpmD、rpsE、rplR、rplF、rpsH、rpsN、rplE、rplX、rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ft sE, ftsY, yhhQ, bcsB, glyQ, gpsA, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spoT, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, g The gene may be selected from one or more of the list consisting of lmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, yihA, ftsN, murI, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, and dnaC.

[0132] In other embodiments, the cell may comprise a genome comprising five or fewer natural occurrences of the first and / or second type of sense codons. The genome may be derived from a parent genome and may comprise less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% of the occurrences of the first and / or second type of sense codons compared to the parent genome. The genome may comprise 100 or more, 200 or more, or 1000 or more genes that do not comprise natural occurrences of the first and / or second type of sense codons. In particular, all or substantially all genes in the genome may not have natural occurrences of the first and / or second type of sense codons. Thus, the genome of the cell may comprise five, four, three, two, one, or none of the natural occurrences of the first type of sense codons, and five, four, three, two, or one, or none of the natural occurrences of the second type of sense codons.

[0133] The genome may be derived from a parent genome and may or may not include five or fewer (e.g., five, four, three, two, one) natural occurrences of a first and / or second type of naturally occurring sense codon. In certain embodiments, the genome is derived from a parent genome and does not include natural occurrences of a first and second type of naturally occurring sense codon. In some embodiments, the genome includes 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1500 or more, or 2000 or more recoded genes. In some embodiments, the genes are ones for which there is evidence of translation and / or predicted protein product. For example, a genome may contain 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1500 or more, or 2000 or more recoded genes for which there is evidence of translation and / or predicted protein products.

[0134] In one embodiment, all annotated coding regions in the genome do not have natural occurrences of the first and second types of sense codons. The cell may be a bacterial cell, preferably E. coli, whose genome may not contain natural occurrences of the first and second types of sense codons annotated in GenBank Accession No. CP040347.1.

[0135] In certain embodiments, genes encoding proteins do not have natural occurrences of sense codons of the first and second types. In certain embodiments, proteins are not translated from any of the remaining natural occurrences of sense codons of the first and / or second types, and / or genes that include the remaining natural occurrences of sense codons of the first and / or second types are putative or non-coding genes. In some embodiments, translation of genes that include the remaining natural occurrences of sense codons of the first and / or second types is reduced and / or suppressed (e.g., the genes may include stop codons in the 5' sequences).

[0136] Any remaining natural occurrences of sense codons may be necessary to ensure the viability of the genome. For example, one or more, particularly all, of the remaining natural occurrences of the first and / or second types of sense codons in the genome may be in regulatory elements of essential genes; and / or one or more, particularly all, of the remaining natural occurrences of the first and / or second types of sense codons may be in genes for which there is no evidence for translation or predicted protein product (i.e., putative or non-coding genes).

[0137] As used herein, a "sense codon" is a nucleotide triplet that encodes an amino acid. Thus, sense codons can be identified in a genome by gene prediction, i.e., by identifying the genome that encodes a protein (i.e., a gene) and the region of the genome that encodes the corresponding open reading frame (ORF). Typically, a genome naturally contains 61 sense codons: GCT, GCC, GCA, GCG, CGT, CGC, CGA, CGG, AGA, AGG, AAT, AAC, GAT, GAC, TGT, TGC, CAA, CAG, GAA, GAG, GGT, GGC, GGA, GGG, CAT, CAC, ATT, ATC, ATA, TTA, TTG, CTT, CTC, CTA, CTG, AAA, AAG, ATG, TTT, TTC, CCT, CCC, CCA, CCG, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, TGG, TAT, TAC, GTT, GTC, GTA, and GTG (reading 5' to 3' on the coding strand of DNA). The standard genetic code uses 61 triplet codons to encode the 20 canonical amino acids. 18 out of 20 amino acids are coded for by more than one synonymous codon. The sense codons of the first or second type may be natural sense codons, i.e., sense codons present in the parent genome.

[0138] The 61 sense codons in DNA are transcribed into corresponding mRNAs and then decoded by one or more tRNAs. The tRNAs carry amino acids to ribosomes directed by the sense codons in the mRNA. The tRNAs can recognize one or more sense codons via complementary anticodons. The sequence of the sense codons is then translated into a polypeptide (i.e., a sequence of amino acids). The interaction between codons and anticodons in the E. coli genome is shown in Figure 17 of WO 2020 / 229592 (incorporated herein by reference).

[0139] Genome-wide removal of sense codons of the first and / or second type without removing other sense codons results in the deletion of cognate tRNAs corresponding to said first or second type of sense codons without eliminating the ability to decode the remaining sense codons in the genome.

[0140] The recoded sense codons may be selected from: TCG, TCA, TCT, TCC, AGT, or AGC. In certain embodiments, the first and second types of sense codons are TCA and TCG.

[0141] To achieve the removal of sense codons, they can be replaced with synonymous sense codons. This is preferable to ensure that the encoded protein sequence is not altered. For example, a cell can have a genome in which 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of the occurrences of the first or second type of sense codons in the parent genome are replaced with synonymous sense codons. Those skilled in the art can deduce appropriate synonymous sense codon replacements. For example, in E. coli, TCG, TCA, TCT, TCC, AGT, and AGC typically all encode serine.

[0142] In some embodiments, the replacement is a defined replacement, i.e., a sense codon is replaced with a single synonymous sense codon. Preferably, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of the natural occurrences of the first or second type of sense codon in the parental genome are replaced with a defined (i.e., single) synonymous sense codon.

[0143] For example, the defined replacement can be: TCG replaced with any one of TCT, TCC, AGT, or AGC; or TCA replaced with any one of TCT, TCC, AGT, or AGC. In particular, the replacement is selected from one or more of: TCG to any one of AGT or AGC; or TCA to any one of AGT or AGC. In certain embodiments, TCG is replaced with AGC and TCA is replaced with AGT.

[0144] Preferably, none of these codon replacements affect the ribosome binding site (AGGAGG), a highly conserved regulatory sequence in E. coli. Selected codon replacements can be tested in a small test region (e.g., a 20 kb region of the genome rich in both the essential target gene and the target codon) to assess viability. If the codon replacements are not viable in the small test region, they can be ignored.

[0145] If the replacement of the sense codon in the parent genome containing the defined replacement synonymous sense codon does not produce viable cells, alternative replacement synonymous sense codons can be used.For example, 99.9% of the occurrences of the first and / or second type of sense codon in the parent genome can be replaced with a defined (i.e., single) synonymous sense codon, and the remaining 0.1% can be replaced with an alternative synonymous sense codon.For example, 99.9% of the natural occurrences of TCG can be replaced with AGC, and 0.1% can be replaced with TCT, TCC, AGT or AGC; and / or 99.9% of the occurrences of TCA can be replaced with AGT, and 0.1% can be replaced with TCT, TCC, AGT or AGC.

[0146] In some cases, certain occurrences of sense codons cannot be replaced with any of the potential synonymous sense codons without affecting viability.To maintain viability, sense codons can be replaced with non-synonymous sense codons that do not affect viability.For example, 99.9% of the occurrences of the first and / or second type of sense codons in the parent genome can be replaced with a defined (i.e., single) synonymous sense codon, and the remaining 0.1% can be replaced with an alternative non-synonymous sense codon.

[0147] Stop codon recoding This section further describes exemplary embodiments involving recoding of stop codons and is applicable to all aspects disclosed herein.

[0148] In some cases, the first type of stop codon has been re-encoded in the genome of the cell such that the first endogenous terminator is not required and the cell does not express the first endogenous terminator.

[0149] The first endogenous termination factor can be removed in the recoded cell so that the genome eliminates the occurrence of the first type of stop codon. The removed stop codon can be replaced with a synonymous codon. The deleted endogenous termination factor is a factor that is unnecessary from the perspective of recoding.

[0150] In certain instances, the cell does not express the first endogenous tRNA, the second endogenous tRNA, and the first endogenous terminator; and the genome has been recoded to remove multiple sense codons to which the first and second endogenous tRNAs are cognate, and to remove multiple stop codons to which the first endogenous terminator is cognate.

[0151] An endogenous terminator is not expected to be expressed unless it is present in a form that allows it to decode its cognate codon. Thus, an endogenous terminator can be removed using any means that suppresses the production of a functional form of the endogenous terminator in cells. For example, the endogenous gene may be deleted, or a portion of the gene may be deleted to suppress expression. Regulatory sequences may be deleted or altered to suppress expression. Alternatively, nonsense, frameshift, or missense mutations may suppress the expression of the terminator in a functional form.

[0152] As used herein, a "stop codon" is a nucleotide triplet that codes for the termination of translation into a protein. Typically, a genome naturally contains three stop codons: TAA ("ochre"), TGA ("opal" or "umber"), and TAG ("amber").

[0153] The number of natural occurrences of the first type of stop codon to be removed is suitable to allow removal of the cognate terminators corresponding to the stop codons while maintaining cell viability. Thus, in some cases, essential genes of the cell do not contain occurrences of the first type of stop codon. Essential genes can be any of those discussed herein, particularly those discussed in relation to the removal of the first or second type of sense codons. In certain examples, the genome contains 100 or more, 200 or more, or 300 or more essential genes that do not contain natural occurrences of the first type of stop codon. For example, all or substantially all essential genes in the genome may not contain occurrences of the first type of stop codon.

[0154] For example, a genome may contain 100 or more, 200 or more, or 300 or more essential genes that do not contain natural occurrences of a first type of sense codon, a second type of sense codon, and a first type of stop codon. In particular, all or substantially all essential genes in the genome may not contain natural occurrences of a first type of sense codon, a second type of sense codon, and a first type of stop codon.

[0155] In some embodiments, the genome contains no more than 10, no more than 5, or no natural occurrences of a first type of stop codon, e.g., 5, 4, 3, 2, 1, or 0 natural instances of a first type of stop codon.

[0156] In particular examples, the first type of stop codon is TAG and the first endogenous termination factor is RF-1. In such examples, there may be no more than 10, no more than 5, or no natural occurrences of amber stop codons (TAG). In other examples, 90% or more, 95% or more, 98% or more, 99% or more, or all occurrences of TAG in the parental genome are replaced with TAA (ochre stop codons). In particular embodiments, the genome may contain no occurrences of amber stop codons (TAG), and all occurrences of TAG in the parental genome may be replaced with TAA (ochre stop codons).

[0157] In one embodiment, all annotated translated regions in the genome have no occurrences of the first type of stop codon. The cell of the present disclosure may be a bacterial cell, such as E. coli, whose genome may not contain occurrences of the first type of stop codon annotated in GenBank Accession No. CP040347.1.

[0158] In some cases, a gene encoding a protein does not have a natural occurrence of a first type of stop codon. In particular cases, a protein is not translated from any of the remaining occurrences of a first type of stop codon, and / or the gene including the remaining occurrences of a first type of stop codon is a putative, or non-coding, gene. In some cases, translation of a gene including the remaining occurrences of a first type of stop codon is reduced and / or suppressed (e.g., the gene may include a stop codon in the 5' sequence).

[0159] Any remaining occurrences of the first type of stop codon may be necessary to ensure the genome is viable. For example, one or more, particularly all, of the remaining natural occurrences of the first type of stop codon in the genome may be in regulatory elements of essential genes; and / or one or more, particularly all, of the remaining occurrences of the first type of stop codon may be in genes for which there is no evidence for translation or a predicted protein product (i.e., putative or non-coding genes).

[0160] Genome recoded for sense codons and stop codons This section further describes an exemplary embodiment of recoding, which is applicable to all aspects disclosed herein.

[0161] Thus, in some instances, the genome does not contain occurrences of the first and second types of sense codons, and does not contain occurrences of one type of stop codon, preferably amber stop codon (TAG). In particular instances, the genome does not contain occurrences of the sense codons TCG and TCA, and does not contain occurrences of amber stop codon (TAG), and TCG, TCA, and TAG in the parent genome may be replaced with synonymous codons, for example, 99.9% or more of the occurrences of TCG in the parent genome are replaced with AGC, 99.9% or more of the occurrences of TCA in the parent genome are replaced with AGT, and all occurrences of TAG in the parent genome are replaced with TAA.

[0162] In a particular example, the cell's genome is recoded such that the sense codon TCG is replaced with AGC, the sense codon TCA is replaced with AGT, the stop codon TAG is replaced with TAA, and a sufficient number of said codons are recoded such that two cognate tRNAs and one cognate terminator are not required.

[0163] In certain examples, the cells of the present disclosure comprise a tRNA Ser UGA , tRNA Ser CGA and E. coli cells that do not express RF-1, and the appearance of the sense codon TCA is Ser UGA is recoded so that it is unnecessary (e.g., occurrences of TCA in essential genes of the parent strain are replaced with AGT), and occurrences of the sense codon TCG are replaced with tRNA Ser CGA is recoded to be dispensable (e.g., occurrences of TCG in essential genes of the parent strain are replaced with AGC), and occurrences of the stop codon TAG are recoded to be dispensable (e.g., occurrences of TAG in essential genes of the parent strain are replaced with TAA).

[0164] In some embodiments, the genome of a cell of the present disclosure comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identical to a sequence provided in GenBank Accession No. CP040347.1, and the genome comprises a tRNA Ser UGA and tRNA Ser CGA The genome was further modified so that RF-1 is not functionally expressed (e.g., by deleting serT and serU). The genome can also be further modified so that RF-1 is not functionally expressed (e.g., by deleting prfA). The E. coli strain containing the genome according to GenBank Accession No. CP040347.1 is referred to as Syn61 WT in the examples disclosed herein.

[0165] In some embodiments, the genome of a cell of the present disclosure comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identical to SEQ ID NO:1, and the genome comprises a tRNA Ser UGA and tRNA Ser CGA The genome has been further modified so that RF-1 is not functionally expressed (e.g., by deleting serT and serU). The genome may also be further modified so that RF-1 is not functionally expressed (e.g., by deleting prfA). An E. coli strain comprising a genome according to SEQ ID NO: 1 may be designated Syn61(ev1).

[0166] In some embodiments, the genome of a cell of the present disclosure comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identical to SEQ ID NO:2, and the genome comprises a tRNA Ser UGA and tRNA Ser CGA The genome has been further modified so that RF-1 is not functionally expressed (e.g., by deleting serT and serU). The genome can also be further modified so that RF-1 is not functionally expressed (e.g., by deleting prfA). An E. coli strain comprising a genome according to SEQ ID NO: 2 can be designated Syn61(ev2).

[0167] In some embodiments, the genome of the cell comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 3. An E. coli strain comprising a genome according to SEQ ID NO: 3 may be designated Syn61Δ3.

[0168] In some embodiments, the genome of the cell comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4. An E. coli strain comprising a genome according to SEQ ID NO: 4 may be designated Syn61Δ3(ev3).

[0169] In some embodiments, the genome of the cell comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5. An E. coli strain comprising a genome according to SEQ ID NO: 5 may be designated Syn61Δ3(ev4).

[0170] In some embodiments, the genome of the cell comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identical to SEQ ID NO:6 (also provided as GenBank Accession No. CP071799.1). An E. coli strain comprising a genome according to SEQ ID NO:6 may be designated Syn61Δ3(ev5).

[0171] Provided herein are prokaryotic cells comprising a genome at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identical to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The prokaryotic cell may be a bacterium, such as E. coli. In some embodiments, the calculation of the percent sequence identity excludes any sequences inserted to further modify the cell. The calculation of the percent sequence identity may further exclude any exogenous sequences further introduced into the genome, such as any additional tRNAs, selectable markers, modifications to genes required for viability according to the present disclosure, constructs for industrial expression of peptide or protein products, etc.

[0172] cell type The cells of the present disclosure, including all embodiments disclosed herein, can be prokaryotic cells. Bacterial cells can be of any species suitable for heterologous protein production, particularly for the production of polypeptides. Suitable bacterial host cells include Escherichia coli (e.g., Escherichia coli), Caulobacter (e.g., Caulobacter crescentus), photosynthetic bacteria (e.g., Rhodobacter sphaeroides), psychrophilic bacteria (e.g., Pseudoalteromonas haloplanktis, Shewanella species strain Ac10), Pseudomonas (e.g., Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa), halophilic bacteria (e.g., Halomonas elongate, Chromohalobacter salexigens), and the like. salexigens), Streptomycetes (e.g., Streptomyces lividans, Streptomyces griseus), Nocardia (e.g., Nocardia lactamdurans), Mycobacteria (e.g., Mycobacterium smegmatis), Corynebacterium (e.g., Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum), Bacillus (e.g., Bacillus subtilis, Bacillus virevis), brevis, Bacillus megaterium, Bacillus licheniformisExamples of bacteria include Bacillus licheniformis, Bacillus amyloliquefaciens, Vibrio bacteria (e.g., Vibrio cholera, Vibrio natriegens), and Lactic acid bacteria (e.g., Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri). In some cases, the bacteria are gram-negative.

[0173] In particular examples, the bacterium is Escherichia coli, Salmonella enterica, or Shigella dysenteriae. More preferably, the cell is Escherichia coli. Suitable Escherichia coli cells include K-12, MG1655, BL21, BL21(DE3), AD494, Origami, HMS174, BLR(DE3), HMS174(DE3), Tuner(DE3), Origami2(DE3), Rosetta2(DE3), Lemo21(DE3), NiCo21(DE3), T7 Express, SHuffle Express, C41(DE3), C43(DE3), and m15 pREP4 or derivatives thereof (Rosano, GL and Ceccarelli, EA, 2014. Frontiers in microbiology, 5, p. 172). In particular, the cell may be MG1655 or BL21 or a derivative thereof. MG1655 is considered the wild-type strain of E. coli. The GenBank ID for the genome sequence of this strain is U00096. BL21 is widely available commercially. For example, it can be purchased from New England BioLabs under catalog number C2530H.

[0174] The cells may contain genomes that are from the same species or strain, or may be from different species, for example, if the cell is E. coli, the genome may be the E. coli genome.

[0175] The cells of the present disclosure, including all embodiments disclosed herein, may be biologically contained cells, whereby the cells of the present disclosure are only viable or capable of growing under conditions not found in nature. Such cells may be considered to comprise a biological containment system.

[0176] For example, cells can only survive or grow in the presence of a drug not found in their natural environment. Such cells can be cultured in the presence of the drug, but will not persist as a population of cells when placed in an environment lacking the drug. An example of such a drug includes unnatural amino acids that may be required for functional translation of one or more essential genes. Other examples include ligands required for expression or activity of essential genes / proteins. In another example, a cell may contain a gene that suppresses its ability to survive or proliferate, the gene being inactive in the presence of an agent not found in its natural environment. This gene is termed a "kill switch" and may, for example, encode a toxin.

[0177] Polymer production As disclosed herein, the cells of the present disclosure may be suitable for polymer production. Thus, in a seventh aspect of the present disclosure, there is provided a use of any of the cells disclosed herein for polymer production.

[0178] In one embodiment, a method for making a polymer is provided, the method comprising: culturing a cell disclosed herein, providing the cell comprising a nucleic acid sequence encoding the polymer, and obtaining the polymer.

[0179] The polymer can be a polypeptide. The polymer can be a heterologous protein. The polymer can include monomers that can be incorporated by tRNAs that charge canonical amino acids, natural amino acids, unnatural amino acids, beta amino acids, hydroxy acids, alpha hydroxy acids, etc.

[0180] More Information Sequence comparisons can be performed using readily available sequence comparison programs. These publicly and commercially available computer programs can calculate the sequence identity between two or more sequences.

[0181] Those skilled in the art understand how to calculate the percent identity between two nucleic acid sequences. To calculate the percent identity between two nucleic acid sequences, the alignment of the two sequences must first be prepared, followed by calculating the sequence identity value. The percent identity for two sequences can vary depending on: (i) the method used to align the sequences, such as the Needleman-Wunsch algorithm (e.g., as applied by Needle (EMBOSS) or Stretcher (EMBOSS), the Smith-Waterman algorithm (e.g., as applied by Water (EMBOSS)), or the LALIGN application (e.g., Matcher (EMBOSS)); and (ii) the parameters used by the alignment method, such as local versus global alignment, the matrix used, and the parameters applied to gaps.

[0182] Once aligned, there are many different ways to calculate the identity percentage between two sequences.For example, one can divide the number of identicals by: (i) the length of the shortest sequence; (ii) the length of alignment; (iii) the average length of sequences; (iv) the number of non-gap positions; or (iv) the number of equivalent positions excluding overhangs.It is also understood that identity percentage is also strongly dependent on length.Therefore, the shorter the sequence pair, the higher the sequence identity can be expected to occur by chance.

[0183] A calculation of the percentage identity between two nucleic acid sequences can then be calculated from the alignment such as (N / T)*100, where N is the number of positions at which the sequences share identical residues and T is the total number of positions being compared including gaps excluding overhangs.

[0184] The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In one example, the identity between two amino acid sequences may be calculated using the service Needle (EMBOSS) set to default parameters, such as matrix (BLOSUM62), gap initiation (10), gap extension (0.5), end gap penalty (false), end gap initiation (10), and end gap extension (0.5). In another example, the identity between two amino acid sequences may be calculated using the service Matcher (EMBOSS) set to default parameters, such as matrix (BLOSUM62), gap initiation (14), gap extension (4), and alternative matches (1). In one example, identity between two nucleic acid sequences can be calculated using the service Needle (EMBOSS) set to default parameters, such as matrix (DNAfull), gap initiation (10), gap extension (0.5), end gap penalty (false), end gap initiation (10), and end gap extension (0.5). In another example, identity between two nucleic acid sequences can be calculated using the service Matcher (EMBOSS) set to default parameters, such as matrix (DNAfull), gap initiation (16), gap extension (4), alternative matches (1).

[0185] Every feature described in this specification (including any accompanying claims, abstract and drawings) and / or every step of any method or process disclosed may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0186] For a better understanding of the present invention, and to show how embodiments of the present invention may be practiced, reference will now be made to examples which are not intended to limit the invention in any way.

[0187] Some embodiments of the present invention are defined by the following clauses.

[0188] 1. A cell comprising a genome in which at least a first type of sense codon has been recoded such that a first endogenous tRNA is unnecessary; The cells do not express the first endogenous tRNA; the cell expresses a first modified tRNA capable of decoding a first type of sense codon, the first modified tRNA being charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon; A cell, wherein the cell contains a gene necessary for survival, the gene contains at least one occurrence of a first type of sense codon, and the cell is viable if the first type of sense codon in the gene is decoded as a first amino acid.

[0189] 2. The cell of clause 1, wherein the cell is non-viable when a first type of sense codon in a gene required for viability is decoded according to the canonical genetic code, or wherein a first type of sense codon in a gene required for viability is decoded according to the canonical genetic code, and at least partially contributes to reduced viability.

[0190] 3. The cell of clause 1 or clause 2, wherein the gene required for survival is an essential gene or a positive selectable marker.

[0191] 4. The cell of any one of clauses 1-3, wherein the first amino acid is a naturally occurring amino acid.

[0192] 5. The cell of any one of clauses 1-4, wherein a second type of sense codon is recoded in the genome; the second endogenous tRNA may be unnecessary and the cell does not express the second endogenous tRNA; and the cell may express a second modified tRNA that can decode the second type of sense codon, wherein the second modified tRNA is charged with a second amino acid that is not the naturally cognate amino acid of the second type of sense codon.

[0193] 6. The cell of clause 5, wherein a gene required for viability contains at least one occurrence of a second type of sense codon, and the cell is viable if the second type of sense codon in said gene is decoded as a second amino acid.

[0194] 7. The cell of clause 6, wherein the cell is non-viable when the second type of sense codon in the gene required for viability is decoded according to the normal genetic code, or wherein the second type of sense codon contributes at least in part to reduced viability when decoded according to the normal genetic code.

[0195] 8. The cell of any one of clauses 5 to 7, wherein the second amino acid is a naturally occurring amino acid.

[0196] 9. The cell of any one of clauses 1 to 8, wherein the cell is viable when its genes are decoded by the modified tRNA and is non-viable when its genes are decoded at least in part according to the canonical genetic code.

[0197] 10. A cell having increased resistance to horizontal gene transfer or mobile genetic elements, wherein the cell has been modified to reassign at least one type of sense codon to an amino acid that is not associated with a sense codon in the normal genetic code, and wherein the cell contains a gene necessary for survival that is functional when decoded according to the reassigned genetic code and is not functional when decoded according to the normal genetic code.

[0198] 11. A cell comprising a genome in which a first type of sense codon and a second type of sense codon are recoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; the cell expresses a first anticodon-swapped tRNA derived from a naturally occurring first parent tRNA, the first anticodon-swapped tRNA being charged with a first amino acid, the first parent tRNA being an isoacceptor for the first amino acid, and the first amino acid is not the naturally cognate amino acid of the first type of sense codon; the cell expresses a second anticodon-swapped tRNA derived from a naturally occurring second parent tRNA, the second anticodon-swapped tRNA being charged with a second amino acid, the second parent tRNA being an isoacceptor for the second amino acid, and the second amino acid is not the naturally cognate amino acid of the second type of sense codon; A cell, wherein the first and / or second modified tRNA cannot decode any type of codon other than the first type of sense codon and / or the second type of sense codon.

[0199] 12. The first and second types of sense codons are normally decoded by the same tRNA or overlapping tRNAs due to wobble base pairing, and the first anticodon-swapped tRNA cannot decode any type of codon other than the first type of sense codon, and / or the second anticodon-swapped tRNA cannot decode any type of codon other than the second type of sense codon, and / or the first type of sense codon and the second type of sense codon have the formula XXN, the first anticodon-swapped tRNA cannot decode the second type of sense codon, and the second anticodon-swapped tRNA cannot decode the first type of sense codon; Article 11 Cells.

[0200] 13. The first amino acid and the second amino acid are different types of amino acids. A cell under Article 11 or Article 12.

[0201] 14. The cell of any one of clauses 11-13, wherein the first and second parent tRNAs are derived from the same cell type as the cell of clause 11; and wherein the first and / or second anticodon-swapped tRNAs optionally contain an identity element recognized by an aminoacyl-tRNA synthetase endogenous to the cell.

[0202] 15. The cell of any one of clauses 11-14, wherein the first and second types of sense codons normally encode serine, or the first and second types of sense codons normally encode alanine, or the first and second types of sense codons normally encode leucine.

[0203] 16. The cell of any one of clauses 11 to 15, wherein the first and / or second anticodon-swapped tRNA does not decode a TCC or TCT codon.

[0204] 17. The cell of any one of clauses 11-16, wherein the first and / or second amino acid is a naturally occurring amino acid, and the first amino acid may be any one of alanine, histidine, leucine, and proline; and / or the second amino acid may be any one of alanine, histidine, leucine, and proline.

[0205] 18. A cell comprising a genome in which a first type of sense codon and a second type of sense codon are recoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; the cell expresses a first modified tRNA capable of decoding a first type of sense codon, the first modified tRNA being charged with a first amino acid that is not the naturally cognate amino acid of the first type of sense codon; the cell expresses a second modified tRNA capable of decoding a second type of sense codon, the second modified tRNA being charged with a second amino acid that is not the naturally cognate amino acid of the second type of sense codon; i) the first amino acid is alanine and the second amino acid is alanine; ii) the first amino acid is alanine and the second amino acid is histidine; iii) the first amino acid is alanine and the second amino acid is leucine; iv) the first amino acid is alanine and the second amino acid is proline; v) the first amino acid is histidine and the second amino acid is alanine; vi) the first amino acid is histidine and the second amino acid is histidine; vii) the first amino acid is histidine and the second amino acid is leucine; viii) the first amino acid is histidine and the second amino acid is proline; ix) the first amino acid is leucine and the second amino acid is alanine; x) the first amino acid is leucine and the second amino acid is histidine; xi) the first amino acid is leucine and the second amino acid is proline; xii) the first amino acid is proline and the second amino acid is alanine; xiii) the first amino acid is proline and the second amino acid is histidine; xiv) the first amino acid is proline and the second amino acid is leucine; or xv) A cell, wherein the first amino acid is proline and the second amino acid is proline.

[0206] 19. The first modified tRNA cannot decode a second type of sense codon, and / or the second modified tRNA cannot decode a first type of sense codon, and / or 19. The cell of clause 18, wherein the first modified tRNA is incapable of decoding any type of codon other than the first type of sense codon, and / or the second modified tRNA is incapable of decoding any type of codon other than the second type of sense codon.

[0207] 20. The cell of clause 18 or clause 19, wherein the first modified tRNA is an anticodon-swapped tRNA that is normally associated with a first amino acid, and / or the second modified tRNA is an anticodon-swapped tRNA that is normally associated with a second amino acid.

[0208] 21. The first modified tRNA is derived from a tRNA that is endogenous to the cell and is an isoacceptor for the first amino acid, or is derived from a tRNA found in a mobile genetic element and is an isoacceptor for the first amino acid; and / or the second modified tRNA is derived from a tRNA that is endogenous to the cell and is an isoacceptor for the second amino acid, or is derived from a tRNA that is found in a mobile genetic element and is an isoacceptor for the second amino acid; and / or 21. The cell of any one of clauses 18-20, wherein the first and / or second modified tRNA comprises an identity element recognized by an aminoacyl-tRNA synthetase endogenous to the cell.

[0209] 22. The cell of any one of clauses 18 to 21, wherein the first and second types of sense codons normally encode serine.

[0210] 23. The cell of any one of clauses 18 to 22, wherein the first type of sense codon is TCA and / or the second type of sense codon is TCG.

[0211] 24. A cell according to any preceding clause which is a prokaryotic cell, a bacterial cell or an E. coli cell.

[0212] 25. A method for increasing the resistance of a cell to mobile genetic elements or horizontal gene transfer, wherein the cell is modified to reassign at least one type of sense codon to an amino acid that is not associated with a sense codon in the normal genetic code, comprising: the method comprising modifying a gene required for survival to include at least one occurrence of a reassigned sense codon; the cell is viable if the reassigned sense codon in said gene is decoded as the reassigned amino acid; A method wherein the reassigned sense codons in the gene, when decoded according to the normal genetic code, cause the cell to be non-viable, or the reassigned sense codons in the gene, when decoded according to the normal genetic code, contribute at least in part to reduced survival. [Example]

[0213] overview A near-universal genetic code defines the correspondence between codons in genes and amino acids in proteins. It is widely hypothesized that refactoring the structure of the genetic code could generate organisms with novel properties and create genetic firewalls that limit the leakage of genetic information from synthetic organisms. However, it has not been possible to test these hypotheses. Here, we create a refactored genetic code / decoder system that—unlike code-compressed organisms—exhibits semantic and functional orthogonality with respect to the code / decoder system for the regular code. We thereby create an orthogonal, and mutually orthogonal, horizontal gene transfer system that allows the transfer of genetic information between organisms using the same genetic code but restricts the transfer of genetic information between cells using different genetic codes. Furthermore, we show that locking the orthogonal code into a synthetic organism completely blocks invasion by mobile genetic elements that successfully invade code-compressed organisms.

[0214] To elaborate, we demonstrate that code-compressed genes are read in natural cells, such that code compression cannot limit gene leakage from engineered organisms into the biosphere. Furthermore, we demonstrate that mobile genetic elements using the canonical genetic code and carrying the tRNA decoders necessary to complement tRNAs not present in recipient cells can enter Syn61Δ3 cells. We reassign sense codons to alternative canonical amino acids in Syn61Δ3, thereby refactoring the structure of the genetic code; we create 16 refactored codes with properties not found in nature. We demonstrate that code reassignment enables the creation of synthetic genes written in novel codes that are read correctly in synthetic organisms using their cognate decoders but are read incorrectly in natural cells. We also demonstrate that genes written in canonical codes that are read correctly in natural organisms are read incorrectly in synthetic organisms. The genetic code-decoder system in synthetic organisms exhibits semantic and functional orthogonality with respect to the code-decoder system for the canonical code. We exploit this orthogonality to create an orthogonal, and mutually orthogonal, horizontal gene transfer system that allows horizontal transfer of genetic information between cells using the same genetic code but restricts horizontal transfer of genetic information between cells using different genetic codes. Furthermore, we show that locking the orthogonal code into synthetic organisms completely blocks invasion by mobile genetic elements that successfully invade code-compressed organisms. [Example]

[0215] The compressed code is non-orthogonal The spectinomycin resistance gene (SpecR WT) written in the canonical genetic code was correctly read in cells containing the full complement of tRNAs for reading the canonical code, conferring spectinomycin resistance to WT cells (Syn61 WT). However, consistent with previous observations (W.E. Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)), SpecR WT did not confer spectinomycin resistance to Syn61Δ3 cells because Syn61Δ3 does not read all codons in the canonical genetic code (Figure 1).

[0216] We created a spectinomycin resistance gene (recSpecR(ΔTCG,TCA)) written using the compressed genetic code we used to create Syn61 (TCG and TCA codons were replaced with AGC and AGT, respectively, and the TAG stop codon was replaced with TAA). recSpecR(ΔTCG,TCA) conferred spectinomycin resistance to Syn61Δ3 cells, which use the same codon compression scheme as recSpecR(ΔTCG,TCA) in their genomes (Figure 1). The recSpecR(ΔTCG,TCA) gene also conferred spectinomycin resistance to cells reading the regular genetic code; this was predicted because the compressed genetic code uses a subset of codons used in the regular genetic code. We obtained similar observations using codon-compressed and wt-hygromycin resistance genes (Figure S1).

[0217] These experiments demonstrated that genetic information written in the regular code can be read in WT cells but not in cells that are genome-wide code-compacted and lack the cognate tRNA. However, code-compacted genes can be read both in cells whose genomes are code-compacted and lack the cognate tRNA, and in WT cells. The codons used in the compacted genetic code are not orthogonal with respect to the tRNA decoder in WT cells. Therefore, there are no barriers limiting the genetic information from engineered biological cells—using the compacted genetic code—to being read by natural forms of life that use the regular code. Creating orthogonal genetic codes with effective barriers that limit the transfer of genetic information from engineered biological systems to natural systems remains an important and unaddressed challenge. [Example]

[0218] tRNAs enable the invasion of codon-compressed organisms The WT F plasmid (F(WT)), which uses the canonical genetic code, was efficiently transmitted to WT cells. In contrast, as expected, F(WT) was not transmitted to Syn61Δ3 (Figure 1). However, in selecting for the conjugation of F(WT) from WT cells to Syn61Δ2 cells (Syn61 cells lack serU and serT but contain prfA), we obtained two viable colonies in which the recipient cells had received F(WT) (Figure S2). These colonies represented rare events and were 10 times more numerous than the colonies resulting from the conjugation of F(WT) to WT cells. 6 This occurs at a frequency of 1 in 1. Sequencing of two clones revealed that they had acquired serT-containing sequences from donor cells. This provided direct experimental evidence that selection for the propagation of mobile genetic elements that use the canonical code into cells allows selection for the tRNA genes necessary to read the canonical genetic code.

[0219] To track the effects of introducing serT into recipient cells in a reproducible system, we generated a mobile genetic element, F(WT+serT), a variant of F(WT) that contains serT. We demonstrated that F(WT+serT) can propagate in Syn61Δ3 cells and that this propagation is dependent on serT (Fig. 1). We concluded that acquisition of serT is sufficient to circumvent the genetic isolation caused by codon compression and cognate tRNA deletion in Syn61Δ3. These experiments highlight the significant challenge of generating a system that actively blocks invasion by mobile genetic elements carrying their own decoders. [Example]

[0220] Refactoring the code structure tRNA CGA Ser SerU, and tRNA encoding UGA Ser serT, encoding Ala (tRNA αα), both decodes TCG and TCA codons and incorporates serine into proteins in Syn61Δ3 (Figure S3). To reassign the TCG and TCA codons to distinct natural amino acids in Syn61Δ3, we generated variants of isoacceptor tRNAs for the canonical amino acids; for each isoacceptor, we changed the anticodon to CGA or UGA. We measured the activity of these chimeric tRNAs for decoding the TCG or TCA codon at position 3 (a known permissive site) of the sfGFP gene in Syn61Δ3 (Figure S3). We also measured the activity of Ala (tRNA αα). CGA Ala , tRNA UGA Ala ), His(tRNA CGA His , tRNA UGA His ), Leu(tRNA CGA Leu , tRNA UGA Leu ) and Pro(tRNA CGA Leu, tRNA UGA Leu We found that chimeric tRNAs for sfGFP and ubiquitin specifically directed the incorporation of amino acids defined by the parent isoacceptor tRNA in response to their cognate codons (TGC or TCA) at position 3 of sfGFP or position 11 of ubiquitin in Syn61Δ3 (Figures 2, 8, 12-21), producing good yields of protein (Figure S3). UGA Leu We note that the fidelity of tRNAs was lower than that of other tRNAs (Figure 13). Alanine and leucine tRNAs were investigated because their anticodons are not identity elements for their cognate aminoacyl-tRNA synthetases and therefore they were predicted to be tolerant to anticodon mutations; other tRNAs were identified using screening (Figure 22). We have identified tRNAs CGA Ser and tRNA UGA Ser Unlike other chimeric tRNAs, our chimeric tRNAs specifically decode the Watson-Crick complement of their anticodon sequences; e.g., tRNA CGA Ala decodes the TCG codon rather than the TCA codon, and tRNA UGA Ala We also found that Syn61Δ3 decodes the TCA codon rather than the TCG codon (Figure S3, Figure S13). These tRNAs are also specific for other TCN codons (Figures S14, S15), and most reassigned strains grew as well as the parent strain (Figure S23). Therefore, we independently reassigned the TCA and TCG codons to Ala, His, Leu, or Pro in Syn61Δ3, thereby generating 16 novel genetic codes (Figure S3, Data File S1, Figure 2B). In each novel genetic code, we changed the identity of the canonical amino acid encoded by a specific sense codon relative to the canonical code and the other 15 codes we created (Figure 2C).

[0221] Overall, we have refactored the structure of the genetic code. Our new genetic code increases the number of codons used to encode Ala and Pro (from four to six), doubles the number of codons used to encode His (from two to four), and increases the number of codons used to encode Leu (from six to eight); this is more codons than are used to encode any amino acid in the canonical code. These experiments also show that the UCN codon box, which encodes serine in the canonical code, can be split to encode additional canonical amino acids. [Example]

[0222] Orthogonal code and orthogonal decoder pair Genes written using the normal genetic code, in which the TCG and TCA codons encode serine, will make the correct protein product in natural cells that read these codons as serine. However, these genes will produce incorrect—and possibly non-functional—protein products in cells that decode these codons to incorporate amino acids other than serine.

[0223] Similarly, synthetic genes—in which we use the Syn61 recoding scheme to compress the genetic code and replace codons with TCG and TCA codons for specific natural amino acids—make the correct protein product in cells that decode the TCG and TCA codons to incorporate the correct amino acid, but these synthetic genes produce incorrect—and likely nonfunctional—protein products in cells that read the natural genetic code ( FIG. 3 ).

[0224] We converted all 27 GCN codons (which encode alanine in the canonical code) to TCG codons and all 6 CAT / C codons (which encode histidine in the canonical code) to TCA codons in recSpecR(ΔTCG,TCA). This created the orthogonal resistance gene O-SpecR(TCG-Ala,TCA-His). We also used Syn61Δ3(tRNA CGA Ala , tRNA UGA His We demonstrated that O-SpecR (TCG-Ala, TCA-His) can be decoded in Syn61 cells and confer spectinomycin resistance to them. We further demonstrated that O-SpecR (TCG-Ala, TCA-His) did not confer spectinomycin resistance to Syn61 WT cells, in which TCG and TCA are decoded as Ser, as in the canonical genetic code. Finally, we demonstrated that SpecR WT, in which serine is encoded using TCG and TCA codons, conferred spectinomycin resistance to Syn61Δ3 (tRNA CGA Ala , tRNA UGA His ) cells (Fig. 3). We extended this approach to five other reassignment schemes and other genes (Fig. 9). We obtained similar results using the wt hygromycin resistance gene and a hygromycin resistance gene in which all Ala codons were converted to TCG and all histidine codons were converted to TCA (O-HygR(TCG-Ala, TCA-His)) (Fig. S4B).

[0225] These experiments demonstrated that we can create genetic code-decoder pairs for synthetic genes that are functionally orthogonal with respect to the canonical genetic code-decoder pairs for natural genes. The orthogonal codes (TCG-Ala, TCA-His) written in the synthetic genes are functionally orthogonal to the orthogonal decoders (tRNA CGA Ala , tRNA UGAHis ), but is correctly read by the regular decoder (tRNA UGA Ser The canonical code written in the native gene (TCG-Ser, TCA-Ser) is correctly read by the canonical decoder, but not by the orthogonal decoder.

[0226] The functional orthogonality of genes in cells with altered decoders depends on the frequency of rearranged codons and the functional consequences of the codon rearrangements. The consequences of amino acid substitutions—the results of codon rearrangements—can be globally and roughly correlated with differences in amino acid polarity and hydrophobicity (EV Koonin, AS Novozhilov, Origin and evolution of the genetic code: the universal enigma. IUBMB Life 61, 99-111 (2009); M. Schmidt, V. Kubyshkin, How To Quantify a Genetic Firewall? A Polarity-Based Metric for Genetic Code Engineering. Chembiochem 22, 1268-1284 (2021)).The consequences of amino acid substitutions at specific sites in proteins can be predicted using computational approaches that use evolutionary lineage and / or structural information (DS Marks, SW Michnick, Democratizing the mapping of gene mutations to protein biophysics. Nature 604, 47-48 (2022); S. Teng, AK Srivastava, CE Schwartz, E. Alexov, L. Wang, Structural assessment of the effects of amino acid substitutions on protein stability and protein-protein interaction. Int J Comput Biol Drug Des 3, 334-349 (2010); V. Parthiban, MM Gromiha, D. Schomburg, CUPSAT: prediction of protein stability upon point mutations. Nucleic Acids Res 34, W239-242 (2006); PC Ng, S. Henikoff, Predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet 7, 61-80 (2006)). While the composition of natural genes is fixed, the codon usage in synthetic genes written in standard or any orthogonal code can be easily designed to maximize the number of codons available for reassignment, thereby maximizing the functional orthogonality of the synthetic gene. [Example]

[0227] Orthogonal horizontal gene transfer Next, based on the orthogonal genetic code-decoder pair, we created an orthogonal horizontal gene transfer (O-HGT) system consisting of an orthogonal decoder and a mobile genetic element that uses the orthogonal genetic code. WT cells can transfer WT mobile genetic elements among themselves, but cannot transfer WT mobile genetic elements to cells containing the orthogonal decoder. Cells containing the O-HGT system can transfer their mobile genetic elements to cells containing a compatible orthogonal decoder, but cannot transfer their mobile genetic elements to cells containing an incompatible orthogonal decoder or to WT cells.

[0228] A mobile genetic element (F plasmid, F(WT)) using the canonical genetic code was propagated into WT cells (Syn61 WT) as expected. We found that F(WT) is a tRNA plasmid containing Syn61Δ3 (tRNA Δ3), in which the TCG codon is read as Ala and the TCA codon is read as His. CGA Ala , tRNA UGA His ) cells (Fig. 4).

[0229] Next, we investigated horizontal gene transfer using mobile genetic elements containing altered genetic codes. We synthesized the mobile genetic element O-F1 (TCG-Ala, TCA-His). The genetic codes in all annotated translation regions of this F plasmid were compressed using the Syn61 scheme, and the GCN codon (encoding alanine in the canonical coding) and CAT / C codon (encoding histidine in the canonical coding) were converted to TCG and TCA codons, respectively, within the trfA gene, which is essential for replication of the mobile genetic element.

[0230] O-F1 (TCG-Ala, TCA-His) is Syn61Δ3 (tRNA CGA Ala , tRNA UGA His) cells. We further demonstrated that O-F1 (TCG-Ala, TCA-His) is not horizontally transmitted to cells that read the normal genetic code (Figure 4). These experiments demonstrated that we were able to create an O-HGT system.

[0231] Next, we created a mutually orthogonal HGT system that is orthogonal to the natural gene system and to each other. We created a novel mobile genetic element, O-F2 (TCG-His, TCA-Ala). The genetic code in all annotated translation regions of this F plasmid was compressed using the Syn61 scheme, and the GCN codon (which encodes alanine in the canonical coding) and the CAT / C codon (which encodes histidine in the canonical coding) were also converted to TCA and TCG codons, respectively, within the trfA gene.

[0232] The present inventors have demonstrated that O-F2 (TCG-His, TCA-Ala) is a tRNA decoded by Syn61Δ3 (TCG-His, TCA-Ala), in which TCG is decoded as His and TCA is decoded as Ala. CGA His , tRNA UGA Ala In contrast, O-F2 (TCG-His, TCA-Ala) was not propagated into Syn61 cells, which use the normal genetic code to decode the TCG and TCA codons as Ser. O-F2 (TCG-His, TCA-Ala) was not propagated into Syn61Δ3 (tRNA CGA Ala , tRNA UGA His Furthermore, we found that the WT mobile genetic elements (F (WT; TCG-Ser, TCA-Ser) and O-F1 (TCG-Ala, TCA-His) were not propagated in Syn61Δ3 (tRNA CGA His , tRNA UGA Ala) cells (Figure 4). Further experiments demonstrating the HGT system are illustrated in Figure 24. Overall, we have demonstrated the scalability of our approach through the creation of five mutually orthogonal horizontal gene transfer systems.

[0233] These experiments demonstrated that we were able to create orthogonal and mutually orthogonal HGT systems. [Example]

[0234] Orthogonal code locking blocks intrusive codes We hypothesized that replacing codons for specific natural amino acids in essential genes with TCA and TCG codons and adding tRNAs that reassign these codons to specific natural amino acids (Figure 5) would disrupt the serT-mediated HGT we observed in Syn61Δ3 (Figure 1).

[0235] tRNA CGA Ala and tRNA UGA His In recipient cells, tRNAs are expressed in a manner that reduces the production of functional proteins from mobile genetic elements. UGA Ser Since it competes with Syn61Δ3 (tRNA CGA Ala , tRNA UGA His , O-SpecR (TCG-Ala, TCA-His)) was prevented in the absence of spectinomycin (10 4However, this disruption was insufficient to completely eliminate the propagation of mobile genetic elements. Addition of spectinomycin, which renders O-SpecR (TCG-Ala, TCA-His) essential in cells, makes the decoding of TCG codons as alanine and TCA codons as histidine essential. Under these conditions, the propagation of mobile genetic elements was completely eliminated (Figure 5). Similar results were obtained with other refactored codes and other essential genes (Figures 25 and 26).

[0236] To extend our approach to viral infection, we identified pools of Kam River phage capable of infecting Syn61Δ3 (Methods). From these pools, we identified phage with identical tRNA sequences. UGA Ser We isolated two distinct phages (12 and 06, both T-4-like phages) that carry the gene and infect Syn61Δ3 (Figure 27); it is well known that some viruses carry their own tRNAs and other translation factors to augment the cellular pool of translation factors and assist in the translation of codons within their own genes. As expected, expression of this tRNA in Syn61Δ3 is sufficient to confer susceptibility to infection by the (otherwise noninfectious) T4 phage (Figure 28). We demonstrated that, unlike Syn61Δ3, several refactored, code-locked strains were completely resistant to infection with phage 6 and phage 12 (Figures 5, 29).

[0237] Our results demonstrate that writing essential genes in orthogonal code and reading these genes with cognate orthogonal decoders creates cells locked into the orthogonal code, which resist invasion by mobile genetic elements using competing codes. [Example]

[0238] Genetic code-locking enables stable phage resistance in synthetic organisms The experiments discussed in the previous examples show that seryl-tRNA (tRNA Ser UGA We identified naturally occurring phage encoding Syn61Δ3 and showed that such phage could infect Syn61Δ3. These experiments also show that we were able to eliminate infection by these phage using codelocking (Figure 34). However, we also show that reassignment is sufficient to eliminate plaque formation, as opposed to conjugative spread. Further experiments were performed to determine the reason for this.

[0239] We theorize that genomic differences may explain the phenotypic differences; one possible explanation is the number of TCG and TCA codons in each genome. The genome of the phage investigated here is considerably larger than F'(WT+serT), with the total number of target codons more than three-fold greater (Fig. 30a). Because more positions are affected by amino acid misincorporation, deleterious effects are even more likely.

[0240] This may be due to the genomic frequency of the target codon. Compared to F'(W+serT), the phage genomes show an approximately 25% increase in the frequency of the target codon in their genomes (Figure 30b). With the increased frequency of amino acid misincorporation, the likelihood of the translated region being translated non-functionally increases.

[0241] These two effects may add up. Not only are many more genes affected in phage genomes, but these genes are affected to a greater extent on average. Therefore, we predict that codon reassignment has a greater effect on phage infection than on conjugative spread. While in principle it could also be the relative usage of TCG and TCA codons in the genomes of phages 12, 6, and F'(WT+serT), we believe this is unlikely because reassigning both of these codons to leucine results in differences between phage infection and conjugative spread.

[0242] Mechanistic differences may explain the phenotypic differences. The two modes of horizontal gene transfer, conjugative transfer and phage infection, are essentially different processes. For successful phage infection and plaque formation, the entire phage life cycle must be completed. This includes cell attachment, viral genome injection, viral protein production, phage genome replication, and phage particle maturation (Figure 30c). Plaques can only form if mature phage particles are formed and successfully infect neighboring cells. Completion of the entire life cycle is always a complex process requiring multiple parts to function together under strict temporal control. For T-4-like phages, such as phages 12 and 6, multiple genes are essential for this process. Deletion of one of these genes results in a loss of plaque formation.

[0243] In contrast, conjugative transfer and subsequent colony formation is a simpler process. After donor cell attachment to the recipient, ssDNA is transferred to the recipient through the conjugative channel. In the recipient cell, the DNA is then recircularized to form a stable dsDNA plasmid. Importantly, all proteins involved in this process are either expressed from the recipient cell genome or transferred from the donor along with the DNA. Subsequently, only replication of the plasmid and its proper segregation during cell division need be ensured for successful colony formation (Figure 30d). This process requires functional expression of very few genes from the conjugative elements.

[0244] Decoding ambiguity through codon reassignment is predicted to be even more detrimental to phage infection, since more genes need to be functionally expressed from the horizontally transmitted DNA for successful plaque formation. If disruption of one essential gene is sufficient so that the product is not functional, plaque formation is eliminated.

[0245] A further explanation is dominant-negative effects resulting from ambiguous decoding of certain genes. Dominant-negative effects are more likely for proteins that form complex interactions. Some mutations in viral envelope proteins are known to exhibit dominant-negative phenotypes. Mutations in envelope subunits likely disrupt oligomerization and correct particle assembly. In T-4-like phages, the major capsid protein (gp23) forms a hexamer that is the basis for particle assembly. In phages 06 and 12, there are three surface-exposed serine residues encoded by TCA (Figure 32). Misincorporation of an amino acid at one of these positions in a subset of gp23 can disrupt capsid assembly, thereby having a dominant-negative effect on plaque formation.

[0246] The inventors realized that an advantage of code locking is the long-term maintenance of alternative genetic codes. The tRNAs involved in code refactoring can be inactivated through various mechanisms, e.g., mutation, deletion, or silencing. This essentially reverts cells with the refactored genetic code back to codon-compressed cells with the original decoder deletion (e.g., Syn61Δ3), making them susceptible to infection by phages carrying the appropriate tRNA genes. However, once the code is locked, the tRNAs involved in refactoring are essential and cannot be inactivated. This ensures the stability of the refactored code over time and up-to-date phage resistance.

[0247] We modeled the stability of alternative genetic codes in the presence and absence of code locking. tRNAs involved in alternative decoding of TCG and TCA codons were encoded on a low-copy-number plasmid carrying hygromycin resistance. A second plasmid carried the spectinomycin resistance gene (Spec R ) variants; respectively, for cells without coding: recSpec R(ΔTCG, ΔTCA), for cells with codelocking: oSpec R (TCG: Ala, TCA: His) and oSpec R (TCG:Leu, TCA:Leu). Cells were serially passaged in the presence of spectinomycin and in the absence of hygromycin (no endogenous pressure to maintain the plasmid). At each passage, we measured the percentage of cells maintaining the tRNA-encoding plasmid. We found that code locking acts to stabilize the alternative code and maintain the code (Figure 31a).

[0248] As a result, code-locked cells retain phage resistance for extended periods, while unlocked cells lose resistance. We exposed cells with and without locked codes to phages 12 and 06 over the time course described above. We observed that cells with locked genetic codes retained resistance to phage infection, while cells that had lost the tRNAs involved in code refactoring were susceptible to phage infection (Figure 31b).

[0249] These experiments also show that plasmids can be stably maintained by making them essential to the host based on their genetic code, for example, by their requirement for tRNAs and essential genes on the plasmid, which may find utility in circumventing antibiotic remittance in biotechnological applications. [Example]

[0250] Phage proliferation assay OD of cells from overnight cultures 600The cells were diluted to approximately 0.3 and inoculated with phage 12 (MOI = 0.001). After 24 hours of incubation in a volume of 3 mL (2 × ty), the phage titer was assessed by serial dilution (7.5 μL spots on a layer of top agar) and plaque formation assay with a permissive strain (Syn61 WT). The control was empty medium (2 × ty) without cells. The detection limit for this assay is one plaque per 7.5 μL (133.3 PFU / mL).

[0251] The results, shown in Figure 33, demonstrate that phage 12 replicates successfully in Syn61WT and Syn61Δ3 cells, but not in the refactored, code-locked cells (Syn61Δ3(alaTCGA, hisRtga) and Syn61Δ3(leuQcga, leuQtga)). Compared to the cell-free control (ctrl.), phage propagation in code-locked cells resulted in lower phage titers, likely because the phage adsorbs to these cells but is unable to replicate. Experiments were performed in biological triplicate.

[0252] Consideration We created 16 synthetic genetic codes; in each novel codon, a subset of sense codons was reassigned to amino acids different from the canonical code. Code reassignment refactors the structure of the genetic code and directly alters the number and types of amino acids that can be evaluated by point mutation (figs. S5 and S6). Previous experimental studies have shown that although the choice of synonymous codons in individual genes and viruses can alter their robustness and evolvability (BA Renda, MJ Hammerling, JE Barrick, Engineering reduced evolutionary potential for synthetic biology. Mol Biosyst 10, 1668-1678 (2014); G. Moratorio et al., Attenuation of RNA viruses by redirecting their evolution in sequence space. Nat Microbiol 2, 17088 (2017); JR Coleman et al., Virus attenuation by genome-scale changes in codon pair bias. Science 320, 1784-1787 (2008)), such approaches are limited to exploring a subset of canonical codons.Despite extensive theoretical research, the number of in vitro experiments examining the relationship between the structure of the genetic code and its robustness and evolvability has been limited (G. Pines, J.D. Winkler, A. Pines, R.T. Gill, Refactoring the Genetic Code for Increased Evolvability. mBio 8, (2017); J. Calles, I. Justice, D. Brinkley, A. Garcia, D. Endy, Fail-safe genetic codes designed to intrinsically contain engineered organisms. Nucleic Acids Res 47, 10439-10451 (2019)). Therefore, it has been impossible to experimentally investigate the resulting hypotheses in living cells. Refactoring the structure of the genetic code offers new opportunities to experimentally explore how altered codes affect the robustness and evolvability of protein and cellular functions. In future studies, we aim to utilize genetic code refactoring to accelerate directed evolution.

[0253] We experimentally demonstrated the creation of semantic orthogonality between organisms that use distinct reassignment codes. We clearly demonstrated that semantic orthogonality creates functional orthogonality for the genes examined; mismatches between the genetic code used to write genes and the decoder used to read them result in mis-synthesized proteins that are non-functional.

[0254] The inventors have created multiple mutually orthogonal HGT systems in which genes are correctly read and propagated only by cells with their cognate decoders. Each cell type, with a distinct code-decoder system, executes a distinct, refactored genetic code. These systems enable experimental investigation of the role of HGT in fixing a universal genetic code through competition between pools of genotypes written in different codes (K. Vetsigian, C. Woese, N. Goldenfeld, Collective evolution and the genetic code. Proc Natl Acad Sci USA 103, 10696-10701 (2006)).

[0255] Shielding synthetic organisms from environmental genetic elements could be valuable for industrial-scale biotechnology applications, where contamination with mobile genetic elements, including viruses, could cause economic losses and disrupt essential supply chains (PW Barone et al., Viral contamination in biologic manufacture and implications for emerging therapies. Nat Biotechnol 38, 563-572 (2020)). The resistance of natural genes to genome code compaction and horizontal transfer to organisms with tRNA deletions can be circumvented by reacquiring the deleted tRNAs and by mobile genetic elements carrying these tRNAs. Indeed, mobile genetic elements, including viruses, possess their own tRNAs and other translation factors that augment the cellular pool of translation factors and assist in the translation of codons within their own genes (AL Borges et al., Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes. Nat Microbiol 7, 918-927 (2022); P. Alamos et al., Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium. RNA Biol 15, 518-527 (2018); T. Tuller et al., Association between translation efficiency and horizontal gene transfer within microbial communities. Nucleic Acids Res 39, 4743-4755 (2011)).

[0256] Synthetic organisms with essential genes written in orthogonal genetic code and decoders that correctly read the orthogonal code confer complete resistance to the spread of mobile genetic elements written in the canonical code, even when the mobile genetic elements contain tRNAs that enable the cell to correctly read the canonical code. This defines a paradigm for creating organisms that actively resist invasion by foreign codes.

[0257] Novel strategies to limit the transmission of genetic information from synthetic organisms to natural organisms could form the basis of a genetic firewall that isolates synthetic genetic systems from the environment. This is an important complementary challenge to managing the survival and proliferation of synthetic organisms for biocontainment, especially when considering the use of engineered organisms outside the laboratory (JW Lee, CTY Chan, S. Slomovic, JJ Collins, Next-generation biocontainment systems for engineered organisms. Nat Chem Biol 14, 530-537 (2018)). All compressed genetic codes are subsets of natural codes and are correctly read by full-code decoders; genetic systems written in compressed genetic codes are correctly read by natural organisms. Therefore, compressed genetic codes cannot be used to genetically isolate synthetic organisms from natural organisms. The ability to refactor the structure of the genetic code and write genes that are correctly read in synthetic organisms but incorrectly read in natural organisms provides the basis for a powerful strategy to disrupt the transmission of genetic information from synthetic organisms to natural organisms. Importantly, this strategy is generally applicable to any gene or genetic system in addition to synthetic organisms: given the near-universal conservation of the genetic code, we anticipate that the principles we have established can be applied to a wide range of other organisms.

[0258] References and Notes 1. F. H. Crick, L. Barnett, S. Brenner, R. J. Watts-Tobin, General nature of the genetic code for proteins. Nature 192, 1227-1232 (1961). 2. M. W. Nirenberg, J. H. Matthaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A 47, 1588-1602 (1961). 3. R. J. Hall, F. J. Whelan, J. O. McInerney, Y. Ou, M. R. Domingo-Sananes, Horizontal Gene Transfer as a Source of Conflict and Cooperation in Prokaryotes. Front Microbiol 11, 1569 (2020). 4. K. Vetsigian, C. Woese, N. Goldenfeld, Collective evolution and the genetic code. Proc Natl Acad Sci U S A 103, 10696-10701 (2006). 5. D. de la Torre, J. W. Chin, Reprogramming the genetic code. Nat Rev Genet 22, 169-184 (2021). 6. E. V. Koonin, A. S. Novozhilov, Origin and evolution of the genetic code: the universal enigma. IUBMB Life 61, 99-111 (2009). 7. M. Kollmar, S. Muhlhausen, Nuclear codon reassignments in the genomics era and mechanisms behind their evolution. Bioessays 39, (2017). 8. J. Ling et al., Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria. Nucleic Acids Res 42, 499-508 (2014). 9. A. L. Borges et al., Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes. Nat Microbiol 7, 918-927 (2022). 10. M. A. Santos, A. C. Gomes, M. C. Santos, L. C. Carreto, G. R. Moura, The genetic code of the fungal CTG clade. C R Biol 334, 607-611 (2011). 11. D. J. Taylor, M. J. Ballinger, S. M. Bowman, J. A. Bruenn, Virus-host co-evolution under a modified nuclear genetic code. PeerJ 1, e50 (2013). 12. Y. Shulgina, S. R. Eddy, A computational screen for alternative genetic codes in over 250,000 genomes. Elife 10, (2021). 13. D. G. Gibson et al., Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215-1220 (2008). 14. D. G. Gibson et al., One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci U S A 105, 20404-20409 (2008). 15. J. Fredens et al., Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514-+ (2019). 16. F. J. Isaacs et al., Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348-353 (2011). 17. M. J. Lajoie et al., Genomically recoded organisms expand biological functions. Science 342, 357-360 (2013). 18. W. E. Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021). 19. G. Pines, J. D. Winkler, A. Pines, R. T. Gill, Refactoring the Genetic Code for Increased Evolvability. mBio 8, (2017). 20. J. Calles, I. Justice, D. Brinkley, A. Garcia, D. Endy, Fail-safe genetic codes designed to intrinsically contain engineered organisms. Nucleic Acids Res 47, 10439-10451 (2019). 21. M. Schmidt, V. Kubyshkin, How To Quantify a Genetic Firewall? A Polarity-Based Metric for Genetic Code Engineering. Chembiochem 22, 1268-1284 (2021). 22. D. S. Marks, S. W. Michnick, Democratizing the mapping of gene mutations to protein biophysics. Nature 604, 47-48 (2022). 23. S. Teng, A. K. Srivastava, C. E. Schwartz, E. Alexov, L. Wang, Structural assessment of the effects of amino acid substitutions on protein stability and protein protein interaction. Int J Comput Biol Drug Des 3, 334-349 (2010). 24. V. Parthiban, M. M. Gromiha, D. Schomburg, CUPSAT: prediction of protein stability upon point mutations. Nucleic Acids Res 34, W239-242 (2006). 25. P. C. Ng, S. Henikoff, Predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet 7, 61-80 (2006). 26. B. A. Renda, M. J. Hammerling, J. E. Barrick, Engineering reduced evolutionary potential for synthetic biology. Mol Biosyst 10, 1668-1678 (2014). 27. G. Moratorio et al., Attenuation of RNA viruses by redirecting their evolution in sequence space. Nat Microbiol 2, 17088 (2017). 28. J. R. Coleman et al., Virus attenuation by genome-scale changes in codon pair bias. Science 320, 1784-1787 (2008). 29. P. W. Barone et al., Viral contamination in biologic manufacture and implications for emerging therapies. Nat Biotechnol 38, 563-572 (2020). 30. P. Alamos et al., Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium. RNA Biol 15, 518-527 (2018). 31. T. Tuller et al., Association between translation efficiency and horizontal gene transfer within microbial communities. Nucleic Acids Res 39, 4743-4755 (2011). 32. JW Lee, CTY Chan, S. Slomovic, JJ Collins, Next-generation biocontainment systems for engineered organisms. Nat Chem Biol 14, 530-537 (2018).

[0259] Materials and Methods KK Throughout the specification, Syn61 WT refers to Syn61(ev2) (WE Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)), and Syn61Δ3 refers to Syn61Δ3(ev4) (WE Robertson et al., Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062 (2021)).

[0260] Gene recoding For all genes and plasmids used in experiments with Syn61Δ3-derived cells, the genetic code had to be compressed according to the Syn61 recoding rules (TCG and TCA codons were replaced with AGC and AGT, respectively, and the TAG stop codon was replaced with TAA). We recoded the translated regions as previously described for Syn61 (J. Fredens et al., Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514-+ (2019)). The plasmids used in this study are provided in data file S2 of Zurcher et al. "Refactored genetic codes enable bidirectional genetic isolation," Science, and are provided herein as Table 1.

[0261] Construction of tRNA plasmids for decoding TCG and TCA codons in Syn61Δ3 To incorporate amino acids in response to TCG and TCA codons, we used the pSC101-Kan and pSC101-Hyg plasmids (which confer resistance to kanamycin and hygromycin, respectively) into which we cloned genes encoding one or more relevant tRNAs. Because all tRNAs used in this study are acylated by endogenous E. coli aaRSs, no exogenous aaRSs were used. For tRNAs that incorporate amino acids other than serine in response to TCG and TCA codons, we designed genes in which the anticodons of the relevant isoacceptor tRNAs were replaced with CGA and UGA, respectively. We constructed pSC101-based tRNA plasmids using multi-fragment HiFi assembly. Two different constructs of tRNA plasmids were used: i) tRNAs were expressed under the lpp promoter, and the pSC101 backbone contained a pheS-HygR dual selection cassette expressed under the EM7 promoter, and ii) tRNAs were expressed using the native expression context of serT in the E. coli genome, and the pSC101 backbone contained a kan expression cassette expressed under the T3 promoter. R When two tRNAs were expressed from one plasmid, they were expressed as an operon using the intergenic region between the alaX and alaW tRNA genes in the E. coli genome. The backbone fragments were generated using PCR. tRNAs and tRNA operons were ordered as oligos (Merck) or gBlocks (IDT). All cloning was performed in Syn61Δ3.

[0262] Construction of recoded antimicrobial plasmids for evaluation of genetic code orthogonality To evaluate the functionality of antimicrobial genes encoded according to different genetic codes, we used pMB1-based plasmids containing codon-compressed antibiotic resistance genes into which we cloned genes encoding hygromycin or spectinomycin resistance (Data File S2). We constructed pMB1-based tRNA plasmids using HiFi assembly of multiple fragments. Backbone fragments were generated using PCR. Recoded spectinomycin and hygromycin resistance genes were ordered as gBlocks (IDT).

[0263] Construction of recoded mobile genetic elements The intermediate, F(ΔTCG,TCA,TAG), was constructed from synthetic DNA (TWIST Bioscience) using yeast assembly (W.E. Robertson et al., Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS. Nat Protoc 16, 2345-2380 (2021)). F(ΔTCG,TCA,TAG) was designed by recoding all annotated coding regions in the RK2 conjugative plasmid as previously described (J. Fredens et al., Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514-+ (2019)). Recoding of trfA to generate O-F1 (TCG-Ala, TCA-His) and O-F2 (TCG-His, TCA-Ala) was performed by Lambda Red recombination (KC Murphy, Lambda Recombination and Recombineering. EcoSal Plus 7, (2016)). Recoded versions of trfA were synthesized as gBlocks (IDT). All modifications were performed in E. coli Dh10B. To enable replication of F plasmids lacking the trfA gene or encoding trfA in a genetic code not decoded by Dh10B, a pMB1-based helper plasmid was used, expressing WT trfA in the endogenous context in the RK2 conjugative plasmid. This plasmid was used in the amplicon of ... R amp expressed under the promoter R It contains the gene and was assembled by HiFi assembly from fragments generated by PCR.

[0264] sfGFP expression measurement We expressed the sfGFP-His6 gene containing a single TCG or TCA codon at position 3 in Syn61Δ3 cells harboring a plasmid encoding a tRNA or tRNA operon. We electroporated 50 μL of Syn61Δ3 cells with the pBAD_sfGFP reporter plasmid (100 ng) and allowed the cells to recover in 1 mL of SOB for 90 minutes at 37°C with shaking at 1050 rpm. We then inoculated the recovery culture (1 mL) into 5 mL of prewarmed 2xYT medium containing 50 μg / mL apramycin and incubated the cells overnight at 37°C with shaking at 220 rpm before preparing electrocompetent cells. We electroporated pSC101-based tRNA plasmids (100 ng) into Syn61Δ3 cells containing pBAD_sfGFP and allowed the cells to recover in 500 μL SOB for 90 minutes in a deep-well 96-well plate. We then inoculated one-tenth (50 μL) of the recovered culture into 450 μL of prewarmed 2xYT medium supplemented with 200 μg / mL hygromycin and 50 μg / mL apramycin. After 36 hours of recovery at 37°C and 750 rpm, we set up expression in a 96-well microtiter plate format and inoculated 1:50 of the overnight culture into 500 μL of prewarmed 2xYT medium containing hygromycin (200 ng / μL), apramycin (50 ng / μL), and L-arabinose (0.2%). The expression was incubated for 16 hours at 37°C with shaking at 750 rpm. The plate was centrifuged at 3200 g for 10 minutes. We resuspended the cell pellet in 150 μL of PBS and transferred 100 μL of the suspension to a Costar clear 96-well flat-bottom plate. We measured the OD of the plate using a PHERAstar FS plate reader (BMG LABTECH). 600 and GFP fluorescence (λ ex :485nm;λ em : 520 nm) measurements were recorded (gain setting 0, focus adjustment 00 mm).

[0265] To determine protein yield, sfGFP(WT) was expressed in Syn61Δ3 (16 h, 37°C, 5 mL of 2xTY + 0.2% arabinose) and purified as described below (three elutions of 100 μL each). Protein concentrations after purification were determined by nanodrop (elution 1: 0.77 mg / mL; elution 2: 0.09 mg / mL; elution 3: approximately 0.0 mg / mL). The measured amount was a total of 0.086 mg of protein extract from 5 mL of culture, corresponding to a protein yield of approximately 17 mg per L of culture.

[0266] Purification of sfGFP-His6× and ubiquitin-His6× proteins Syn61Δ3 cells harboring pSC101-based tRNA plasmids and pBAD_sfGFP (or ubiquitin) plasmids were grown for 16 hours in 5 mL (20 mL for ubiquitin) 2xYT medium containing 200 μg / mL hygromycin, 50 μg / mL apramycin, and 0.2% L-arabinose at 37°C with shaking at 220 rpm. After expression, cells were centrifuged and resuspended in 1 mL lysis buffer (1x Bugbuster Protein Extraction Reagent (Novagen), 1x PBS, 50 μg / mL DNase 1, 20 mM imidazole, and 100 μg / mL lysozyme) and incubated at 4°C for 1 hour. The resulting lysate was centrifuged (16,000 × g) for 30 minutes at 4°C. The supernatant was then lysed in 50 μL of Ni 2+ The Ni2+-NTA beads were transferred to a 1.5 mL microcentrifuge tube containing Ni2+-NTA slurry (Qiagen) and incubated for 1 h at 4°C with tumbling. The Ni2+-NTA beads were collected by gravity filtration on a fritted column and washed three times in 500 μL wash buffer (1× PBS, 40 mM imidazole). Finally, the protein was eluted in 100 μL elution buffer (1× PBS, 300 mM imidazole, pH 8) and collected in a new microcentrifuge tube using centrifugation (100 × g, 4°C, 1 min).

[0267] Mass spectrometry of intact proteins ESI-MS analysis of proteins (ubiquitin, Figure 13 and sfGFP, Figure 8) was performed using a Waters Xevo G2 mass spectrometer coupled to a modified nanoAcquity LC system. The purified samples (as described above) were separated on a BEH C4 UPLC column (1.7 μm; 1.0 × 100 mm; Waters) for 20 min at a flow rate of 50 μL / min using a water / acetonitrile gradient from 2% v / v to 80% v / v. The eluted samples were then coupled to the mass spectrometer (Waters) using a Zspray electrospray ionization source. Data were acquired in positive ion mode in the m / z range of 300–2000 with an applied cone voltage of 30 V. Spectra were deconvoluted in MassLynx software (Waters) using the MaxEnt1 function. To calculate the predicted molecular mass, the predicted mass of the wild-type protein was determined using GPMAW (Lighthouse Data) and then manually edited to accommodate changes in the encoded amino acids.

[0268] ESI-MS analysis of the protein (sfGFP, Figure 2B, Figures 14, and 15) was performed using a Waters Vion IMS Qtof mass spectrometer coupled to a modified nanoAcquity LC system. The purified sample (as described above) was separated on an Acuity UPLC protein BEH C4 column (1.7 μm; 2.1 × 50 mm; Waters) for 7 min at a flow rate of 200 μL / min using a water / acetonitrile gradient from 5% v / v to 100% v / v. The eluted sample was then coupled to a mass spectrometer (Waters) using a Zspray electrospray ionization source. Data were acquired in positive ion mode in the m / z range of 100–2000. Spectra were deconvoluted in Unifi software (Waters) using the MaxEnt1 function. To calculate the predicted molecular mass, the predicted mass of the wild-type protein was determined using GPMAW (Lighthouse Data) and then manually edited to accommodate changes in the encoded amino acids.

[0269] Mass spectra of ubiquitin in the screening of anticodon-modified tRNAs (Figure 18) were acquired on an Agilent 1200 LC-MS system equipped with a 6130 quadrupole analyzer. Proteins were eluted from a Phenomenex Jupiter C4 column (150 x 2 mm, 5 μm). Reverse-phase HPLC was performed using Buffer A (0.2% formic acid in water) and Buffer B (0.2% formic acid in acetonitrile (MeCN)). Mass spectra were acquired in positive ion mode and analyzed using MS Chemstation software (Agilent Technologies). The deconvolution program provided with the software was used to acquire the entire mass spectrum.

[0270] Signal-to-noise calculation and fidelity measurement in ESI-MS spectra For the ESI-spectrum of sfGFP, we calculated the mean signal intensity and the standard deviation of the intensity between 20,000 Da and 27,000 Da. We defined the noise as the mean signal intensity plus two times the standard deviation for this mass window. The limit of fidelity measurement was calculated as: (1-(N / S)) x 100. (Note: Due to the presence of a degradation product peak around 27,000 Da, the baseline signal for the spectrum in Figure S3 was determined to be between 20,000 Da and 26,500 Da.) This calculation determines the maximum fidelity that can be obtained from the spectrum; we note that true biological fidelity may be even higher.

[0271] tRNA CGA XXX and tRNA UGA YYYTo determine the specificity for decoding the TCG codon in the presence of both XXX and YYY, where XXX and YYY are distinct amino acids, we divided the intensity of the signal at the peak resulting from the incorporation of XXX at TCG by the intensity of the signal at the mass predicted for the incorporation of YYY at TCG, which was determined as the maximum signal in a 2 Da window around the theoretically calculated mass.

[0272] tRNA CGA XXX and tRNA UGA YYY To determine the specificity for decoding the TCA codon in the presence of both XXX and YYY, where XXX and YYY are distinct amino acids, we divided the intensity of the signal at the peak resulting from the incorporation of YYY at TCA by the intensity of the signal at the mass predicted for the incorporation of XXX at TCA, which was determined as the maximum signal in a 2 Da window around the theoretically calculated mass.

[0273] Western blot of cell lysates from ubiquitin expression experiments Syn61Δ3 cells harboring the pSC101-based tRNA plasmid and the pBAD_ubiquitin plasmid were grown in 20 mL of 2xTY medium containing 200 μg / mL hygromycin, 50 μg / mL apramycin, and 0.2% L-arabinose for 16 h at 37°C with shaking at 220 rpm. The culture was normalized to an OD of 1.0. 500 μL of the normalized culture was solubilized with sample buffer (Nupage buffer, 10% beta-mercaptoethanol, PMSF) and vortexed vigorously to shear the DNA. Samples were separated by SDS-PAGE (4-12% NuPAGE in MES buffer) and transferred to polyvinylidene difluoride (PVDF) membranes using an iBlot 2 dry blotting system (Thermo Fisher Scientific). The membranes were blocked with Odyssey Blocking Buffer (cat. no. 927-40000, Li-Cor) in PBS for 30 minutes at room temperature. The membranes were then incubated overnight at 4°C with anti-His tag primary antibody (Abcam, cat. no. ab18184) in primary antibody solution (1:1000 dilution in Odyssey T20 (PBS) antibody diluent (927-75001, Li-Cor). All incubations were performed on a platform shaker. The membranes were washed three times with PBST (PBS supplemented with 0.1% Tween-20 (v / v)) and then incubated with the secondary antibody goat anti-mouse IRDye. The cells were incubated with 680RD 925-68070 (1:15,000 (v / v) in PBS blocking buffer supplemented with 0.2% Tween-20 (v / v) and 0.01% SDS) for 1 hour at room temperature. After washing three times with PBST and once with PBS, immunoreactive proteins were visualized on a Typhoon Trio phosphorimager (GE Life Sciences). Samples analyzed by Western blot were also separated by SDS-PAGE, and the gels were stained with InstantBlue (Expedeon) for 30 minutes, followed by a water rinse.

[0274] MS / MS of ubiquitin variants The solution samples were reduced with dithiothreitol at 37°C and alkylated with chloroacetamide in the dark at room temperature. The samples were digested with LysC (Promega) for 4 hours at 37°C, followed by overnight trypsin (Promega) digestion at 37°C. The peptide mixture was acidified and desalted using a homemade C18 (3M Empore) stage tip containing 3 μl of Poros Oligo R3 (Thermo Fisher Scientific) resin. Bound peptides were eluted from the stage tip using 30–80% acetonitrile (MeCN) and partially dried in a Speed Vac (Savant).

[0275] Peptides were separated on an Ultimate 3000 RSLC nano System (Thermo Scientific) equipped with a 75 μm x 25 cm nanoEase C18 T3 column (Waters) using mobile phase buffer A (2% MeCN, 0.1% formic acid) and buffer B (80% MeCN, 0.1% formic acid). Eluted peptides were directly injected into a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) using a nanospray ion source. The mass spectrometer was operated in data-dependent mode. MS1 spectra were acquired from 380 to 1600 m / z at a resolution of 70,000, followed by MS2 acquisition of the 15 most intense ions at a resolution of 17,500 and an NCE of 27%. An MS target value of 1e6 and an MS2 target value of 1e5 were used. Dynamic exclusion was set to 30 s.

[0276] The acquired raw data files were searched against the E. coli UniProt Fasta database (downloaded September 2022), which contains an additional 20 ubiquitin sequences (each sequence with a different canonical amino acid at position 11), using the integrated Andromeda search engine (v.1.6.17.0) and MaxQuant. Cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation was set as a variable modification. Enzyme specificity was set to trypsin / p, allowing for a maximum of two cleavage defects.

[0277] Preparation and electroporation of electrocompetent Syn61Δ3 cells Inoculate 250 mL of pre-warmed 2xYT medium with 5 mL of Syn61Δ3 overnight culture and measure the OD with shaking (220 rpm) at 37 °C. 600 The cells were grown to approximately 0.5% dsDNA. The cells were chilled on ice for 10 minutes and harvested by centrifugation (4000 rpm, 10 minutes, 4°C). The cell pellet was washed three times in 50 mL of ice-cold 20% glycerol, resuspended in a final volume of 500 μL of ice-cold 20% glycerol, and frozen in 100 μL aliquots in liquid nitrogen. For electroporation, the frozen cells were thawed on ice, and 50 μL of cells were mixed with 100 ng of plasmid DNA. The mixture was placed in an electroporation cuvette (2 mm gap; SLS Scientific) and electroporated using an Eppendorf e-porator (2500 V). The cells were immediately resuspended in 1 mL of prewarmed SOB growth medium, transferred to a 2 mL microcentrifuge tube, and incubated at 37°C for 90 minutes with shaking (1050 rpm). We then inoculated the recovery medium (1 mL) into 5 mL of pre-warmed 2xYT medium containing the appropriate antibiotics and incubated the cells overnight at 37°C with shaking at 220 rpm.

[0278] Mating assay Donor and recipient cells for mating assays were grown overnight in 5 mL 2xYT in the presence of the appropriate antibiotic (50 μg / mL kanamycin for recipients; 20 μg / mL chloramphenicol for donors). The OD of the cultures was 600 Determine the OD of the culture 600 The culture was normalized to a % RI = 2.0. 400 μL of the culture was then transferred to a 2 mL microcentrifuge tube and washed twice with 2xYT. After washing, the pellet was resuspended to a final volume of 200 μL. For conjugation, 100 μL of donor and 100 μL of recipient were mixed and spotted onto a TYE plate in a 5 μL drop. The plate was then incubated at 37°C for 2 hours. The cells were then washed off the plate using 2 mL 2xYT and transferred to a new 2 mL microcentrifuge tube. The cells were pelleted by centrifugation (1 minute, 3000 x g), resuspended in 1 mL HO, and serially diluted (1:10). 10 0 ~10 -7 Dilutions ranging from 0.01 to 0.01 were spotted (3 μL drops) onto 2xYT agar plates containing 50 μg / mL kanamycin and 20 μg / mL chloramphenicol. Plates were incubated at 37°C for 24-36 hours, and colonies were manually counted to determine the number of successful exconjugants. For experiments using cordlocking, the appropriate antimicrobial (200 μg / mL hygromycin or 75 μg / mL spectinomycin) was added to the 2xTY agar plates.

[0279] Doubling time measurement Cells were seeded from high-density overnight cultures (1:100 ratio) in 200 μL 2xTY containing 200 μg / mL hygromycin in Costar clear 96-well flat-bottom plates. Cells were grown at 37°C with shaking (880 rpm) on a TECAN infinite M200 Pro. Every 5 minutes over 24 hours, we measured OD to determine cell density. 600 Measurements were taken. A sliding window of 10 time points was used to determine the region of the growth curve with the steepest slope. Doubling times were determined from this region.

[0280] Phage enrichment from environmental samples Water samples were collected from various locations along the River Cam (Cambridge, UK). After filtration through a 0.22 μm filter, 4 mL of water sample was mixed with 4 mL of 2×LB and 200 μl of an overnight culture of E. coli, followed by incubation for 48 hours at 37°C on a rotating wheel. The culture was then centrifuged at 4500 × g for 15 minutes, and the filtered supernatant was kept as the phage concentrate.

[0281] Note: Location A: Cambridge Water Treatment Plant Outflow (52°13'55.3"N 0°10'15.3"E); B: Grassland (52°13'21.3"N 0°10'00.0"E); C: Coffee Temple (52°13'07.7"N 0°09'01.5"E); D: Green Dragon Bridge (52°13'02.9"N 0°08'44.8"E); D: Jesus Green's Rock (52°12'45.8"N 0°07'15.4"E); E: Scudamore, Granta Place (52°12'04.6"N 0°06'56.8"E)

[0282] Plaque purification and phage lysate preparation To purify phage plaques, the phage concentrate was serially diluted (10-fold) in LB, and 10 μl of each dilution was added to a bijou bottle containing 200 μL of an overnight culture of the bacterial host for evaluation. Next, 4 mL of molten top agar (0.35% agarose) was added, mixed, and poured onto an LB agar plate containing the appropriate antibiotic. The resulting plate was incubated overnight at 37°C. We used a sterile toothpick to pick individual phage plaques and resuspended them in 100 μl of LB. The mixture was spun down, and the supernatant was diluted and used for further rounds of purification as described above. This process was repeated three times to ensure phage purity.

[0283] Phage lysates were collected from bacterial lawns that showed nearly confluent lysis after infection with pure phage isolates. The top agar was scraped into a glass universal bottle containing 3 ml of LB and homogenized using a sterile pipette. The suspension was then centrifuged (4500 × g, 4 °C, 20 min). The resulting supernatant was filtered through a 0.22 μm filter and stored in a bijou bottle at 4 °C. Phage titers were estimated by counting the number of plaques obtained from phage lysate dilutions plated as described above.

[0284] Phage DNA extraction Phage genomic DNA was added to 450 μL of high-titer phage lysate (approximately 10 10 PFU / mL) using the standard phenol / chloroform method described by Chen et al. (2017) (Non-Patent Document 43).

[0285] Efficiency of the plaque formation assay Phage lysates were serially diluted (10-fold) in LB. Dilutions were spotted (7.5 μL per spot) onto freshly poured and dried overlay lawns (200 μL of overnight culture mixed with 4 mL of top agar overlaid onto LB agar plates containing 200 μg / mL hygromycin and 75 μg / mL spectinomycin) and incubated overnight at 37°C. Images of the spots were taken with an iPhone 8 and converted to grayscale in Adobe Illustrator. For concentrations where a single plaque was expected, the entire overlay lawn was poured at a given concentration (200 μL of overnight culture mixed with 10 μL of phage lysate at the desired concentration and 4 mL of top agar overlaid onto an LB agar plate containing 200 μg / mL hygromycin and 75 μg / mL spectinomycin) for better assessment of plaque-forming units. All plaque counts displayed in the bar graphs are from the entire overlay lawn. Soluble titer (>10 6For each PFU / mL, the entire overlying lawn was poured as described above to avoid lysis. The maximum titers used for infection with phage 06 and phage 12 were approximately 7.5 x 10 9 PFU / mL and approximately 1.1 × 10 10 The strains used in this experiment contain different versions of the spectinomycin resistance gene: Syn61 WT contains SpecR WT, Syn61Δ3 contains recSpecR, and Syn61Δ3(tRNA CGA Ala , tRNA UGA His ) contains O-SpecR (TCG-Ala, TCA-His) and Syn61Δ3 (tRNA CGA Ala , tRNA UGA Leu ) contains O-SpecR (TCG-Ala, TCA-Leu) and Syn61Δ3 (tRNA CGA Leu , tRNA UGA Leu ) contains O-SpecR (TCG-Leu, TCA-Leu) and Syn61Δ3 (tRNA CGA Pro , tRNA UGA Leu ) contains O-SpecR (TCG-Pro, TCA-Leu).

[0286] electron microscope Phage samples were collected in 10 μL of high-titer phage lysate (>10 9 Cells were prepared by adsorption of 1000 PFU / mL (1000 PFU / mL) onto charged copper grids and staining with 2% (w / v) uranyl acetate. Transmission electron microscopy (TEM) images were taken at the Cambridge Advanced Imaging Centre (CAIC), University of Cambridge, using an FEI Tecnai G2 series transmission electron microscope (accelerating voltage: 200.0 kV; direct magnification: 50,000×).

[0287] Phage genome sequencing and de novo assembly Purified phage DNA was prepared for NGS using the Nextera XT DNA Library Preparation Kit. Libraries were paired-end sequenced on a MiSeq (Illumina, Reagent Kit v3 (150 cycles)). De novo assembly of the phage genome was performed using Unicycler in short read mode and with default selections. Sequence coverage across the phage genome is expressed as median sequencing coverage in 250-bp windows.

[0288] tRNA screening A summary of the sequences used in the tRNA screen is provided in SEQ ID NOs: 7-68. These sequences are, in order, ArgX (anticodon modified to CGA), ArgX (anticodon modified to TGA), ArgW (anticodon modified to CGA), ArgW (anticodon modified to TGA), ileT (anticodon modified to CGA), ileT (anticodon modified to TGA), PheU (anticodon modified to CGA), PheU (anticodon modified to TGA), AspT (anticodon modified to CGA), AspT (anticodon modified to TGA), AsnT (anticodon modified to CGA). AsnT (anticodon modified to TGA), GltU (anticodon modified to CGA), GltU (anticodon modified to TGA), ValV (anticodon modified to CGA), ValV (anticodon modified to TGA), ThrT (anticodon modified to CGA), ThrT (anticodon modified to TGA), ThrU (anticodon modified to CGA), ThrU (anticodon modified to TGA), GlyU (anticodon modified to CGA), GlyU (anticodon modified to TGA), GlyT( Anticodon modified to CGA), GlyT (anticodon modified to TGA), GlnU (anticodon modified to CGA), GlnU (anticodon modified to TGA), GlnV (anticodon modified to CGA), GlnV (anticodon modified to TGA), MetV (anticodon modified to CGA), MetV (anticodon modified to TGA), MetY (anticodon modified to CGA), MetY (anticodon modified to TGA), ThrV (anticodon modified to CGA), ThrV (anticodon modified to TGA anticodon), valW (anticodon modified to CGA), valW (anticodon modified to TGA), ArgQ (anticodon modified to CGA), ArgQ (anticodon modified to TGA), ArgV (anticodon modified to CGA), ArgV (anticodon modified to TGA), CysT (anticodon modified to CGA), CysT (anticodon modified to TGA), HisR (anticodon modified to CGA), HisR (anticodon modified to TGA), ileX (anticodon modified to CGA),ileX (anticodon modified to TGA), LysQ (anticodon modified to CGA), LysQ (anticodon modified to TGA), ProK (anticodon modified to CGA), ProK (anticodon modified to TGA), ProL (anticodon modified to CGA), ProL (anticodon modified to TGA), ProM (anticodon modified to CGA), ProM (anticodon modified to TGA), TrpT (anticodon modified to CGA), TrpT (anticodon modified to TGA), TyrV (anticodon modified to CGA), TyrV (anticodon modified to TGA), AlaT (anticodon modified to CGA), AlaT (anticodon modified to TGA), LeuQ (anticodon modified to CGA), LeuQ (anticodon modified to TGA).

[0289] [Table 1] JPEG2025525561000003.jpg227147JPEG2025525561000004.jpg227128

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Claims

1. A cell containing a genome in which at least a first type of sense codon is re-encoded such that a first endogenous tRNA is not required; The cells do not express the first endogenous tRNA; The cells express a first modified tRNA capable of decoding the first type of sense codon, the first modified tRNA being charged with a first amino acid that is not a naturally occurring congener of the first type of sense codon; and The cell contains a gene necessary for survival, the gene contains the appearance of at least one sense codon of the first type, and the cell is viable when the sense codon of the first type in the gene is decoded as the first amino acid; The aforementioned cells.

2. The cell according to claim 1, wherein the first modified tRNA is an anticodon swap tRNA that normally associates with the first amino acid.

3. The cell according to claim 1 or 2, wherein the first modified tRNA is derived from a tRNA that is endogenous to the cell and is an isoacceptor for the first amino acid, or is derived from a tRNA found in a mobile gene that is an isoacceptor for the first amino acid.

4. A cell according to claim 1 or 2, If the first type of sense codon in the genes necessary for survival is decoded according to the regular genetic code, the cell is unable to survive, or If the first type of sense codon in a gene necessary for survival is decoded according to the regular genetic code, it contributes at least partially to the reduction in survival. The aforementioned cells.

5. The cell according to claim 1 or 2, wherein the gene necessary for survival is an essential gene or a positively selectable marker.

6. The cell according to claim 1 or 2, wherein the first amino acid is a naturally occurring amino acid.

7. The cell according to claim 1, wherein a second type of sense codon is re-encoded within the genome.

8. The cell according to claim 7, wherein a second endogenous tRNA is not required and does not express the second endogenous tRNA.

9. A cell according to claim 7 or 8, expressing a second modified tRNA capable of decoding a second type of sense codon, wherein the second modified tRNA carries a second amino acid that is not naturally cognate with the second type of sense codon.

10. A cell according to claim 9, wherein a gene necessary for survival includes the appearance of at least one sense codon of a second type, and the cell is viable when the sense codon of the second type in the gene is decoded as a second amino acid.

11. A cell according to claim 10, If the aforementioned cell is unable to survive when the second type of sense codon in the gene necessary for survival is decoded according to the regular genetic code, If the second type of sense codon is decoded according to the regular genetic code, it contributes at least partially to the reduction in survival. The aforementioned cells.

12. The cell according to claim 10, wherein the second modified tRNA is an anticodon swap tRNA that normally associates with the second amino acid.

13. A cell according to claim 10, The second modified tRNA is derived from a tRNA that is endogenous to the cell and is an isoacceptor for the second amino acid, or It was found in mobile genetic elements and is derived from tRNA, which is an isoacceptor for the second amino acid. The aforementioned cells.

14. The cell according to claim 10, wherein the second amino acid is a naturally occurring amino acid.

15. The cell according to claim 10, wherein the first and second amino acids are either the same type of amino acid or different types of amino acids.

16. The cell according to claim 7 or 8, wherein the first type of sense codon is TCA and the second type of sense codon is TCG.

17. The cell according to claim 1 or 7, wherein the first type of sense codon is TCA or TCG.

18. The cell according to claim 1 or 7, wherein the gene is viable when decoded by a modified tRNA, and is inviolable when the gene is decoded at least partially according to the regular genetic code.

19. A cell comprising a genome in which a first type of sense codon and a second type of sense codon are re-encoded such that a first endogenous tRNA and a second endogenous tRNA are unnecessary; the cell does not express the first endogenous tRNA and the second endogenous tRNA; The cells express a first anticodon-swap tRNA derived from a naturally occurring first parental tRNA, the first anticodon-swap tRNA carries a first amino acid, the first parental tRNA is an isoacceptor for the first amino acid, and the first amino acid is not an amino acid that is naturally congenerated with the first type of sense codon; The cells express a second anticodon-swap tRNA derived from a naturally occurring second parental tRNA, the second anticodon-swap tRNA is charged with a second amino acid, the second parental tRNA is an isoacceptor for the second amino acid, and the second amino acid is not an amino acid that is naturally congenerated with the second type of sense codon; The first and / or second modified tRNA cannot decode any codon other than the first type of sense codon and / or the second type of sense codon; The aforementioned cells.

20. The cell according to claim 19, wherein the first and second types of sense codons normally encode the same amino acid.

21. The cell according to claim 19 or 20, wherein the first and second types of sense codons are normally decoded by the same tRNA or tRNA duplicated due to a fluctuating base pair, the first anticodon swap tRNA is unable to decode any type of codon other than the first type of sense codon, and / or the second anticodon swap tRNA is unable to decode any type of codon other than the second type of sense codon.

22. The cell according to claim 19 or 20, wherein the first type of sense codon and the second type of sense codon have the formula XXN, the first anticodon swap tRNA cannot decode the second type of sense codon, and the second anticodon swap tRNA cannot decode the first type of sense codon.

23. The cell according to claim 19 or 20, wherein the first amino acid and the second amino acid are different types of amino acids.

24. The cell according to claim 19 or 20, wherein the first and second parental tRNAs are derived from the same cell type as the cell described in claim 19.

25. The cell according to claim 19 or 20, wherein the first and / or second anticodon swap tRNA comprises an identity element recognized by an aminoacyl-tRNA synthetase endogenous to the cell.

26. The cell according to claim 19 or 20, wherein the first and second types of sense codons normally encode serine, or the first and second types of sense codons normally encode alanine, or the first and second types of sense codons normally encode leucine.

27. The cell according to claim 19 or 20, wherein the first and / or second anticodon swap tRNA does not decode a TCC or TCT codon.

28. The cell according to claim 19 or 20, wherein the first type of sense codon is TCA and / or the second type of sense codon is TCG.

29. The cell according to claim 19 or 20, wherein the first and / or second amino acid is a naturally occurring amino acid.

30. The cell according to claim 19 or 20, wherein the first amino acid is one of alanine, histidine, leucine, and proline; and / or the second amino acid is one of alanine, histidine, leucine, and proline.

31. The cell according to claim 19 or 20, wherein the first and / or second anticodon swap tRNA is derived from a parent tRNA encoded by ArgQ, ArgU, GltU, HisR, ProK, ProL, ProM, TrpT, ThrU, ThrT, TyrU, TyrV, AlaT, or LeuQ.

32. The cell according to claim 31, wherein the first and / or second anticodon swap tRNA is derived from a parental tRNA encoded by HisR, ProM, AlaT, or LeuQ.

33. The cell according to claim 19 or 20, wherein the parental tRNA is E. coli tRNA.

34. In order to eliminate the first endogenous tRNA and the second endogenous tRNA, the first type of ce A cell comprising a genome re-encoded with a senscodon and a second type of senscodon; the cell does not express the first endogenous tRNA and the second endogenous tRNA; The cells express a first modified tRNA capable of decoding the first type of sense codon, and the first modified tRNA is charged with a first amino acid that is not naturally congenerated with the first type of sense codon; The cells express a second modified tRNA capable of decoding the second type of sense codon, and the second modified tRNA is charged with a second amino acid that is not naturally congenerated with the second type of sense codon; i) Whether the first amino acid is alanine and the second amino acid is alanine; ii) whether the first amino acid is alanine and the second amino acid is histidine; iii) whether the first amino acid is alanine and the second amino acid is leucine; iv) Whether the first amino acid is alanine and the second amino acid is proline; v) whether the first amino acid is histidine and the second amino acid is alanine; vi) whether the first amino acid is histidine and the second amino acid is histidine; vii) Whether the first amino acid is histidine and the second amino acid is leucine ； viii) Whether the first amino acid is histidine and the second amino acid is proline; ix) Whether the first amino acid is leucine and the second amino acid is alanine; x) Whether the first amino acid is leucine and the second amino acid is histidine; xi) Whether the first amino acid is leucine and the second amino acid is proline; xii) Whether the first amino acid is proline and the second amino acid is alanine; xiii) Whether the first amino acid is proline and the second amino acid is histidine; xiv) Whether the first amino acid is proline and the second amino acid is leucine; or xv) The first amino acid is proline, and the second amino acid is proline; The aforementioned cells.

35. The cell according to claim 34, wherein the first modified tRNA is unable to decode a second type of sense codon, and / or the second modified tRNA is unable to decode a first type of sense codon.

36. The cell according to claim 34 or 35, wherein the first modified tRNA cannot decode any codon of any kind other than the first type of sense codon, and / or the second modified tRNA cannot decode any codon of any kind other than the second type of sense codon.

37. The cell according to claim 34 or 35, wherein the first modified tRNA is an anticodon swap tRNA that normally associates with a first amino acid, and / or the second modified tRNA is an anticodon swap tRNA that normally associates with a second amino acid.

38. A cell according to claim 34 or 35, The first modified tRNA is derived from a tRNA that is endogenous to the cell and is an isoacceptor for the first amino acid, or is derived from a tRNA found in a mobile gene that is an isoacceptor for the first amino acid; and / or The second modified tRNA is derived from a tRNA that is endogenous to the cell and acts as an isoacceptor for the second amino acid, or is derived from a tRNA found in a mobile gene that acts as an isoacceptor for the second amino acid. The aforementioned cells.

39. The cell according to claim 34 or 35, wherein the first and / or second modified tRNA comprises an identity element that is recognized by an aminoacyl-tRNA synthetase endogenous to the cell.

40. The cell according to claim 34 or 35, wherein the first and second types of sense codons normally encode serine.

41. The cell according to claim 7, 19, or 34, wherein the first type of sense codon is TCA and / or the second type of sense codon is TCG.

42. The cell according to claim 7, 19, or 34, wherein the essential genes of the genome do not contain naturally occurring examples of the second type of sense codon, and the second endogenous tRNA is a tRNA of the same family as the second type of sense codon.

43. The cell according to claim 7, 19, or 34, wherein the genome comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 naturally occurring examples of a second type of sense codon, and the second endogenous tRNA is a tRNA of the same family as the second type of sense codon.

44. The second type of sense codon is TCG, and the second endogenous tRNA is tRNA. Ｓｅｒ ＣＧＡ The cell according to claim 7, 19, or 34.

45. The cell according to claim 1, 19, or 34, wherein the essential genes of the genome do not contain any naturally occurring examples of the first type of sense codon, and the first endogenous tRNA is a tRNA of the same family as the first type of sense codon.

46. The cell according to claim 1, 19, or 34, wherein the genome comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 naturally occurring examples of a first type of sense codon, and the first endogenous tRNA is a tRNA of the same family as the first type of sense codon.

47. The first type of sense codon is TCA, and the first endogenous tRNA is tRNA. Ｓｅｒ ＵＧＡ Either the first type of sense codon is TCG, or the first endogenous type is tRNA. Ｓｅｒ ＣＧＡ The cell according to claim 1, 19, or 34.

48. The cell according to claim 1, 19, or 34, wherein multiple naturally occurring instances of the TCA codon are replaced with AGT, and / or multiple naturally occurring instances of the TCG codon are replaced with AGC.

49. Cells having increased resistance to horizontal gene transfer or mobile genetic factors, wherein the cells are modified to reassign at least one type of sense codon to an amino acid that does not associate with a sense codon in the normal genetic code, and the cells contain a gene necessary for survival that is functional when decoded according to the reassigned genetic code, but is not functional when decoded according to the normal genetic code.

50. A cell according to any one of claims 1, 19, 34, or 49, wherein the genome of the cell is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identical to any one of sequence numbers 1 to 6.

51. The cell according to claim 1, 19, 34, or 49, which is a prokaryotic cell, a bacterial cell, or an Escherichia coli cell.

52. A method for increasing the resistance of cells to mobile genetic factors or horizontal gene transfer, wherein the cells are modified to reassign at least one type of sense codon to an amino acid that does not associate with a sense codon in the normal genetic code: The method includes modifying a gene necessary for survival such that it includes the appearance of at least one reassigned sense codon. The cell is viable if the reassigned sense codon in the gene is decoded as a reassigned amino acid. The method wherein, if the reassigned sense codon in the gene is decoded according to the regular genetic code, the cell is unviable, or the reassigned sense codon in the gene is decoded according to the regular genetic code, which contributes at least partially to a reduction in survival.

53. A kit comprising a first cell recoded according to a first orthogonal coding scheme and a second cell recoded according to a second orthogonal coding scheme, wherein the first and second coding schemes are orthogonal to each other.

54. The first cell is one described in claim 1, 19, 34, or 49, and / or The second cell is one of the cells described in claim 1, 19, 34, or 49. The kit according to claim 53.

55. The kit according to claim 53, wherein a first cell utilizes a reassignment scheme illustrated in Figure 2C, and a second cell utilizes a different reassignment scheme illustrated in Figure 2C.

56. Movable genetic elements recoded according to an orthogonal coding scheme.

57. The mobile genetic element according to claim 56, wherein at least one, multiple, or all examples of a particular type of sense codon in at least one gene necessary for horizontal transfer of genetic information are replaced with sense codons that normally encode different amino acids.

58. The mobile genetic factor according to claim 57, wherein the substitution of a specific type of sense codon follows the reassignment scheme illustrated in Figure 2C.

59. A method for suppressing horizontal transfer of genetic information between a mobile genetic factor and a first cell, comprising incubating the mobile genetic factor and the first cell, The method wherein the mobile genetic element is one of the claims 56 to 58, and the first cell comprises a tRNA that decodes codons according to a regular genetic code or according to a coding scheme that is orthogonal to that of the mobile genetic element.

60. A method for altering the susceptibility of a gene to mutations that alter the encoded amino acid sequence, wherein: i) the step of identifying the target gene; and ii) A step of incubating cells containing the target gene, wherein the cells contain tRNA capable of decoding at least one sense codon for a reallocated amino acid. The method, including the method described above.

61. The method according to claim 60, wherein the cells are those described in claim 1, 19, 34, or 49.

62. The method according to claim 60, wherein the reallocated amino acids are illustrated in Figure 2C.

63. The method according to claim 60, wherein the genome of the cells is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identical to any one of sequence numbers 1 to 6, and the cells contain anticodon swap tRNA.

64. Use of the cells according to claim 1, 19, 34, or 49 for the production of polymers.

65. A method for producing polymers, To culture the cells according to claim 1, 19, 34, or 49, To provide cells having a nucleic acid sequence encoding a polymer, and To obtain the aforementioned polymer The method, including the method described above.

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