Aminoacyl-trna synthetase sets

Abstract

The invention relates to aminoacyl-tRNA synthetases (aRSs), classes of pyrrolysyl-tRNA synthetases (PylRSs), sets of aRSs, and cells expressing said aRSs and / or sets of aRSs. The invention also relates to methods of generating sets of aRS and methods of generating cells comprising exogenous sets of aRSs. Furthermore, the invention relates to methods and uses of cells for generating polymers.

Description

[0001] AMINOACYL-tRNA SYNTHETASE SETS FIELD OF THE INVENTION The invention relates to acyl-tRNA synthetases (aRSs), classes of pyrrolysyl-tRNA synthetases (PylRSs), sets of aRSs, and cells expressing said aRSs and / or sets of aRSs. The invention also relates to methods of generating sets of aRS and methods of generating cells comprising exogenous sets of aRSs. Furthermore, the invention relates to methods and uses of cells for generating polymers. BACKGROUND OF THE INVENTION The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of non-canonical amino acids (ncAAs) and hydroxy acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides.1-4These advances are underpinned by the discovery of aminoacyl-tRNA synthetases (aaRSs) and tRNAs that are orthogonal – in their aminoacylation specificity – with respect to the synthetases and tRNAs of the host organism, and mutually orthogonal with respect to each other. These pairs, including Methanococcus janaschii (Mj) tyrosyl–tRNA synthetase (TyrRS) / MjtRNATyr, Archaeoglobus fulgidus (Af) TyrRS / AftRNATyr, Methanococcus maripaludis (Mmp) phosphoseryl–tRNA synthetase (SepRS) / MjtRNASep, Saccharomyces cerevisiae (Sc) tryptophanyl–tRNA synthetase (TrpRS) / SctRNATrp), Methanosarcina mazei (Mm) or Methanosarcina barkeri (Mb) pyrrolysyl–tRNA synthetase (PylRS) / MmtRNAPylor MbtRNAPyl, and engineered mutually orthogonal PylRS / tRNAPyl pairs such as Candidatus Methanomethylophilus sp.1R26 (1R26)PylRS / Candidatus Methanomethylophilus alvus (Alv)tRNAPyl-8and Methanomassiliicoccus luminyensis 1 (Lum1)PylRS / Candidatus Methanomassiliicoccus intestinalis (Int)tRNAPyl-17C10,5-16have been altered to recognise distinct amino acids. While initial work incorporated ncAAs in response to the amber codon, recent work has taken advantage of other codons including additional stop codons,17quadruplet codons,18-21codons containing non-canonical bases22-24and sense codons in organisms with genomic code compression and tRNA deletion.3,25,26Mutually orthogonal pairs provide a foundation for incorporating combinations of ncAAs and encoded cellular polymer synthesis and, despite recent progress, the discovery of such pairs remains an outstanding challenge.1,2,5,6,17,18,27-30The pyrrolysyl-tRNA synthetase PylRS / tRNAPylpairs are the most widely used systems for genetic code expansion.2These pairs enable the site-specific incorporation of ncAAs in all domains of life;31the anticodon of the pyl tRNAs tested can be mutated to decode diverse codons,6,19,21,32,33as it is not a recognition element for PylRS enzymes;34and the PylRS active site does not recognise canonical amino acids and can accept, or be evolved to accept, diverse ncAAs and hydroxy acids.4,10,35-40Most genetic code expansion work with pyrrolysyl systems has focussed on the MmPylRS / MmtRNAPylCUApair and the closely related MbPylRS / MbtRNAPylCUA pair.31The PylRS enzymes of these pairs are composed of two domains: an amino (N)-terminal domain and a carboxy (C)-terminal domain. The C-terminal domain binds the amino acid substrate and catalyses the aminoacylation of the cognate tRNAPyl, and the N-terminal domain contacts the variable and T loops of the tRNAPylto enhance binding affinity and specificity.34,41Both domains are required to create a functional MmPylRS / MmtRNAPyl pair in E. coli, and it was widely thought that all PylRS systems required both domains for activity.42,43We demonstrated that a new group of PylRS enzymes12– ΔN PylRS, lacking an N-terminal domain (in the same polypeptide or in trans) – are active and orthogonal.7These pairs, and their engineered derivatives, were combined with pairs from the canonical +N group to enable the creation of mutually orthogonal Pyl systems. We further showed that PylRS and tRNAPylsequences in the ΔN group clustered into two classes, A and B, on the basis of their sequence identity, and we created triply orthogonal pairs composed of a pair derived from the +N group, a class A pair, and a class B pair.6The discovery of new mutually orthogonal Pyl systems has been combined with strategies for providing codons with which to encode non-canonical monomers, and this has enabled the incorporation of several distinct non-canonical amino acids into a protein and the encoded cellular synthesis of non-canonical polymers and macrocycles.3,7,21,28,44-48Despite these advances there were no criteria with which to effectively search genomic data for mutually orthogonal Pyl systems and we hypothesized that many orthogonal and mutually orthogonal systems remained to be discovered. SUMMARY OF THE INVENTION In an aspect, there is provided a cell comprising an exogenous Class C acyl-tRNA synthetase (aRS) and comprising one, two, three, or four members of the group: an exogenous Class A aRS, an exogenous Class B aRS, an exogenous Class N aRS, and an exogenous Class S aRS, wherein each aRS is a pyrrolysyl-tRNA synthetase (PylRS) or a variant engineered to alter the PylRS acylation specificity. In an aspect, there is provided a cell comprising an exogenous Class S aRS and comprising one, two, three, or four members of the group: an exogenous Class A aRS, an exogenous Class B aRS, an exogenous Class C aRS, and an exogenous Class N aRS, wherein each aRS is a PylRS or is a variant engineered to alter the PylRS acylation specificity. In an aspect, there is provided a method of producing a cell comprising at least two exogenous acyl-tRNA synthetases, wherein the method comprises: i) screening one or more PylRS in order to identify a first PylRS belonging to Class C or S; ii) screening one or more PylRS in order to identify a second PylRS belonging to Class A, B, C, N, or S; optionally iii) modifying the first and / or second PylRS in order to alter the acylation specificity; and iv) generating a cell expressing the first PylRS the second PylRS, wherein the first and the second PylRS are not of the same Class. In an aspect, there is provided a cell obtained or obtainable by any of the methods disclosed herein. In an aspect, there is provided a cell comprising nucleic acid sequence encoding an exogenous protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 29 or SEQ ID NO: 30 and a protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 62. In an aspect, there is provided a cell comprising nucleic acid sequence encoding an exogenous protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31 or SEQ ID NO: 32 and a protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 63. In an aspect, there is provided use of any cell disclosed herein for the production of a polymer comprising at least one unnatural amino acid or monomer that is not an alpha amino acid. In an aspect, there is provided a method for making a polymer comprising at least one unnatural amino acid or monomer that is not an alpha amino acid, the method comprising: culturing any cell disclosed herein, providing the cell with a gene encoding the polymer, and obtaining the polymer. BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 | Selection of candidate PylRS and tRNAPylCUA sequences and partitioning of the Pyl system into five distinct sequence-defined classes. a. Activity of each combination of ΔN PylRSi and ΔN tRNAPylj, measured by production of GFP(150AllocK)His6 from cells bearing a GFP(150TAG)His6 gene in the presence of 4 mM AllocK 1, plotted against the sequence identity between ΔN PylRSi and ΔN PylRSj, where ΔN PylRSj is the synthetase from the same organism as ΔN tRNAPylj. ΔN PylRS proteins with greater than 55% sequence identity (dashed grey line) are predominantly active with each other’s pyl tRNAs (88% of cases). ΔN PylRS proteins with less than 55% sequence identity may or may not be active with each other’s pyl tRNAs. b. Activity of each combination ΔN PylRSi and ΔN tRNAPylj, plotted against the sequence identity between ΔN tRNAPyli and ΔN tRNAPylj, where ΔN tRNAPyli is the tRNAPylfrom the same organism as ΔN PylRSi. ΔN pyl tRNAs with greater than 75% sequence identity (dashed grey line) are predominantly active with each other’s synthetases (93% of cases). ΔN pyl tRNAs with less than 75% sequence identity may or may not be active with each other’s synthetases. c. Clustergram of 351 PylRS C-terminal domain amino acid sequences retrieved from a BLAST search against the AΔ-AlvPylRS protein sequence, with the three groups highlighted in red (+N), blue (ΔN), and green (sN) over the dendrogram. Using a clustering threshold of 55%, 37 clusters were obtained. Unweighted average linkage clustering is performed using the scikit-learn (version 1.0.1) package in the Python programming language (version 3.9.7), with sequence identity scores converted to Euclidian distance measures. The heatmaps display percentage sequence identity scores. d. Dendrogram showing the 37 clusters generated from agglomerative hierarchical clustering of the 351 PylRS C-terminal domain amino acid sequences. The 37 PylRS sequences chosen as cluster representatives are labelled. The radial coordinate represents percentage sequence identity (log scale), with grey contours corresponding to intervals of 20%. The red contour represents 55% sequence identity, the clustering threshold value. e. Clustergram of 35 identified tRNAPylsequences from the same organism as a representative PylRS from each cluster, with the five Pyl system classes highlighted in red (N), purple (A), light blue (B), dark blue (C), and green (S) over the dendrogram. f. Dendrogram showing the eight clusters generated from agglomerative hierarchical clustering of the 35 identified tRNAPylsequences. Coloured labels correspond to the 16 tRNAPylsequences chosen for experimental characterization along with their cognate PylRS enzymes. The radial coordinate represents percentage sequence identity (log scale), with grey contours corresponding to intervals of 20%. The red contour represents 75% sequence identity, the clustering threshold value. g. A representative tRNAPylfrom each class is shown; notable structural differences with respect to the canonical N-MmtRNAPylare highlighted with blue nucleotides. h. Schematic of the three Pyl system groups and their division into five classes. Names of the Pyl systems chosen for characterisation are annotated below each class. For classes A and B, the AΔ-1R26PylRS / A-AlvtRNAPyland BΔ-Lum1PylRS / B-InttRNAPylpairs were used. For all other classes, PylRS / tRNAPylpairs were derived from the same organism. i. Schematic of the interaction network between all five classes. Classes N and S are known to interact (red arrow); interactions between classes N, A and B can be abolished by tRNA engineering (grey arrows). All other interactions between classes are unexplored (pink arrows). Fig.2 | Activity mapping of candidate PylRS enzymes and pyl tRNAs, and discovery of new triply orthogonal, PylRS / tRNAPyl pairs. a. Structure of the amino acid, N6-((allyloxy)carbonyl)-L-lysine (AllocK) 1, used in this work. b. Heatmap displaying the activity of all combinations of the selected pyl tRNAs and PylRS enzymes, measured by production of GFP(150AllocK)His6 from cells bearing a GFP(150TAG)His6 gene in the presence of 4 mM AllocK 1. Values are given as a percentage of expression of wild-type GFP. Only PylRS enzymes and pyl tRNAs that have greater than 30% activity with at least one tRNAPylor PylRS enzyme, respectively, are shown. We note that most active pairs are composed of heterologous combinations of PylRS enzymes and pyl tRNAs. Each heatmap value represents the average of three biological replicates. c. Activity heatmaps of representative sets from each family of doubly orthogonal PylRS / tRNAPylpairs obtained from the activity screen. Orthogonality coefficient (o.c), defined as the quotient of the lowest intra-pair activity over the highest inter-pair cross-reactivity, is shown in grey; the set with the highest o.c. in each family is displayed. Each family shares the same combination of PylRS enzymes but has a different combination of pyl tRNAs. To be considered mutually orthogonal, each intra-pair activity must be greater than 40% of the wtGFP control, and each inter-pair cross-reactivity must be less than 20% of the wtGFP control. In addition, the o.c. of the pair must be at least 2.5. d. Activity heatmap of the set with the highest o.c. from the family of triply orthogonal PylRS / tRNAPylpairs obtained from the activity screen. e. The generation of SΔPylRS variants by deletion of the N-terminal domain from class S PylRS enzymes. We considered SΔPylRS variants as engineered members of the ΔN group; their activity profiles are too diverse to be considered as a distinct class. f. The mutual interaction network between all five PylRS classes based on the activity between the characterised PylRS enzymes and pyl tRNAs. Mutually orthogonal pairs can be found using PylRS enzymes from: classes A and B; classes A and S; classes B and S; classes C and N; and classes C and S (double-headed grey arrows). Therefore, five out of ten possible mutually orthogonal combinations were discovered; the other five each showed one undesired cross reactivity (single-headed red arrows). These orthogonal interactions were identified without engineering to tailor PylRS:tRNAPylinteractions. For no two PylRS classes did all combinations of pairs possess two-sided cross- reactivity. Fig.3 | Screening of engineered pyl tRNAs permits the control of 18 out of 20 cross-reactivities between specific members of the five Pyl classes. a. Schematic of the interactions between five specific PylRS / tRNAPylpairs (one from each class) that represent a logical starting point for the development of quintuply orthogonal pairs through a tRNAPylengineering strategy. For classes N, A, and B, we chose active pairs for which the inter-class cross-reactivities (arrows highlighted in blue) have previously been controlled by tRNAPylengineering. For class C, we chose the pair CΔ-NitraPylRS / C-Therm1tRNAPylpair, for which the tRNAPylis naturally orthogonal to all other PylRS classes. Finally, for class S, we chose the most active intraclass PylRS / tRNAPylpair. b. Activity heatmap of the set of PylRS enzymes and pyl tRNAs chosen as a basis for developing quintuply orthogonal pairs. The pyl tRNAs that require engineering or replacement to control unwanted cross-reactions are labelled in red, while the pyl tRNAs that already satisfy all necessary orthogonality requirements are labelled in green. Green box: the natural orthogonality of C-Therm1tRNAPyl. Blue box: class interactions that have previously orthogonalized by tRNAPylengineering and screening. c. Screening of previously reported class N pyl tRNAs against key active PylRS enzymes from each class (and SΔPylRS variants). Multiple class N pyl tRNAs are highly specific to N+- MmPylRS. Each heatmap value represents the average of three biological replicates. d. Activity heatmap from b, updated based on the results of the class N tRNAPylscreen. N-MettRNAPyl, the most orthogonal tRNA from the class N tRNAPylscreen with respect to the chosen PylRS enzymes, is paired with N+-MmPylRS. N-MettRNAPylsatisfies all orthogonality requirements (green box). e. Screening of previously reported A-AlvtRNAPylmutants against key active PylRS enzymes from each class (and SΔPylRS variants). Multiple mutants are highly specific to AΔ-1R26PylRS. Each heatmap value represents the average of three biological replicates. f. Activity heatmap from d, updated based on the results of the A-AlvtRNAPylscreen. A-AlvtRNAPyl-21, the most orthogonal tRNAPylfrom the class A tRNAPylscreen, is paired with AΔ-1R26PylRS. A-AlvtRNAPyl-21satisfies all orthogonality requirements (green box). g. Screening of previously reported B-InttRNAPylmutants against key active PylRS enzymes from each class (and SΔPylRS variants). The activity of BΔ-Lum1PylRS with class B pyl tRNAs is closely matched by SΔ-ClosPylRS, SΔ-I2PylRS, and – most problematically – CΔ-NitraPylRS. Each heatmap value represents the average of three biological replicates. h. Activity heatmap from f, updated based on the results of the class B tRNAPylscreen. B-InttRNAPyl-17C10, the most orthogonal tRNAPylfrom the class B tRNAPylscreen, does not satisfy all necessary orthogonality requirements due only to cross-reactivity with CΔ-NitraPylRS (red box). i. Schematic summarizing the key results of the N, A, and B tRNAPylscreens. By screening of natural and engineered pyl tRNAs, 18 out of 20 interactions – which need to be orthogonalised to generate quintuply orthogonal PylRS / tRNAPylpairs – could be controlled (left diagram, arrows highlighted in blue show interactions that were successfully controlled). Three fully orthogonal pyl tRNAs (labelled in green with their cognate PylRS enzymes) were identified and for each of the remaining two pyl tRNAs (in red) only one cross reaction remains to be controlled. Fig.4 | Unique activity patterns of SΔ PylRS enzymes enable the development of quadruply orthogonal pairs. a. Schematic representation of the strategy of replacing a class B or C PylRS enzyme with a SΔPylRS variant to resolve the undesired cross-reactivity between class C PylRS and class B tRNAPyl. The diverse activities of SΔPylRS variants mean that a different variant can be found to substitute for either class B (e.g. SΔ-ClosPylRS for BΔ-Lum1PylRS, as labelled) or class C (e.g. SΔ-I2PylRS for CΔ-NitraPylRS, as labelled) PylRS enzymes. b. Activity heatmaps of the highest o.c. sets from each family of triply orthogonal PylRS / tRNAPylpairs obtained following the results of the N, A, and B tRNAPylscreens. The substitution of a B or C class PylRS with different SΔPylRS variants (labelled in two shades of blue) allows the generation of many of the new families. Gold box: representative set from the previously reported triply orthogonal N+-MmPylRS, AΔ-1R26PylRS, BΔ-Lum1PylRS family. Silver box: representative set from the only triply orthogonal family found prior to the N, A, and B tRNAPylscreens. Orthogonality coefficient, o.c. is shown in grey. Each family shares the same combination of PylRS enzymes but has a different combination of pyl tRNAs. To be considered mutually orthogonal, each intra- pair activity must be greater than 40% of the wtGFP control, and each inter-pair cross-reactivity must be less than 20% of the wtGFP control. In addition, the o.c. of the pair must be at least 2.5. c. Activity heatmaps of the highest o.c. sets from each family of quadruply orthogonal PylRS / tRNAPylpairs obtained following the results of the class N, A, and B tRNAPylscreens. The substitution of a class B or C PylRS with different SΔPylRS variants (labelled in two shades of blue) allows the generation of all such families. d. Activity heatmaps of the two quadruplet families with a single SΔPylRS variant substituting for a class B or class C PylRS, shown along with the most orthogonal fifth pair from the final class (class N). The pyl tRNAs that require engineering or replacement to abolish unwanted cross-reactions are labelled in red, while the pyl tRNAs that already satisfy all necessary orthogonality requirements are labelled in green. e. Schematic of the interactions between the quadruply orthogonal set with the highest o.c. (class B PylRS substituted by SΔB, o.c.3.9) and the most orthogonal fifth pair from the final class (class N). The cross-reaction between class C PylRS and class B tRNAPylhas been abrogated by substitution of the class B pair with a pair formed with a SΔPylRS variant (green arrow). However, two other cross-reactivities must be abolished to yield quintuply orthogonal pairs (red arrows). f. Schematic of the interactions between the quadruply orthogonal set with the lowest o.c. (class C PylRS substituted by SΔC, o.c.2.5) and the most orthogonal fifth pair from the final class (class N). The cross-reaction between class C PylRS and class B tRNAPylhas been abrogated by substitution of the class C pair with a pair formed with a SΔPylRS variant (green arrow). However, two other cross-reactivities must be abolished to yield quintuply orthogonal pairs (red arrows). g. Activity heatmap of the two (overlapping) quadruplet families with both class B and class C PylRS enzymes substituted by SΔPylRS variants (labelled in two shades of blue), shown along with the most orthogonal fifth pair from the final class (class N or class S). The tRNAPylthat requires engineering or replacement to abolish unwanted cross-reactions is labelled in red, while the pyl tRNAs that already satisfy all necessary orthogonality requirements are labelled in green. h. Schematic of the interactions within the quadruplets from g (both o.c.2.9). For both, cross-reactivity between class N and class S must be eliminated to yield quintuply orthogonal pairs (red arrow). In addition, although the problematic cross-reaction between class C PylRS and class B tRNAPylhas been diminished by substitution of both class B and C pairs by pairs formed with a SΔPylRS variant, the remaining cross-reactivity limits the o.c. of these sets (yellow arrow). Fig.5 | Quintuply orthogonal PylRS / tRNAPyl pairs via directed evolution. a. Activity heatmap combining the quadruplet with the highest o.c. and the most orthogonal fifth pair from the final class. Cross-reactivities are boxed in red; pyl tRNAs to be replaced with quintuply orthogonal evolved variants are labelled in red. b. Schematic showing the library used for evolution of quintuply orthogonal pyl tRNAs from the S-I2tRNAPylscaffold. The cloverleaf structure of S-I2tRNAPylis shown, with randomised nucleotides depicted as blue circles. c. Heatmap showing the activity of hits obtained from positive selection of the library with SΔB-ClosPylRS followed by successive negative screening with the PylRS enzymes from classes N, A, C and S. Each heatmap value represents the average of three biological replicates. d. Activity heatmap from a, updated based on the results of the directed evolution in c. S-I2tRNAPyl-B32, the most orthogonal tRNA from the directed evolution, satisfies all necessary orthogonality requirements (green box). As a result, the quintuply orthogonal SΔB- ClosPylRS / S-I2tRNAPyl-B32pair substitutes effectively for class B and requires no further engineering. e. Heatmap showing the activity of the tRNAPylhit S-I2tRNAPyl-S52obtained from positive selection of the library with S+- DebPylRS followed by successive negative screening with the PylRS enzymes from the other classes. This tRNAPylis exclusively active with S+-DebPylRS. Each heatmap value represents the average of three biological replicates. f. Activity heatmap from d, updated based on the results of the directed evolution in e. S-I2tRNAPyl-S52satisfies all necessary orthogonality requirements (green box). As a result, the S+-DebPylRS / S-I2tRNAPyl-S52pair requires no further engineering, and completes a quintuply orthogonal set of pairs (o.c.4.0). g. Schematic of the overall tRNAPylevolution strategy and resulting pairs. The cross-reactions between class N and class B (or its SΔequivalent), and between class N and class S are successively destroyed to yield a set of five pairs where all twenty cross-reactions are minimized. h. Activity heatmaps from each family of quadruply orthogonal PylRS / tRNAPylpairs obtained following the tRNAPyldirected evolution strategy; the quadruplets with the highest o.c. are shown. Dark grey box: quadruply orthogonal families discovered in Fig.4. Light grey box: quadruply orthogonal family discovered in Fig.4, but which have a higher o.c. when incorporating a newly evolved tRNAPyl. Orthogonality coefficient, o.c., is shown in grey. i. Activity heatmaps from two families of quintuply orthogonal pairs that incorporate the evolved pyl tRNAs; the quintuplets with the highest o.c. are shown. j. Schematic of the interactions within the quintuply orthogonal set of pairs with the highest o.c. (5.4), which is formed with one PylRS from each class. k. Schematic highlighting the successful division of pyrrolysine systems into five mutually orthogonal functional classes: N, A, B or SΔB, C or SΔC, and S. Fig.6 | Alignment of PylRS C-terminal domains. The sequences within Figure 6 are SEQ ID NOs: 2, 4, 6, 9, 12, 15, 18, 21, 24, 27, 29, 31, and 33. The figure demonstrates the alignment of C-terminal domains, as performed during average linkage clustering. DETAILED DESCRIPTION The inventors provide herein methods of categorising pyrrolysine tRNA-synthetases (PylRSs) into one of five categories: Class A, Class B, Class C, Class N, and Class S. Classes A, B, and N are already known from the inventors’ previous work (Dunkelmann et al., Nature Chemistry 12, 535-544 (2020) and Willis et al. Nature Chemistry 10, 831-837 (2018); each of which is incorporated by reference in their entirety). The inventors have identified that PylRSs from these classes have a surprising degree of natural orthogonality to each other. Thus, if one PylRS is chosen from each of these classes, they may be used to form mutually orthogonal systems. The PylRSs may be engineered or modified to change the acylation specificity. For instance, such that the resultant acyl-tRNA synthetase (aRS) is able to charge a tRNA with an alternative canonical amino acid or a non-canonical monomer. For instance, the non-canonical monomer may be an unnatural alpha amino acid or a monomer that is not an alpha amino acid (referred to herein as a “non-alpha-amino acid”). The monomer may, for instance, be a hydroxy acid or a beta amino acid. When paired with appropriate tRNAs, the inventors demonstrate that the five PylRS classes enable the formation of quintuply orthogonal PylRS / tRNA pairs within a host cell. Thus, in an aspect, there is provided a cell comprising an exogenous Class C aRS and comprising one, two, three, or four members of the group: an exogenous Class A aRS, an exogenous Class B aRS, an exogenous Class N aRS, and an exogenous Class S aRS, wherein each aRS is a PylRS or a variant engineered to alter the PylRS acylation specificity. In a particular embodiment, there is provided a cell comprising an exogenous Class C aRS and comprising one, two, or three members of the group: an exogenous Class A aRS, an exogenous Class B aRS, and an exogenous Class N aRS, wherein each aRS is a PylRS or a variant engineered to alter the PylRS acylation specificity. In another aspect, there is provided a cell comprising an exogenous Class S aRS and comprising one, two, three, or four members of the group: an exogenous Class A aRS, an exogenous Class B aRS, an exogenous Class C aRS, and an exogenous Class N aRS, wherein each aRS is a PylRS or a variant engineered to alter the PylRS acylation specificity. As used herein, an “exogenous” aRS is an aRS that is present in a cell that does not naturally express said aRS. For instance, the exogenous aRS may be encoded on an episomal replicon such as a plasmid. Alternatively, sequence encoding the exogenous aRS may have been inserted into the genome of the host cell. In an embodiment, a Class A, B, C, N, or S PylRS can be defined by comparing the amino acid sequence of the C- terminal domain of a candidate PylRS with the amino acid sequences of representative PylRS enzymes known to belong all five Classes. The sequences may be compared using unweighted average linkage clustering. For instance, using python (version 3.9.7) a matrix of percentage identities can be calculated from the multiple sequence alignment of C terminal domains for all pairs of PylRS sequences in a database. The database may be Table 1 (Example 11) herein. This matrix may then be used to perform unweighted average linkage agglomerative hierarchical clustering (UPGMA) of aligned PylRS C terminal domain sequences with a cluster merging threshold of 55% sequence identity, using the biopython (version 1.79) and the scikit-learn (version 1.0.1) python libraries. Thus, in an embodiment, a Class A, Class B, or Class C aRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the host cell, that clusters with a PylRS from Class A, Class B, or Class C (respectively) in Table 1 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. This definition is referred to as a “sequence-dependent” definition herein. In a particular embodiment, a Class A, Class B, or Class C aRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the host cell, that clusters with a PylRS from Class A, Class B, or Class C (respectively) in Figure 6, or with a PylRS referred to as a cluster representative for the respective Class in Table 1, after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C- terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. The Class A aRS may cluster with SEQ ID NO: 35 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. The Class B aRS may cluster with one of SEQ ID NOs: 36, 375, 392, 399, or 400 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. The Class C aRS may cluster with one of SEQ ID NOs: 8, 11, 14, 17, 20, 23 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. The Class N aRS may cluster SEQ ID NO: 331 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C- terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. The Class S aRS may cluster with one of SEQ ID NOs: 26, 29, 31, 33, 149, 174, 187, 198, 200, 272, 277, 278, 285, 288, 291, 294, 376, 377, 381, 382, 384, 388, 393, 394, 397 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. Similarly, in an embodiment, a Class N aRS is a PylRS that clusters with a PylRS from Class N in Table 1 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. In a particular embodiment, a Class N aRS is a PylRS that clusters with a PylRS from Class N in Figure 6, or with a PylRS referred to as a Class N cluster representative in Table 1, after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. Furthermore, in an embodiment, a Class S aRS is a PylRS that clusters with a PylRS from Class S in Table 1 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. In a particular embodiment, a Class S aRS is a PylRS that clusters with a PylRS from Class S in Figure 6, or with a PylRS referred to as a Class S cluster representative in Table 1, after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%. The sequence of a PylRS C-terminal domain is defined as starting from the amino acid that is aligned with S186 of MmPylRS (SEQ ID NO: 1) in a multiple sequence alignment. The multiple sequence alignment may be performed using Clustal Omega with default parameters (Dealign Input Sequences: No, MBED-Like Clustering Guide-Tree: Yes, MBED-Like Clustering Iteration: Yes, Number of Combined Iterations: default (0), Max Guide Tree Iterations: default, Distance Matrix: No, Guide Tree: yes, Order: aligned). The percentage identity between two sequences is defined as the quotient of the number of identical positions and the total number of aligned positions, expressed as a percentage. All Class A, B, and C aRSs lack an N-terminal domain and so the host cell does not express any associated N- terminal domain, either as a part of the protein containing the aRS C-terminal domain or separately. The Class A, B, and C aRSs may be derived from an archaeal species. As an alternative, the Class A, B, or C aRSs may be derived from a bacterial species wherein a naturally occurring N-terminal domain is not expressed by the host cell. In such situations, the aRSs are not defined according to the sequence-dependent definition disclosed herein and instead are defined according to the functionality-dependent definition. The functionality-dependent definition is discussed further below. The Class A, B, or C aRS may be of a sequence that is the same as a wild-type sequence of a naturally occurring PylRS. Alternatively, the Class A, B, or C aRS may be an engineered variant of a naturally occurring PylRS. For instance, the PylRS active site may be modified to recognise a substrate that is not pyrrolysine. In addition, or as an alternative to the sequence-dependent and / or functionality-dependent definitions, a Class N aRS may be defined as a PylRS derived from an archaeal species and wherein the PylRS comprises a PylRS N- terminal domain as a part of the same polypeptide as a PylRS C-terminal domain. The Class N aRS may be of a sequence that is the same as a wild-type sequence of a naturally occurring PylRS. Alternatively, the aRS may be an engineered variant of a naturally occurring PylRS. For instance, the PylRS active site may be modified to recognise a substrate that is not pyrrolysine. This definition is referred to as a “origin-dependent” definition herein. In addition, or as an alternative to the sequence-dependent and / or functionality-dependent definitions, a Class S aRS may be defined as a PylRS derived from a bacterial species and wherein the PylRS N-terminal domain is separately encoded. The N-terminal domain is also expressed as an exogenous protein in the host cell. The Class S aRS may be of a sequence that is the same as a wild-type sequence of a naturally occurring PylRS. Alternatively, the aRS may be an engineered variant of a naturally occurring PylRS. For instance, the PylRS active site may be modified to recognise a substrate that is not pyrrolysine. This definition is referred to as a “origin-dependent” definition herein. In a particular embodiment, a Class A, Class B, or Class C aRS is a PylRS derived from an archeal species and with no N terminal domain, that clusters with a PylRS from Class A, Class B, or Class C (respectively) in Figure 6 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%; a Class S aRS is a PylRS derived from a bacterial species wherein the PylRS N-terminal domain is separately encoded and also expressed as an exogenous protein in the host cell; and a Class N aRS is a PylRS derived from an archaeal species and wherein the PylRS comprises a PylRS N-terminal domain as a part of the same polypeptide as a PylRS C-terminal domain. In another embodiment, a Class A, B, C, N, or S aRS can be identified by a functional comparison. In such a comparison, the candidate aRS is tested to ensure that it is capable of charging the same tRNA as a particular Class of PylRS enzymes and the candidate aRS is tested to ensure that it is not capable of charging the same tRNAs as each of the other four Classes of PylRS enzymes. As an example of a suitable assay, a test cell is modified to express a candidate aRS and a test tRNA. The cell also comprises a marker gene that is only capable of being decoded if the test tRNA is charged. For instance, the marker gene may comprise a stop codon to which the test tRNA is specific, and the stop codon may be situated within the gene such that termination of translation at said stop codon does not lead to expression of the marker. The level of expression of the marker may be compared to the level of expression of a control gene, which is a gene that is identical to the marker gene but that is capable of being decoded by endogenous tRNAs within the test cell. An example of such an assay is provided in the Examples section and in the cited references, where an amber stop codon is positioned within a Green Fluorescent Protein (GFP) gene such that termination at the amber stop codon does not lead to expression of a fluorescent protein, whereas read through of the amber stop codon does lead to functional GFP expression (the marker). If the candidate aRS can charge the test tRNA with an appropriate amino acid, then the stop codon will be read as a sense codon and functional GFP will be expressed because the test tRNA comprises an anticodon that can recognise the amber stop codon. Candidate aRS enzymes not capable of charging the test tRNA will not lead to GFP expression. The control gene is a wild type GFP gene that is identical to the marker gene but has the wild-type codon in the place of the experimental stop codon. To functionally categorise a candidate aRS it may be tested in the above-mentioned assays. DNA sequences encoding test tRNAs for use in such assays are listed below. A test tRNA for use in testing for Class A functionality is encoded by: GGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCataagCGGGGTTCGACcCCCCGGTCTCTCGCCA SEQ ID NO: 37. A test tRNA for use in testing for Class B functionality is encoded by: GGGGTGTTGATCGGATTGATCGCGTGGACTCTAAATCCGCGGTAGACGGGTGAAACTCCCGTACACCTCACCA SEQ ID NO: 38. A test tRNA for use in testing for Class C functionality is encoded by: GGGGGGCTGGTCGGGTGGCCAAGGGGGCTCTAAACCCTCGGTTGCCGGGTTCAACTCCCGGGCTCCCCACCA SEQ ID NO: 39. A test tRNA for use in testing for Class N functionality is encoded by: GGAGACTTGATCATGTAGATCGAACGGACTCTAAATCCGTTCAGCCGGGTTAGATTCCCGGAGTTTCCGCCA SEQ ID NO: 40. A test tRNA for use in testing for Class S functionality is encoded by: GGGGCGTTGATCGGATTGATCGCGTGGACTCTAAATCCGCGGCCGACGGGTGAAACTCCCGTACACCTCTCCA SEQ ID NO: 41. In such embodiments, any aRS is defined as a Class A aRS if, when paired with a tRNA encoded by SEQ ID NO: 37, it leads to expression of the marker gene at greater than or equal to 40% of expression for a control gene, and, when paired with each individually of a tRNA encoded by SEQ ID NO: 38, a tRNA encoded by SEQ ID NO: 39, a tRNA encoded by SEQ ID NO: 40, and a tRNA encoded by SEQ ID NO: 41, it leads to expression of the marker gene at less than 20% of expression for a control gene. Thus, in such embodiments, a Class A aRS has above- threshold activity with a tRNA that defines Class A but has below-threshold activity with all four tRNAs that define each of the other Classes. This is referred to herein as a “functionality-dependent” definition. Classes B, C, N, and S may be functionally categorised in the same manner, wherein a Class B aRS has above- threshold activity with a tRNA encoded by SEQ ID NO: 38, a Class C aRS has above-threshold activity with a tRNA encoded by SEQ ID NO: 39, a Class N aRS has above-threshold activity with a tRNA encoded by SEQ ID NO: 40, and a Class S aRS has above-threshold activity with a tRNA encoded by SEQ ID NO: 41, and wherein each aRS has below-threshold activity with the tRNA that defines each of the other classes. In some embodiments, the cell comprises aRSs defined according to the sequence-dependent definition disclosed herein. In some embodiments, the cell comprises aRSs defined according to the sequence-dependent definition and / or the origin-dependent definitions. In some embodiments, the cell comprises aRSs defined according to the functionality-dependent definition disclosed herein. In other embodiments, the cell may comprise at least one aRS defined according to the sequence-dependent definition and at least one aRS defined according to the functionality-dependent definition disclosed herein. In a further embodiment, the cell comprises at least one aRS defined according to the sequence-dependent definition and / or at least one aRS defined according to the functionality-dependent definition disclosed herein, and a Class N and / or Class S aRS defined according to the origin-dependent definition. In yet another embodiment, the cell comprises aRSs that meet both the sequence- dependent definition and the functionality-dependent definition for the respective Class. The cell may comprise a Class A and a Class C aRS derived from an archeal species and with no N terminal domain, that clusters with a PylRS from Class A or Class C (respectively) in Figure 6 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%; a Class B aRS meeting the functional definition, and a Class N aRS derived from an archaeal species and wherein the aRS comprises a PylRS N- terminal domain as a part of the same polypeptide as a PylRS C-terminal domain. The cell may comprise a Class A and a Class B aRS derived from an archeal species and with no N terminal domain, that clusters with a PylRS from Class A or Class B (respectively) in Figure 6 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of the aRS sequences in Table 1, with a sequence identity lower threshold of 55%; a Class C aRS meeting the functional definition, and a Class N aRS derived from an archaeal species and wherein the aRS comprises a PylRS N- terminal domain as a part of the same polypeptide as a PylRS C-terminal domain. The cell may comprise a Class C aRS meeting the sequence-dependent definition or functionality dependent definition, optionally a Class A aRS meeting the sequence-dependent definition, optionally a Class B aRS meeting the sequence-dependent definition and optionally a Class N aRS meeting the origin-dependent definition. The cell may comprise a Class C aRS meeting the sequence-dependent definition or functionality dependent definition, optionally a Class A aRS meeting the sequence-dependent definition, optionally a Class B aRS meeting the functionality-dependent definition, and optionally a Class N aRS meeting the origin-dependent definition. The aRSs expressed within the cell are aRSs that are active within the host cell. An active aRS is one that can be expressed without rendered in the host cell non-viable and that can acylate a tRNA. In an example, the cell is an E. coli cell and each exogenous aRS expressed by the E. coli cell is compatible with and active within E. coli cells. An example of a Class N PylRS from Methanosarcina mazei (Mm) is provided below: MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSD EDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASV STSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRK KDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPN LYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDS CMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRARSESYYNGISTNL (SEQ ID NO: 1). The C-terminal domain of the above sequence is: SAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLE IKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKE HLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGI DKPWIGAGFGLERLLKVKHDFKNIKRARSESYYNGISTNL (SEQ ID NO: 2) An example of an engineered Class N PylRS from Mm is provided below: MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSD EDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASV STSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRK KDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPXLXPN XXNYXRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLXFXQMGSGCTRENLESIITDFLNHLGIDFKIVGDS CMVXGDTLDVMHGDLELSSAXVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRARSESYYNGISTNL (SEQ ID NO: 3) An example of a Class A PylRS from Candidatus Methanomethylophilus alvus (Alv) is provided below: MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLMNDIADALVA EGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPMLAPNLYSVMRDLRDHTDGPVKIFEMGSCFRKE SHSGMHLEEFTMLNLVDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESDVYKETIDVEINGQEVCSAAVGPHY LDAAHDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKIN (SEQ ID NO: 4) The C-terminal domain of the above sequence is as above. An example of an engineered Class A PylRS from Alv is provided below: MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMIANPSRHGLTQLMNDIADALVA EGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKRALRPXLXPNXXSVXRDLRDHTDGPVKIFEMGSCFRKE SHSGMHLEEFTMLXLXDMGPRGDATEVLKNYISVVMKAAGLPDYDLVQEESDVXKETIDVEINGQEVCSAXVGPHY LDAAHDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKIN (SEQ ID NO: 5) An example of a Class B PylRS from Candidatus Methanomassiliicoccus intestinalis (Int) is provided below: MPVEWTASQKQRLKELGIPAEADRIFNDTKEREEVFKDITSEHLSKVRKDIKHMLDYPERHQLSQIESILAQALVD NGFIEVKTPSIISRSALEKMGIDRSHPLHEQVFWLDEKRCLRPMLAPNLYFMMRHMYRYSKGPLRLFEIGSCFRKE SKGSNHLEEFTMLNLVEMAPDNDPADQLLVHIKTIMDALGLEYSLVECESDVYVKTLDVEIDGVEVASGAVGPHKL DPAHGITQSWAGVGFGLERLSMMKYGMDNIKKSGRSLIYLRGVRLDI (SEQ ID NO: 6) The C-terminal domain of the above sequence is as above. An example of an engineered Class B PylRS from Int is provided below: MPVEWTASQKQRLKELGIPAEADRIFNDTKEREEVFKDITSEHLSKVRKDIKHMLDYPERHQLSQIESILAQALVD NGFIEVKTPSIISRSALEKMGIDRSHPLHEQVFWLDEKRCLRPXLXPNXXFMXRHMYRYSKGPLRLFEIGSCFRKE SKGSNHLEEFTMLXLXEMAPDNDPADQLLVHIKTIMDALGLEYSLVECESDVXVKTLDVEIDGVEVASGXVGPHKL DPAHGITQSWAGVGFGLERLSMMKYGMDNIKKSGRSLIYLRGVRLDI (SEQ ID NO: 7) An example of a Class C PylRS from Candidatus Bathyarchaeota archaeon (Bathy) is provided below: MGNNGLQKLPRSRMENQKILEGKKSSMKDCQFTLSQKRRLKELGADSYVNLTFKNEKERDNAFDNLATILERKHKE ALLNLLTLTKRPLIRQLESKLIEALTSAGFVEVNTPFIIPRKFIECMGIRETHKLWKQIHWLRNGRCLRPMLAPNL YHIMRLLRKFTKPVSIFEIGPCFRKESKGREHVEEFTMLNVVELAPNQDPFERLKEIINIVTKTVELPHYRLRNVK SEIYGETMDVIVDEHEIASAVVGPHSLDKNWGIFEAWAGVGFGIERIAMVKKDIKRIRHVARSLTYLDGASLDVQ (SEQ ID NO: 8) The C-terminal domain of the above sequence is: KDCQFTLSQKRRLKELGADSYVNLTFKNEKERDNAFDNLATILERKHKEALLNLLTLTKRPLIRQLESKLIEALTS AGFVEVNTPFIIPRKFIECMGIRETHKLWKQIHWLRNGRCLRPMLAPNLYHIMRLLRKFTKPVSIFEIGPCFRKES KGREHVEEFTMLNVVELAPNQDPFERLKEIINIVTKTVELPHYRLRNVKSEIYGETMDVIVDEHEIASAVVGPHSL DKNWGIFEAWAGVGFGIERIAMVKKDIKRIRHVARSLTYLDGASLDVQ (SEQ ID NO: 9) An example of an engineered Class C PylRS from Bathy is provided below: MGNNGLQKLPRSRMENQKILEGKKSSMKDCQFTLSQKRRLKELGADSYVNLTFKNEKERDNAFDNLATILERKHKE ALLNLLTLTKRPLIRQLESKLIEALTSAGFVEVNTPFIIPRKFIECMGIRETHKLWKQIHWLRNGRCLRPXLXPNX XHIXRLLRKFTKPVSIFEIGPCFRKESKGREHVEEFTMLXVXELAPNQDPFERLKEIINIVTKTVELPHYRLRNVK SEIXGETMDVIVDEHEIASAXVGPHSLDKNWGIFEAWAGVGFGIERIAMVKKDIKRIRHVARSLTYLDGASLDVQ (SEQ ID NO: 10) An example of a Class C PylRS from Nitrososphaeria archaeon (Nitra) is provided below: MSKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKNLKNIKDFLEQRRKPLVRVIEEKLRTTA LRLGFSEVVTPIIIPRLFIKRMGIDEGDPLWKQVMLIDDKRALRPMLAPNLYVLMAKLSNIVRPVKIFEIGPCFRR ETGGRYHLEEFTMFNMVELAPEGDPKERLLDYIDTIMRDIGLNYTISVEPSNVYGETLDVVVNGIEVASAAIGPKP IDANWGVREPWIGVGFGVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (SEQ ID NO: 11) The C-terminal domain of the above sequence is: SKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKNLKNIKDFLEQRRKPLVRVIEEKLRTTAL RLGFSEVVTPIIIPRLFIKRMGIDEGDPLWKQVMLIDDKRALRPMLAPNLYVLMAKLSNIVRPVKIFEIGPCFRRE TGGRYHLEEFTMFNMVELAPEGDPKERLLDYIDTIMRDIGLNYTISVEPSNVYGETLDVVVNGIEVASAAIGPKPI DANWGVREPWIGVGFGVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (SEQ ID NO: 12) An example of an engineered Class C PylRS from Nitra is provided below: MSKIRFTRGQIHRLIELGAEPTELERDFETEAERDKEFNKIAENLARKNLKNIKDFLEQRRKPLVRVIEEKLRTTA LRLGFSEVVTPIIIPRLFIKRMGIDEGDPLWKQVMLIDDKRALRPXLXPNXXVLXAKLSNIVRPVKIFEIGPCFRR ETGGRYHLEEFTMFXMXELAPEGDPKERLLDYIDTIMRDIGLNYTISVEPSNVXGETLDVVVNGIEVASAXIGPKP IDANWGVREPWIGVGFGVERLAMLVGGYNSIARIAKSLSYLDGSTLSVIKLRW (SEQ ID NO: 13) An example of a Class C PylRS from Candidate division MSBL1 archaeon SCGC-AAA382A20 (SCGC) is provided below: MNLTSSQKQRLRELGWDGSIPDFDNKKERDQFFNKTATKLKNRNKERFLKLLENKVPSWRRVERKLRNIFYELGFV EVQTPSIISPSLLEKMDIGEESKLYNQIYQIKGEKKSLRPMLAPNLYRELRYFSRISDEEVIRLFELGSCFRKENG GERHLNEFKMLNAVEMGNIKDTKKRLDELISNVFSPFANYKVEKEKSTVYEETVDVNIKNTEVASCVIGPHFLDSN WHIDEPWVGLGIGVERLTRVIEGEPSVKPFGKSYVYQDGIRLDIE (SEQ ID NO: 14) The C-terminal domain of the above sequence is: MNLTSSQKQRLRELGWDGSIPDFDNKKERDQFFNKTATKLKNRNKERFLKLLENKVPSWRRVERKLRNIFYELGFV EVQTPSIISPSLLEKMDIGEESKLYNQIYQIKGEKKSLRPMLAPNLYRELRYFSRISDEEVIRLFELGSCFRKENG GERHLNEFKMLNAVEMGNIKDTKKRLDELISNVFSPFANYKVEKEKSTVYEETVDVNIKNTEVASCVI (SEQ ID NO: 15) An example of an engineered Class C PylRS from SCGC is provided below: MNLTSSQKQRLRELGWDGSIPDFDNKKERDQFFNKTATKLKNRNKERFLKLLENKVPSWRRVERKLRNIFYELGFV EVQTPSIISPSLLEKMDIGEESKLYNQIYQIKGEKKSLRPXLXPNXXREXRYFSRISDEEVIRLFELGSCFRKENG GERHLNEFKMLXAXEMGNIKDTKKRLDELISNVFSPFANYKVEKEKSTVXEETVDVNIKNTEVASCXIGPHFLDSN WHIDEPWVGLGIGVERLTRVIEGEPSVKPFGKSYVYQDGIRLDIE (SEQ ID NO: 16) An example of a Class C PylRS from Candidatus Methanohalarchaeum thermophilum 1 (Therm1) is provided below: MELTRSQSQRLRELGYQGEAPTFEDQEERDEFFERKETELQKKNRNKFKKLQRINEPDWKKTEQKLRKNLYESDFT EVQTPHIISMSVLKNKMNISEESNIYNQIYKLDEGNKCLRPMLAPNLYRQMKHFLRISKKDVVKLFELGTCFRKEQ GKNHVREFKMLNAVEVGEIKDKEKRTREMIDEIIGNLVDYKIEEEKSTVYGKTLDIEVNGLEIASSVIGPHPLDAN FSINKPWIGIGIGVERLIQTKNEGNSIKSYARSLSYQDGIRLEIN (SEQ ID NO: 17) The C-terminal domain of the above sequence is: MELTRSQSQRLRELGYQGEAPTFEDQEERDEFFERKETELQKKNRNKFKKLQRINEPDWKKTEQKLRKNLYESDFT EVQTPHIISMSVLKNKMNISEESNIYNQIYKLDEGNKCLRPMLAPNLYRQMKHFLRISKKDVVKLFELGTCFRKEQ GKNHVREFKMLNAVEVGEIKDKEKRTREMIDEIIGNLVDYKIEEEKSTVYGKTLDIEVNGLEIASSVI (SEQ ID NO: 18) An example of an engineered Class C PylRS from Therm1 is provided below: MELTRSQSQRLRELGYQGEAPTFEDQEERDEFFERKETELQKKNRNKFKKLQRINEPDWKKTEQKLRKNLYESDFT EVQTPHIISMSVLKNKMNISEESNIYNQIYKLDEGNKCLRPXLXPNXXRQXKHFLRISKKDVVKLFELGTCFRKEQ GKNHVREFKMLXAXEVGEIKDKEKRTREMIDEIIGNLVDYKIEEEKSTVXGKTLDIEVNGLEIASSXIGPHPLDAN FSINKPWIGIGIGVERLIQTKNEGNSIKSYARSLSYQDGIRLEIN (SEQ ID NO: 19) An example of a Class C PylRS from Candidatus Methanohalarchaeum thermophilum.9096 (Therm2) is provided below: MEFTETQKQRLRELGYKGEFPELDTKEEVNEAYSQLEKKLRKKHRKKLNDLFESKKPTWKNTVENIRQNLQDLGFI EVQTPLIISKNLLKKMKIDQKSDLMNQVYRINDNKVLRPMLAQNLYKELENFSKLSNRDTIQLFEIGTCFRKEKGG KDHLNEFKMLNAVELGNFKDKEKRLKEVISTLFKDFDEYVLEKEKSTVYGETYDVLVNGTELASCAIGPHQLDEKW DINRPWIGIGIGIERFTRELNNSDSTVKAYGRSFVYQDGIRLDIK (SEQ ID NO: 20) The C-terminal domain of the above sequence is: MEFTETQKQRLRELGYKGEFPELDTKEEVNEAYSQLEKKLRKKHRKKLNDLFESKKPTWKNTVENIRQNLQDLGFI EVQTPLIISKNLLKKMKIDQKSDLMNQVYRINDNKVLRPMLAQNLYKELENFSKLSNRDTIQLFEIGTCFRKEKGG KDHLNEFKMLNAVELGNFKDKEKRLKEVISTLFKDFDEYVLEKEKSTVYGETYDVLVNGTELASCAI (SEQ ID NO: 21) An example of an engineered Class C PylRS from Therm2 is provided below: MEFTETQKQRLRELGYKGEFPELDTKEEVNEAYSQLEKKLRKKHRKKLNDLFESKKPTWKNTVENIRQNLQDLGFI EVQTPLIISKNLLKKMKIDQKSDLMNQVYRINDNKVLRPXLXQNXXKEXENFSKLSNRDTIQLFEIGTCFRKEKGG KDHLNEFKMLXAXELGNFKDKEKRLKEVISTLFKDFDEYVLEKEKSTVXGETYDVLVNGTELASCXIGPHQLDEKW DINRPWIGIGIGIERFTRELNNSDSTVKAYGRSFVYQDGIRLDIK (SEQ ID NO: 22) An example of a Class C PylRS from Methanonatronarchaeia archaeon (Tron) is provided below: MEFTVTQKQRLQELGFEGVFPSDFEDVDERNRFFEELVGRLRDRNRKRFERLVGNKIPFWRKVSSDLRNRFYELGF VEVRTPEIISYSLLEKMEISDDLREQVYWLEEDNRCLRPMLAPNLYNELRHFNRISNQSKVRIFEIGTCFRREKSS SEHLNEFTMLNAVEMGDIGDTEERLDRLIEEVFGEFTDYKKVGEESSLYGKTVDVLVDGVEVASCIAGPHPLDSNW SIDQPWVGIGLGVERLAMLLDDGSTAKAYGNSYIYQDGVRLDIK (SEQ ID NO: 23) The C-terminal domain of the above sequence is: MEFTVTQKQRLQELGFEGVFPSDFEDVDERNRFFEELVGRLRDRNRKRFERLVGNKIPFWRKVSSDLRNRFYELGF VEVRTPEIISYSLLEKMEISDDLREQVYWLEEDNRCLRPMLAPNLYNELRHFNRISNQSKVRIFEIGTCFRREKSS SEHLNEFTMLNAVEMGDIGDTEERLDRLIEEVFGEFTDYKKVGEESSLYGKTVDVLVDGVEVASCIA (SEQ ID NO: 24) An example of an engineered Class C PylRS from Tron is provided below: MEFTVTQKQRLQELGFEGVFPSDFEDVDERNRFFEELVGRLRDRNRKRFERLVGNKIPFWRKVSSDLRNRFYELGF VEVRTPEIISYSLLEKMEISDDLREQVYWLEEDNRCLRPXLXPNXXNEXRHFNRISNQSKVRIFEIGTCFRREKSS SEHLNEFTMLXAXEMGDIGDTEERLDRLIEEVFGEFTDYKKVGEESSLXGKTVDVLVDGVEVASCXAGPHPLDSNW SIDQPWVGIGLGVERLAMLLDDGSTAKAYGNSYIYQDGVRLDIK (SEQ ID NO: 25) An example of a Class S PylRS from Clostridiales bacterium (Clos) is provided below: MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGKENIKNLLKEKHITISDEVGNKLSDWLMS KDYTKVLTPTIISKDQLKAMTIDEENHLFSQVFWIDNNKCLRPMLAPNLYIVMRELKRITNEPVKIFEIGSCFRKE SQGARHMNEFTMLNMVELASVEDGKQLDTLKALAHEAMESLGVESYELVIEESAVYGSTLDIEIDGIEVASGSYGP HELDANWDIFDTWVGIGFGIERLAMAINGGSTIKKYGRSINFIDGETMKL (SEQ ID NO: 26) The C-terminal domain of the above sequence is: MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGKENIKNLLKEKHITISDEVGNKLSDWLMS KDYTKVLTPTIISKDQLKAMTIDEENHLFSQVFWIDNNKCLRPMLAPNLYIVMRELKRITNEPVKIFEIGSCFRKE SQGARHMNEFTMLNMVELASVEDGKQLDTLKALAHEAMESLGVESYELVIEESAVYGSTLDIEIDGIEVASGSY (SEQ ID NO: 27) An example of an engineered Class S PylRS from Clos is provided below: MENFTITQTERLKQLNCENDVLELEFEDSEARNSKFREIEIGRVKKGKENIKNLLKEKHITISDEVGNKLSDWLMS KDYTKVLTPTIISKDQLKAMTIDEENHLFSQVFWIDNNKCLRPXLXPNXXIVXRELKRITNEPVKIFEIGSCFRKE SQGARHMNEFTMLXMXELASVEDGKQLDTLKALAHEAMESLGVESYELVIEESAVXGSTLDIEIDGIEVASGXYGP HELDANWDIFDTWVGIGFGIERLAMAINGGSTIKKYGRSINFIDGETMKL (SEQ ID NO: 28) An example of a C-terminal domain of a Class S PylRS from Deltaproteobacteria bacterium (Deb) is provided below: MNSSWTEVQRHRLKELNGAEKDLETAFGDDLQRNRAFQKLEKQLVYQERKRLDRLLDTRFRPLRCELESLLIDALK CEGFTRVETPTIISQNDLERMSIDRSHPFNDQVYRVDSKHCLRPMLAPGLYRLMKDLARIRSGKPVRIFEIGPCFR KETSGARHAGEFTMLNLVEMRIEKGSRRFRIETLAKRIMHAAGIDTYDLVDEPSEVYNTTLDIVCGSDPLEVASCA MGPHPLDAAWGIIDTWVGLGFGLERLLMARENSPGIGKWCKSVSYLDGIRLTL (SEQ ID NO: 29) An example of an engineered C-terminal domain of a Class S PylRS from Deb is provided below: MNSSWTEVQRHRLKELNGAEKDLETAFGDDLQRNRAFQKLEKQLVYQERKRLDRLLDTRFRPLRCELESLLIDALK CEGFTRVETPTIISQNDLERMSIDRSHPFNDQVYRVDSKHCLRPXLXPGXXRLXKDLARIRSGKPVRIFEIGPCFR KETSGARHAGEFTMLXLXEMRIEKGSRRFRIETLAKRIMHAAGIDTYDLVDEPSEVXNTTLDIVCGSDPLEVASCX MGPHPLDAAWGIIDTWVGLGFGLERLLMARENSPGIGKWCKSVSYLDGIRLTL (SEQ ID NO: 30) When expressed in a cell, the N-terminal domain of Deb may also be expressed. The N-terminal domain sequence is: MKETKPAAKRFYRKRVELFRLIDKIKIWPSRTGVLHGIRSVDKRGDIAIITTHCNETFTVRNSRNSRAARWLRNKW FKSVCPACRVPDWKLEKYASTRFKRHFGSDLSRRAD (SEQ ID NO: 62) An example of a C-terminal domain of a Class S PylRS from Gemmatimonadetes bacterium (Gem) is provided below: MGITWSKTQKDRLRALRADGARLADSFEGRPQRDQAFQDLEGALAKARRKELEDLRAGHGRPGLCRLQTTLEGTLV GAGFVQVATPTIMSRGLLAKMGVTKNHDLFEQVFWLDRDRCLRPMLAPHLYYVIKDLLRLWEKPLGIFEVGSCFRK DSQGARHSNEFTMLNLCEFGLPEEDRGGRLREMAEVVTRAAGVHEYELEESASTVYGGTLDVVSVDGLELGSGAMG PHPLDHAWRITDTWVGIGFGLERLLMTVNRETSIGKMGRSLAYLDGIPLSI (SEQ ID NO: 31) An example of an engineered C-terminal domain of a Class S PylRS from Gem is provided below: MGITWSKTQKDRLRALRADGARLADSFEGRPQRDQAFQDLEGALAKARRKELEDLRAGHGRPGLCRLQTTLEGTLV GAGFVQVATPTIMSRGLLAKMGVTKNHDLFEQVFWLDRDRCLRPXLXPHXXYVXKDLLRLWEKPLGIFEVGSCFRK DSQGARHSNEFTMLXLXEFGLPEEDRGGRLREMAEVVTRAAGVHEYELEESASTVXGGTLDVVSVDGLELGSGXMG PHPLDHAWRITDTWVGIGFGLERLLMTVNRETSIGKMGRSLAYLDGIPLSI (SEQ ID NO: 32) When expressed in a cell, the N-terminal domain of Gem may also be expressed. The N-terminal domain sequence is: MSENKKAERERYYRKRVELFRLIDKIKIWPSRKGLLHGIRTTDKMGDVARVTTHCNKTFMVNNSRNSRAARWLRNK WFTGVCPECRIPDWKLAKYSSTHFRRHHGSDLQEGGPPRVDRQPAAPKGAAEVGSGSQEGL (SEQ ID NO: 63) An example of a C-terminal domain of a Class S PylRS from Desulfosporosinus sp. I2 (accession no. WP_045576271.1) is provided below: MGIIWTPIQKQRLQELNASEAQREMCFESQQARDRAFQEQEHSLVVEGKRRLMELRDIKRRPSLSVLEQQLVEALT QQGFVQVVTPTIISKTSLAKMSVSDDHPLFSQVFWLDSKRCLRPMLAPNLYTLWKDLLRLWEKPIRIFEIGTCYRK ESKGSLHLNEFTMLNLTELGLPEDQRHQRLEELASLVMETVGIADYEMELTTSVVYGDTLDVVKGIELGSSAMGPH PLDDQWGIIDPWVGIGFGLERLLMIKEGSQNVQSMGRSLTYLNGVRLNI (SEQ ID NO: 33) An example of an engineered C-terminal domain of a Class S PylRS from I2 is provided below: MGIIWTPIQKQRLQELNASEAQREMCFESQQARDRAFQEQEHSLVVEGKRRLMELRDIKRRPSLSVLEQQLVEALT QQGFVQVVTPTIISKTSLAKMSVSDDHPLFSQVFWLDSKRCLRPXLXPNXXTLXKDLLRLWEKPIRIFEIGTCYRK ESKGSLHLNEFTMLXLXELGLPEDQRHQRLEELASLVMETVGIADYEMELTTSVVXGDTLDVVKGIELGSSXMGPH PLDDQWGIIDPWVGIGFGLERLLMIKEGSQNVQSMGRSLTYLNGVRLNI (SEQ ID NO: 34) An example of a Class A PylRS from Candidatus Methanomethylophilus sp.1R26 (1R26) is provided below: MAEHFTDAQIQRLREYGNGTYKDMEFADVSAREKAFTKLMSDASRDNESALKGMIAHPARQGLSRLMNDIADALVA DGFIEVRTPIIISKDALAKMTITPDKPLFKQVFWIDDKRALRPMLAPSLYTVMRSLRDHTDGPVKIFEMGSCFRKE SHSGMHLEEFTMLNLVDMGPAGDATESLKKYIGIVMKAAGLPDYQLVHEESDVYKETIDVEINGQEVCSAAVGPHY LDAAHDVHEPWAGAGFGLERLLTIRQGYSTVMKGGASTTYLNGAKMD (SEQ ID NO: 35) The 1R26PylRS may be as described in WO2022248061 (herein incorporated by reference). For instance, engineered variants may comprise mutations any one or a combination of substitutions at the following residues: L121, L125, Y126, M129, N166, V168, Y206, and A223, to generate variants with altered selectivity. An example of a Class B PylRS from Methanomassiliicoccus luminyensis 1 (Lum1) is provided below: MDTRLTPAQAQRIREMGGTVDPSLAFSSEAERESAFQRISADLQGANLAKIRRCAEAPERHPIGSLENTLACALAA KGFIEVKTPMMIPADGLVKMGIDESHPLWNQVFWVGPKKALRPMLAPNLYFLMRHLRRSVPAPLLLFEIGPCFRKE SRGSNHLEEFTMLNLVELAPQADATERLKEHIATVMNAVGLPYELVVEGSEVYGTTIDVEVDGVELASGAVGPLPM DKPHGITEPWAGVGFGLERIALMRTKEQNIKKVGRSLVYVNGARIDI (SEQ ID NO: 36) The Lum1PylRS may be as described in WO2022248061. For instance, engineered variants may comprise mutations any one or a combination of substitutions at the following residues: L121, L125, Y126, M129, and V168, to generate variants with altered selectivity. “X” in the above sequences (i.e. any of SEQ ID NOs: 3, 5, 7, 10, 13, 16, 19, 22, 25, 28, 30, 32, and 34) may be any amino acid, any naturally occurring amino acid, or any of the 20 canonical amino acids. The X residues are positioned in common sites that enable the engineering of the PylRS for, e.g., altered substrate specificity. In an embodiment, there is provided a cell comprising two, three, four, or all five members of the group: In an embodiment, there is provided a cell comprising: MmPylRS, 1R26PylRS, Lum1PylRS or SΔ-ClosPylRS, and NitraPylRS or SΔ-I2PylRS. In an embodiment, there is provided a cell comprising two, three, four, or all five members of the group: In an embodiment, there is provided a cell comprising two, three, four, or all five members of the group: In an embodiment, there is provided a cell comprising two, three, four, or all five members of the group: MmPylRS, 1R26PylRS, Lum1PylRS, NitraPylRS, and DebPylS. In an embodiment, there is provided a cell comprising: MmPylRS, 1R26PylRS, Lum1PylRS, and NitraPylRS. Thus, in an embodiment, there is provided a cell comprising an exogenous NitraPylRS or SΔ-I2PylRS and at comprising one, two, three, or four members of the group: an exogenous MmPylRS, an exogenous 1R26PylRS, an exogenous Lum1PylRS or SΔ-ClosPylRS, and an exogenous DebPylS. Thus, in an embodiment, there is provided a cell comprising an exogenous DebPylS and at comprising one, two, three, or four members of the group: an exogenous MmPylRS, an exogenous 1R26PylRS, an exogenous Lum1PylRS or SΔ-ClosPylRS, and an exogenous NitraPylRS or SΔ-I2PylRS. In an embodiment, there is provided a cell comprising two, three, four, or all five members of the group: MmPylRS and a tRNA encoded by SEQ ID NO: 40, 1R26PylRS and a tRNA encoded by SEQ ID NO: 37, Lum1PylRS and a tRNA encoded by SEQ ID NO: 59, NitraPylRS and a tRNA encoded by SEQ ID NO: 39, and DebPylS and a tRNA encoded by SEQ ID NO: 41. In the above embodiments, the MmPylRS, 1R26PylRS, Lum1PylRS, SΔ-ClosPylRS, NitraPylRS, SΔ-I2PylRS, and DebPylS may be engineered, for instance to recognise a substrate that is not pyrrolysine. The anticodon of the tRNAs may be modified to recognise different codons, for instance other triplet or quadruplet codons. In the above embodiments, the MmPylRS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1 or 3. The 1R26PylRS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35. The 1R26PylRS may be engineered, for instance by mutation at any one or any combination of the residues disclosed herein. The Lum1PylRS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 36. The Lum1PylRS may be engineered, for instance by mutation at any one or any combination of the residues disclosed herein. The SΔ- ClosPylRS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 26, 27, or 28. The NitraPylRS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 11, 12, or 13. The SΔ-I2PylRS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 33 or 34. The DebPylS may be of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 29 or 30. The tRNAs may be encoded by a sequence that has 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 37, 39, 40, 41, or 59. Exemplary Class C and Class S PylRSs The inventors provide herein PylRS enzymes and demonstrate functionality of these enzymes wherein expressed exogenously in host cells. In another aspect, there is provided a cell comprising nucleic acid sequence encoding an exogenous protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 30 or 32 or SEQ ID NO: 29 or 31 and a protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 62 or 63. Proteins according to SEQ ID NOs: 29 and 30 may be paired with SEQ ID NO: 62. Proteins according to SEQ ID NOs: 31 and 32 may be paired with SEQ ID NO: 63. The one or both proteins may be engineered to alter the acylation specificity. For instance, such that they are capable of charging a tRNA with an alternative natural amino acid, an unnatural alpha amino acid, or a monomer that is not an alpha amino acid. The proteins may have lost specificity for pyrrolysine. The cells discussed above may have any other features or properties disclosed herein. For instance, the cell may express any other acyl-tRNA synthetase disclosed herein or any combination of acyl-tRNA synthetases disclosed herein. The cell may express any tRNA disclosed herein, any combinations of tRNAs disclosed herein, or any acyl-tRNA synthetase-tRNA pairs disclosed herein. The cell may express two, three, four, five, or more mutually orthogonal acyl-tRNA synthetase-tRNA pairs. The cell may of any type or modified in any manner disclosed herein. Methods of design The inventors provide herein classifications of PylRSs that enable the generation of cells comprising aRS sets with natural orthogonality to each other. As discussed herein, the cells may express two, three, four, or five aRSs each from a different Class. The Classes defined herein are Classes A, B, C, N, and S. Thus, in an aspect, there is provided a method of producing a cell comprising at least two exogenous acyl-tRNA synthetases, wherein the method comprises: i) screening one or more PylRS in order to identify a first PylRS belonging to Class C or S; ii) screening one or more PylRS in order to identify a second PylRS belonging to Class A, B, C, N, or S; optionally iii) modifying the first and / or second PylRS in order to alter the acylation specificity; and iv) generating a cell expressing the first PylRS the second PylRS, wherein the first and the second PylRS are not of the same Class. The Classes A, B, C, N, are as described for the first aspect of the invention. Thus, the screening may be to determine whether the PylRS meets any of the definitions as described herein. The screening one of more PylRS may involve comparing the amino acid sequence of a candidate PylRS with the amino acid sequences of representative PylRS enzymes known to belong all five Classes. The sequences may be compared using unweighted average linkage clustering. Thus, the Class A, Class B, and / or Class C PylRS may be identified using the sequence-dependent definition disclosed herein. Additionally, the Class N and / or Class S PylRS may be identified using the sequence-dependent definition disclosed herein. As discussed herein, all Class A, B, and C aRSs lack an N-terminal domain and so the generated cell does not express any associated N-terminal domain, either as a part of the protein containing the aRS C-terminal domain or separately. The Class A, B, and C PylRS may be derived from an archaeal species. Alternatively, the Class A, B, and C PylRS may be derived from a bacterial species wherein a naturally occurring N-terminal domain is not expressed by the generated cell. The Class of such PylRSs is not defined by the sequence-dependent definition but is defined by the functionality-dependent definition. Alternatively, or in addition, a Class N PylRS may be defined as an PylRS derived from an archaeal species and wherein the PylRS comprises an PylRS N-terminal domain as a part of the same polypeptide as an PylRS C- terminal domain. Alternatively, or in addition, a Class S PylRS may be defined as an PylRS derived from a bacterial species and wherein the generated cell expresses a separately encoded N-terminal domain associated with said Class S PylRS. Alternatively, or in addition, screening one of more PylRS may involve performing an assay to determine the Class of a candidate PylRS. Thus, the Class A, Class B, Class C, Class N, and / or Class S PylRS may be identified using the functionality-dependent definition disclosed herein. In an embodiment, the screening may categorise an PylRS as a Class A, Class B, Class C, Class N, or Class S PylRS if it meets both the sequence-dependent definition / origin-dependent definition and the functionality- dependent definition. In some embodiments, the method identifies the PylRSs defined according to the sequence-dependent definition disclosed herein. In some embodiments, the method identifies the PylRSs defined according to the sequence- dependent definition and / or the origin-dependent definitions. In some embodiments, the method identifies the PylRSs defined according to the functionality-dependent definition disclosed herein. In some embodiments, the method identifies at least one PylRS defined according to the sequence-dependent definition and at least one PylRS defined according to the functionality-dependent definition disclosed herein. In some embodiments, the method identifies at least one PylRS defined according to the sequence-dependent definition and / or at least one PylRS defined according to the functionality-dependent definition disclosed herein, and a Class N and / or Class S PylRS defined according to the origin-dependent definition. In some embodiments, the method identifies PylRSs that meet both the sequence-dependent definition and the functionality-dependent definition for the respective Class. The method may identify a Class C aRS meeting the sequence-dependent definition or functionality dependent definition, optionally a Class A aRS meeting the sequence-dependent definition, optionally a Class B aRS meeting the sequence-dependent definition and optionally a Class N aRS meeting the origin-dependent definition. The method may identify a Class C aRS meeting the sequence-dependent definition or functionality dependent definition, optionally a Class A aRS meeting the sequence-dependent definition, optionally a Class B aRS meeting the functionality-dependent definition, and optionally a Class N aRS meeting the origin-dependent definition. The method may comprise a step, after a PylRS has been identified, of determining whether the PylRS is active within a host cell of choice. For instance, the method may comprise a step wherein it is determined if the candidate can be expressed within a viable E. coli cell and acylate a tRNA. Non-active PylRSs are discarded and the relevant screening step is repeated. The identified PylRSs may be modified in order to alter the acylation specificity. Engineered PylRSs are discussed further herein and the modification may be in order to generate any such engineered PylRS. For instance, mutations known to alter the active site of the PylRS may be transferred into the backbone of the identified PylRS. The method may comprise further screening of one or more PylRS in order to identify a third PylRS, optionally fourth PylRS, and optionally fifth PylRS. Each PylRS may be modified in order to alter the acylation specificity, as discussed herein. A cell may then be generated that expresses the first PylRS, second PylRS, third PylRS, optional fourth PylRS, and optional fifth PylRS, wherein each PylRS belongs to a different Class. The generated cell may be any cell as disclosed herein. For instance, a bacterial cell such as an E. coli cell. Methods of producing polymers Any of the cells disclosed herein may be used to make polymers. Thus, in an aspect, there is provided use of any cell disclosed herein, for the production of a polymer comprising at least one unnatural amino acid or non-alpha- amino acid. In another aspect, there is provided a method for making a polymer comprising at least one unnatural amino acid or non-alpha-amino acid, the method comprising: culturing any cell disclosed herein, providing said cell with a gene encoding said polymer, and obtaining the polymer. The polymers are produced, at least in part, by genetic incorporation of the monomers. The cell expresses an exogenous aRS as part of an aRS-tRNA pair wherein the aRS is capable of charging its tRNA with the unnatural amino acid or non-alpha-amino acid for incorporation into the polymer. The polymer may comprise canonical amino acids. The polymer may comprise at least one unnatural amino acid, such as an unnatural alpha amino acid. The polymer may comprise only unnatural amino acids. The polymer may be a macrocycle. The polymer may comprise at least one non-alpha-amino acid. The polymer may comprise at least one beta amino acid. The polymer may comprise at least one hydroxy acid, such as at least one alpha hydroxy acid. At least one of the monomers may be provided exogenously. At least one of the monomers may be synthesised by the cell. The unnatural amino acid, non-alpha-amino acid, and alpha hydroxy acid may be any as disclosed herein. The cell for the for the production of the polymer may be any disclosed herein. For instance, a prokaryotic cell such as an E. coli cell. Engineered aRS The aRS enzymes disclosed herein may be wild-type or genetically engineered PylRS enzymes. Disclosures of engineered PylRS enzymes and methods of producing said enzymes include: Neumann et al. (Nat Chem Biol 4:232, 2008), Yanagisawa et al. (Chem Biol 2008, 15:1187), de la Torre and Chin (Nat Rev Genet 22, 169–184 (2021)), Wan et al,. (Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1844, Issue 6, June 2014, Pages 1059-1070), WO2009 / 056803 A1, and WO2013 / 171485 (each of which is incorporated herein by reference). The engineered PylRS may be referred to as a mutated PylRS. SEQ ID NOs: 3, 5, 7, 10, 13, 16, 19, 22, 25, 28, 30, 32, and 34 indicate residues that may be mutated in order to alter the acylation specificity of a PylRS. These mutations may also be transferred to alternative aRS backbones, in order to alter the acylation specificity. As the skilled person would appreciate, the sequences disclosed herein may be aligned to the sequence of another PylRS to identify the corresponding residue for mutation. In some examples, only the catalytic region of the PylRS is aligned, in order to simplify the analysis. The PylRS may be mutated such that it can charge a tRNA with a particular natural or canonical amino acid. The PylRS may be mutated such that it can charge a tRNA with an unnatural amino. An “unnatural amino acid” is an amino acid that is not 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, L-tyrosine, L-pyrrolysine, or L-selenocysteine. The PylRS may be mutated such that it can charge a tRNA with a non-canonical amino acid. An “non-canonical amino acid” is an amino acid that is not 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, or L-tyrosine. Suitable unnatural amino acids include those disclosed in Neumann, H., 2012. FEBS letters, 586(15), pp.2057- 2064; and Liu, C.C. and Schultz, P.G., 2010. Annual review of biochemistry, 79, pp.413-444. For example, the unnatural amino acid may be selected from one or more of: p-Acetylphenylalanine, m-Acetylphenylalanine, O- allyltyrosine, Phenylselenocysteine, p-Propargyloxyphenylalanine, p-Azidophenylalanine, p- Boronophenylalanine, O-methyltyrosine, p-Aminophenylalanine, p-Cyanophenylalanine, m-Cyanophenylalanine, p-Fluorophenylalanine, p-Iodophenylalanine, p-Bromophenylalanine, p-Nitrophenylalanine, L-DOPA, 3- Aminotyrosine, 3-Iodotyrosine, p-Isopropylphenylalanine, 3-(2-Naphthyl)alanine, Biphenylalanine, Homoglutamine, D-tyrosine, p-Hydroxyphenyllactic acid, 2-Aminocaprylic acid, Bipyridylalanine, HQ-alanine, p-Benzoylphenylalanine, o-Nitrobenzylcysteine, o-Nitrobenzylserine, 4,5-Dimethoxy-2-nitrobenzylserine, o- Nitrobenzyllysine, o-Nitrobenzyltyrosine, 2-Nitrophenylalanine, Dansylalanine, p-Carboxymethylphenylalanine, 3-Nitrotyrosine, Sulfotyrosine, Acetyllysine, Methylhistidine, 2-Aminononanoic acid, 2-Aminodecanoic acid, Pyrrolysine, Cbz-lysine, Boc-lysine and Allyloxycarbonyllysine. The PylRS may be mutated such that it can charge a tRNA with a non-canonical monomer. For instance, a monomer that is not an alpha amino acid (see Spinck et al. Nature Chemistry volume 15, pages61–69 (2023), herein incorporated by reference). In some embodiments, the non-alpha-amino acid is an alpha hydroxy acid or a beta amino acid. Non-alpha-amino acids are monomers that lack the RCH(NH2)COOH structure of an amino acid, and in particular lack the alpha (NH2) group. Alpha hydroxy acids are exemplary non-alpha-amino acids. Some alpha hydroxy acids are hydroxy variants of canonical amino acids. Alternatively, they may be non-canonical acids. Exemplary alpha-hydroxy acids include hydroxy acids with aromatic side-chain, optionally selected from F-OH, pIF-OH and NapA-OH; and alpha-hydroxy acids with an aliphatic side-chain, optionally selected from BocK-OH, PenK-OH, AllocK-OH, NorK-OH, AlkynK-OH, CbzK-OH, ButK-OH and AcK-OH. Hydroxy-acid analogues of O4BBy, O2beY, pCaaF, pVsaf, pAaF are also contemplated. See (Iannuzzelli and Fasan, Chem. Sci., 2020, 11,6202). In general, alpha-hydroxy acids can be derived from canonical or noncanonical amino acids. For example, alpha- hydroxy acids include, but are not limited to, p-hydroxy-Lphenyllactic acid (the alpha-hydroxy analogue of tyrosine), leucic acid (the alpha-hydroxy analogue of leucine), lactic acid (the alpha-hydroxy analogue of alanine), 2-hydroxy-3-methylbutyric acid (the alpha-hydroxy analogue of valine), 2-hydroxy-3-phenylpropionic acid, the hydroxy derivative of phenylalanine (F-OH), and alpha-hydroxy analogues of other natural and unnatural amino acids. Derivatives of noncanonical amino acids include the hydroxy derivatives of Nε-Alloc-L-lysine (AllocK- OH), p-Iodo-phenylalanine (pIF-OH), Nε-((Prop-2-yn-1-yloxy)carbonyl)-L-lysine (AlkynK-OH), p-Azido- Phenylalanine (pAzF-OH), L-3-(2-Naphthyl)alanine (NapA-OH), Nε-tert-butoxycarbonyl-L-lysine (BocK-OH), N6-Carbobenzyloxy- L-lysine (CbzK-OH) and other lysine derivatives including PenK-OH, NorK-OH, ButK-OH and AcK-OH. tRNAs The PylRS enzymes disclosed herein may be used in concert with exogenous tRNAs and hence may form exogenous PylRS-tRNA pairs. Each exogenous PylRS-tRNA pair may be capable of incorporating a monomer at a codon. For example, the monomer may be a natural amino acid, an unnatural amino acid, or non-alpha-amino acid. For instance, the PylRS may be mutated in order to allow the charging of the tRNA with a particular monomer, such as an alternative natural amino acid, an unnatural amino acid, or a particular hydroxy acid (see the explanation herein of engineered PylRS enzymes). The tRNA may have an altered anticodon for a specific sense codon or stop codon. In embodiments with two, three, four, or five exogenous PylRS-tRNA pairs, they may be capable of directing the incorporation of two, three, four, or five different monomers. The monomers may comprise natural amino acids, unnatural alpha amino acids, and non-alpha-amino acids (such as beta amino acids or hydroxy acids). In embodiments with two, three, four, or five exogenous PylRS-tRNA pairs, they may be capable of directing the incorporation of two, three, four, or five monomers (respectively). The tRNA may be the tRNA that is naturally associated with the PylRS. The tRNA may be an engineered version of the tRNA that is naturally associated with the PylRS. Alternatively, the tRNA, or engineered version thereof, may be one that is not naturally associated with the PylRS. The tRNA may be normally associated with a PylRS from the same or a different class compared to the PylRS with which the tRNA is paired. The tRNA may be from an archaeal species, for instance within the Methanosarcina genus. The tRNA may be from a bacterial species, for instance from a Desulfitobacterium. In some embodiments, two or more of the exogenous tRNAs expressed by the cell have less than 75% sequence identity to each other. In another embodiment, all of the exogenous tRNAs expressed by the call have less than 75% sequence identity to each other. Alternatively, or in additional, two or more of the exogenous tRNAs within the cell differ due to exotic features. Examples of exotic features include: 6-8 base pair D loops, long variable loops, unusual (e.g. adenine or uracil) nucleobases at the discriminator base position, bulges in the anticodon stem, and loops in the anticodon stem. A long variable loop may be more than three nucleotides in length. For instance, a long variable loop may be four nucleotides in length. Exemplary tRNAs include those encoded by the DNA sequences below. Bathy-tRNA: gggggtttggccgaggcggtcgcgagggtactaagctctcgcagccgggttcaactcccgggacccccgcca (SEQ ID NO: 42) Nitra-tRNA: gggggctcggccgaggcggccacaggggctctatacccctgcagccgggttcaactcccggagcccccgcca (SEQ ID NO: 43) SCGC-tRNA: ggggggcaggccgaggatggccagtggggctctaaaccctgcgtctaccgggttcaattcccgggccccccacca (SEQ ID NO: 44) Therm1-tRNA: SEQ ID NO: 39 Therm2- tRNA:ggggggttggtcgggttgaccaaaggaggctctaaaccttctcaagggttcaggcaaatcctgggcctttac cgggttcgactctcgggccccccgcca (SEQ ID NO: 45) Tron-tRNA: ggggggctggtcggggtgaccacggaggccctatacctcccttagccgggttcaactcccgggtccctcgcca (SEQ ID NO: 46) Gem-tRNA: ggggagtggatcgatacaagatcgtgtgggctctaaacccacatagctcggtgtgactccggggctccccacca (SEQ ID NO: 47) Bacc-tRNA: ggagggtgaatcggagtgattatgcggactctaaatccgtacagccgggtgcaactcccggactcttcgcca (SEQ ID NO: 48) I2-tRNA: ggggggtagatcggattgatcgcgtggactctaaatccgcgtagacgggtgaaactcccgtactcctcgcca (SEQ ID NO: 49) Clos-tRNA: ggggaacggatcggatggatcacatagactctaaatctatgtagccgagtgaaactctcgggttcctcgcca (SEQ ID NO: 50) Deb-tRNA: ggggagtggatcggatgagatcgcatgggctctaaacccatgtagccgggtgcgactcccgggcttccctcca (SEQ ID NO: 51) Spi-tRNA: ggggggcggatcgcaagcgagatcgcgcggactctaaatccgcgcagccgggtgcaactcccgggctcctcgcca (SEQ ID NO: 52) Mic-tRNA: ggagtgttgatcgacaacgggatcacatggactctaaatccatgcagccgggtgcaactcccggacactccgcca (SEQ ID NO: 53) Met-tRNA: SEQ ID NO: 40 Bur-tRNA: GGAGACTTGATCATGTAGATCGAACGGACTCTAAATCCTTTCAGCCGGGTTAGATTCCCGGAGTTTCCGCCA (SEQ ID NO: 54) Pro-tRNA: GGAAATCAGATCATGTTGATCGAATGGACTCTAAATCCGTTCAGTCGGGTTAAATTCCCGAGGTTTCCGCCA (SEQ ID NO: 55) Therm-tRNA (intron removed): SEQ ID NO: 39 Tron-tRNA (intron removed, anticodon loop modified): GGGGGGCTGGTCGGGGTGACCACGGAGGCTCTAAACCTCCCTTAGCCGGGTTCAACTCCCGGGTCCCTCGCCA (SEQ ID NO: 56) I2CUAG-tRNA: GGGGGGTAGATCGGATTGATCGCGTGGACTCTAAATCCGCGCTAGACGGGTGAAACTCCCGTACTCCTCGCCA (SEQ ID NO: 57) I2b8-tRNA: GGGGCGTCGATCGGATTGATCGCGTGGACTCTAAATCCGCGCGCAACGGGTGAAACTCCCGTACGCCTCGCCA (SEQ ID NO: 58) I2b32-tRNA: SEQ ID NO: 38 I2b72-tRNA: GGGGTGTAGATCGGATTGATCGCGTGGACTCTAAATCCGCGAACAACGGGTGAAACTCCCGTACACCTCGCCA (SEQ ID NO: 59) I2H52-tRNA: SEQ ID NO: 41 Alv17-tRNA: GGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCgtaagCGGGGTTCGACaCCCCGGTCTCTCGCCA (SEQ ID NO: 60) Alv21-tRNA: SEQ ID NO: 37 Alv22-tRNA: GGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCtcaaggCGGGGTTCGACtCCCCGGTCTCTCGCCA (SEQ ID NO: 61) Thus, the cell may express one, two, three, four, five, or more exogenous tRNAs. The cell may express one, two, three, four, five, or more exogenous PylRS-tRNA pairs. As discussed herein, the cell may express one, two, three, four, or five tRNAs encoded by any combination of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 59, or variants thereof. In particular, variants with altered anticodons. A tRNA may be chosen such that it is active with one of the exogenous PylRS enzymes and such that it is has a lower activity with any other aRS expressed by the host cell. As such, the PylRS-tRNA pair may be orthogonal with regard to any other PylRS-tRNA pair within the host cell. In some embodiments, the host cell may comprise one, two, three, four, five, or more mutually orthogonal exogenous PylRS-tRNA pairs. An orthogonal aRS-tRNA pair is one that leads to expression of a marker gene in a test cell at greater than or equal to 40% of the expression of a control gene, whereas the pairing of the aRS with any other tRNA in the cell or the pairing of the tRNA with any other aRS within the cell leads to expression of the marker gene at less than 20% of the expression of a control gene. Suitable assays are disclosed herein (e.g. wherein the marker gene is a GPF gene with a stop codon positioned within and the control gene is wild-type GFP). An orthogonal set of aRS-tRNA pairs is one where each aRS-tRNA pair meets the above criteria (i.e. each pair achieves greater than 40% of marker gene expression and each cross-reactivity leads to lower than 20% of marker gene expression) and wherein the quotient of the lowest intra-pair activity over the highest inter-pair cross- reactivity is greater than 2.5. Host cells The cell of the present disclosure, including those of all aspects disclosed herein, may be a prokaryotic cell. The cell may be a bacterium. The bacterial cell may be of any species suitable for heterologous protein production, in particular the production of polypeptides. Suitable bacterial host cells include: escherichia (e.g. Escherichia coli), caulobacteria (e.g. Caulobacter crescentus), phototrophic bacteria (e.g. Rodhobacter sphaeroides), cold adapted bacteria (e.g. Pseudoalteromonas haloplanktis, Shewanella sp. strain Ac10), pseudomonads (e.g. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa), halophilic bacteria (e.g. Halomonas elongate, Chromohalobacter salexigens), streptomycetes (e.g. Streptomyces lividans, Streptomyces griseus), nocardia (e.g. Nocardia lactamdurans), mycobacteria (e.g. Mycobacterium smegmatis), coryneform bacteria (e.g. Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum), bacilli (e.g. Bacillus subtilis, Bacillus brevis, Bacillus megaterium, 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 examples, the bacterium is a gram-negative bacterium. In particular examples, the bacterium is an Escherichia coli, Salmonella enterica, or Shigella dysenteriae. More preferably, the cell is an E. coli. Suitable E. 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, G.L. and Ceccarelli, E.A., 2014. Frontiers in microbiology, 5, p.172). In particular, the cell may be MG1655 or BL21, or a derivative thereof. MG1655 is considered as the wild type strain of E coli. The GenBank ID of genomic sequence of this strain is U00096. BL21 is widely available commercially. For example, it can be purchased from New England BioLabs with catalog number C2530H. The host cell may comprise a recoded genome wherein one or more sense codons have been recoded. Examples of such organisms are provided in Fredens et al. (Nature 569, 514-518 (2019)), Robertson et al., Science 372, 1057-1062 (2021), WO2020 / 229592 A1, and WO / 2022 / 248061, each of which is herein incorporated by reference in its entirety). “Recoding” as used herein, is the replacement of an occurrence of a type of codon with a different codon, such that the occurrence of the codon is removed from the genome. A recoded sense codon may be replaced with a synonymous codon to result in different codon usage without changing the encoded polypeptide. Alternatively, a sense codon may be replaced with a non-synonymous codon, for instance if the alteration in the sequence of the encoded polypeptide does not affect viability. The number of occurrences of the type of sense codon that are recoded may be adequate to enable the removal of the cognate tRNA corresponding to said sense codon while maintaining viability of the cell. Stop codons may also be recoded. For instance, by the replacement of occurrences of a first type of stop codon with synonymous codons. The number of occurrences of the type of stop codon that are recoded may be adequate to enable the removal of the cognate release factor while maintaining viability of the cell. In a particular example, the genome of the host cell has been recoded such that the sense codon TCG has been replaced with AGC, the sense codon TCA has been replaced with AGT, and the stop codon TAG has been replaced with TAA, and wherein sufficient numbers of said codons have been recoded such that two cognate tRNAs and a cognate release factor are dispensable. The host cell may be a bacterium, such as E. coli, with a recoded genome. For instance, the cell may be a bacterial cell with a genome recoded with regards to codons TCA and TCG. The bacterial cell may lack tRNASerUGAand tRNASerCGAand, optionally, RF-1. The cell may be Syn61, a strain that is derived from Syn61, or recoded in the same manner as Syn61. The cell may be Syn61Δ3, a strain that is derived from Syn61Δ3, or may be modified in the same manner as Syn61Δ3. The host cell may be recoded to add four types of sense codon, for instance new quadruplet codons, and to recode one type of stop codon. The cells disclosed herein, including those expressing one or more aRS are viable cells. Viable cells are those that are capable of being metabolically active. In a particular embodiment, a viable cell may be capable of growth when cultured in an appropriate media and under appropriate conditions for the particular species or strain. Such cells may be referred to as capable of being cultured. As an example, if the cell is a bacterial cell such as E. coli, the assessment of viability may be performed by culturing said bacteria in a medium comprising LB medium, or on an agar comprising LB agar, at 37oC. The medium or agar may be supplemented with 2% glucose. Growth of the bacteria may be monitored using standard approaches, such as measurement of the OD600. Alternative approaches, or approaches adapted to particular cells, bacteria strains, bacterial species, or in light of the inclusion of marker genes, are known to the skilled person. Sequence comparisons can be conducted with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences. The skilled technician will appreciate how to calculate the percentage identity between two nucleic sequences. In order to calculate the percentage identity between two nucleic sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, 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. as applied by Matcher(EMBOSS); and (ii) the parameters used by the alignment method, for example, local versus global alignment, the matrix used, and the parameters applied to gaps. In a particular embodiment, the sequence identities disclosed herein may be calculated based on a global alignment of the relevant feature, for instance the comparison of a C-terminal domain of a PylRS to the C-terminal domain of another PylRS. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length-dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. A calculation of percentage identities between two nucleic acid sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or GeneWise. In an example, the identity between two amino acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two amino acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (14), gap extend (4), alternative matches (1). In an example, the identity between two nucleic acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1). All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted as prior art to the claimed disclosure. In the case of conflict, the present specification, including definitions, will control. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the Examples, which are not intended to limit the invention in any way. EXAMPLES Summary The discovery of mutually orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pairs provides a foundation for the cellular incorporation of combinations of non-canonical amino acids (ncAAs) into proteins, and encoded non- canonical polymer and macrocycle synthesis. Here we define clusters of pyrrolysine tRNA-synthetase (PylRS) sequences that pass an empirically determined threshold for mutual orthogonality; 84% of the resulting clusters belong to PylRS classes that have not been explored in the search for orthogonal pairs. We identify and cluster tRNAPylsequences from the same organisms for members of 95% of the PylRS clusters, and filter them using an empirical sequence-based threshold for mutual orthogonality and the presence of novel structural features; we thereby define a group of PylRS / tRNAPylpairs within which to search for mutual orthogonality. We identify two new classes of PylRS and tRNAPylsequences and the majority of our new PylRS enzymes and pyl tRNAs are active and orthogonal. We define five classes of PylRS systems that form the basis of quintuply orthogonal pairs. Strikingly, our computational approach directly resolves 20 of the 25 aminoacyl-tRNA synthetase / tRNA pairwise specificities required to make quintuply orthogonal PylRS / tRNAPylpairs; we control the remaining five specificities by tRNAPylengineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS / tRNAPylpairs, 1324 triply orthogonal pairs, 128 quadruply orthogonal pairs, and 8 quintuply orthogonal pairs. These advances will provide a key foundation for encoded polymer synthesis. Introduction Here we leverage experimental PylRS / tRNAPylcross reactivity data to empirically define sequence identity thresholds for mutually orthogonal PylRS enzymes and pyl tRNAs. We then perform agglomerative clustering on 351 PylRS sequences, to define clusters of sequences that pass the empirical thresholds; 84% of the resulting clusters belong to PylRS classes that have not been explored in the search for orthogonal pairs. We identify and cluster tRNAPylsequences from the same organisms for members of 95% of the PylRS sequence clusters. Using both the empirical orthogonality thresholds and the presence of exotic structural features that may confer orthogonality, we select a set of pyl tRNAswhich, along with PylRS enzymes from the same organism, form the starting point of an experimental search for mutually orthogonal pairs. We identify two new classes of PylRS and tRNAPylsequences, which we name class C and class S, and we show that the majority of our new PylRS enzymes and pyl tRNAs are active and orthogonal in E. coli. We explore the specificity of class S and class C systems with respect to each other and with respect to previously characterized class N, A and B PylRS systems. Strikingly our sequence-based approach allows us to control 20 of the 25 aminoacyl-tRNA synthetase / tRNA pairwise specificities required to make a set of quintuply orthogonal PylRS / tRNAPylpairs without additional engineering; we control the remaining five specificities by tRNAPylengineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS / tRNAPylpairs, 1324 triply orthogonal pairs, 128 quadruply orthogonal pairs, and 8 quintuply orthogonal pairs. Example 1 - Cross-reactivity and sequence identity of PylRS / tRNAPyl pairs We previously defined the cross-reactivity profiles of PylRS / tRNAPylpairs belonging to three distinct classes (N, A, and B); we showed that certain non-cognate pairs, drawn from the ΔN PylRS classes A and B, exhibit a surprising degree of natural orthogonality with respect to one another. We postulated that this mutual orthogonality might be related to the sequence identity between the pairs, and we therefore decided to draw on ΔN PylRS / tRNAPylactivity data6to quantify this relationship. Strikingly, we found that if two ΔN PylRS enzymes had a sequence identity of over 55%, then one ΔN PylRS enzyme would show high activity with the tRNAPylthat naturally pair with the other ΔN PylRS enzyme (or vice- versa) in approximately 90% of cases (Fig.1a). Below 55% sequence identity, ΔN PylRS enzymes exhibited a range of activities with the pyl tRNAs of other ΔN PylRS enzymes. Similarly, if two tRNAPylgenes shared a sequence identity of over 75%, then one tRNAPylwould show high activity with the synthetase of the other tRNAPyl(or vice-versa) in approximately 90% of cases (Fig.1b). Below 75% sequence identity, pyl tRNAs exhibited a range of activities with the ΔN PylRS enzymes of other pyl tRNAs. This analysis suggested that the development of new multiply orthogonal pairs should focus on PylRS / tRNAPylpairs whose synthetase and tRNA sequence identities are less than 55% and 75%, respectively. Example 2 - Identification and clustering of PylRS sequences To identify candidate PylRS / tRNAPylcombinations for the development of multiply orthogonal pairs, we first assembled a database of PylRS sequences. By performing a BLAST search for sequence similarity to the Alv ΔN PylRS sequence (class A; henceforth referred to as AΔ-AlvPylRS), we retrieved 351 PylRS protein sequences. Of these, 79 belonged to the archaeal +N group, 66 belonged to the archaeal ΔN group, and 204 belonged to the bacterial (sN) group. In addition, two PylRS genes, despite being classified as archaeal, possessed a separately encoded N-terminal domain – we termed these the archaeal sN group. We performed an agglomerative hierarchical clustering to visualise the sequence diversity among PylRS catalytic domains (Fig.1c). We observed two major groups: a dense cluster consisting of the class N PylRS sequences, and a loose cluster consisting of the bacterial and other archaeal PylRS sequences. The latter cluster itself contained several denser sub-clusters, including those corresponding to the known archaeal class A and B sequences. To discover mutually orthogonal systems we focussed on identifying PylRS sequences with pairwise sequence identities of less than 55% (Fig.1a). To achieve this, we set a linkage distance threshold for the agglomerative clustering such that two clusters would be merged if, and only if, the average of the percentage identities of each PylRS in the two clusters was greater than 55%. This led to 37 clusters (Fig.1d). Three clusters represented the known PylRS classes N, A, and B. By contrast, there were 25 bacterial sN-group clusters and 9 further archaeal clusters (seven ΔN-group and two archaeal sN-group). This analysis demonstrated that substantial sequence diversity among Pyl systems remains to be explored. Example 3 - Identification and clustering of tRNAPyl sequences Next, we chose a representative PylRS enzyme from each of the 37 clusters and aimed to identify the pyl tRNAs from the same organisms. We obtained the DNA sequence of the host genome or metagenomic read containing each PylRS gene, and scanned it for tRNAPylgenes using the tRNA detection program ARAGORN.49In total, we obtained tRNAPylgenes for 35 of the 37 PylRS enzymes. We performed an agglomerative hierarchical clustering of the 35 tRNAPylsequences (Fig.1e). We observed tighter grouping than with the PylRS sequences. Of the nine new archaeal pyl tRNAs, three grouped with class B and six formed a clear albeit more loosely related new grouping, which we termed class C. Meanwhile, bacterial sN pyl tRNAs grouped together strongly; we assigned these to a new class S. Curiously, the pyl tRNAs of the two archaeal sN PylRS enzymes are fairly weakly related and fall into classes B and C, indicating they may not have a common origin. By setting a linkage distance threshold for agglomerative clustering to 75% sequence identity (Fig.1b) we generated eight clusters of tRNAPylgenes (Fig.1f). Of these clusters, three represented the known PylRS classes N, A, and B. There were two bacterial clusters, one of which only contained a single tRNAPyl. The three remaining clusters were from class C, in line with the looser interrelatedness of the members of this class. Some of the newly discovered pyl tRNAs contain exotic structural features that are not observed in canonical class N tRNAPyl(Fig.1g). For instance, certain class S pyl tRNAs contain 6-8 base pair D loops, while several class C pyl tRNAs contain long variable loops. Previous studies have shown that structural elements can strongly influence tRNAPyl:PylRS interactions;7,50indeed, the orthogonality of the PylRS / tRNAPylsystem with respect to endogenous aminoacyl-tRNA synthetases in a variety of host organisms has been attributed to the compact structure of the tRNAPylbody.41Notably, expansions of the tRNAPylvariable loop have previously been used to attenuate cross-reactivity by a non-cognate PylRS class, while maintaining activity with the cognate PylRS class.6,7,44,45Moreover, multiple pyl tRNAs from both classes contain unusual (adenine or uracil) nucleobases at the discriminator base position, which is a known identity element for previously characterised PylRS proteins.32,46Therefore when selecting pyl tRNAs for further characterisation, in addition to ensuring that at least one member from each tRNAPylcluster was picked, we also chose additional pyl tRNAs from certain clusters on the basis of such non-canonical features. We hypothesised that despite their higher (>75%) sequence identities with other selected pyl tRNAs, their structural differences might give rise to mutually orthogonal interactions. In total, we chose 16 pyl tRNAs for further investigation (Fig.1h). This group comprised 13 newly chosen pyl tRNAs, uncharacterised in E. coli (six archaeal class C, and seven bacterial class S), along with three previously characterised pyl tRNAs from classes N, A and B. To finalise our representative set of PylRS / tRNAPylpairs for experimental characterisation, we combined each chosen tRNAPylwith the synthetase from the same organism – with the exception of A-AlvtRNAPyland B-InttRNAPyl, which form highly active heterologous cognate pairs with the previously reported PylRS enzymes AΔ-1R26PylRS and BΔ-Lum1PylRS, respectively (Fig.1h).6We note that, of the ten inter-class relationships, only four have been even partially characterised in E. coli (Fig.1i): N+- MbPylRS is known to interact with S-Desulfitobacterium hafniense (Dh)-tRNAPyl,42while sets of engineered pairs from classes N, A, and B (such as N+-MmPylRS / N- Methanosarcina spelaei (Spe)tRNAPyl, AΔ-1R26PylRS / A- AlvtRNAPyl-8, and BΔ-Lum1PylRS / B- InttRNAPyl-17C10), are known to be triply orthogonal to one another.6,7In preparing the new systems for characterisation, we observed that multiple class S PylRS genes were recalcitrant to cloning, and that even those that could be successfully cloned resulted in reduced growth when expressed in E. coli cells. We hypothesized that these issues might be related to their separately expressed N-terminal domain protein (PylSn), and prepared variants of each class S PylRS system with the PylSn gene removed, which abrogated the toxicity effects. These variants (which we term SΔ) were characterised alongside (or in place of) the wild-type enzymes (which we term S+). Example 4 - New and active PylRS enzymes and pyl tRNAs We measured the activities of each chosen PylRS enzyme with each chosen tRNAPylvia the production of green fluorescent protein (GFP) from a gene coding for GFP containing an amber codon at position 150, in the presence of the non-canonical amino acid N6-((allyloxy)carbonyl)-L-lysine (AllocK 1, Fig.2a) – a known substrate of previously characterised PylRS enzymes (Fig.2b).5115 out of 16 pyl tRNAs gave rise to PylRS-dependent GFP production (at a level at least 30% of that produced from a control GFP gene without an amber stop codon, ‘wtGFP control’) in the presence of at least one PylRS enzyme. This included all class C pyl tRNAs and all but one class S tRNAPyl. In addition, 13 out of 20 PylRS enzymes led to GFP production at a level at least 30% of the wtGFP control, in the presence of at least one tRNAPyl. Moreover, six new PylRS enzymes (CΔ-Nitrososphaeria archaeon (Nitra)PylRS, CΔ- Methanonatronarchaeia archaeon (Tron)PylRS, SΔ-Desulfosporosinus sp. I2 (I2)PylRS, SΔ-Clostridiales bacterium (Clos)PylRS, SΔ-Deltaproteobacteria bacterium (Deb)PylRS, and SΔ-Spirochaetales bacterium (Spi)PylRS) led to GFP production, at a level at least 50% of the wtGFP control, in the presence of the appropriate new class C or S tRNAPyl. Intriguingly, the active class C PylRS enzymes showed considerable specificity towards certain class C pyl tRNAs over class S pyl tRNAs. In particular, C-TronPylRS, which was highly active with the C-TrontRNAPyl(76% of wtGFP control), had less than 10% activity with all but one class S tRNAPyltested. Most active class S PylRS enzymes aminoacylated pyl tRNAs within both class C and class S. However, C-Candidatus Methanohalarchaeum thermophilum 1 (Therm1)tRNAPyl(and to a lesser extent, C-Candidate division MSBL1 archaeon SCGC-AAA382A20 (SCGC)tRNAPyl) was poorly recognised by most active class S PylRS enzymes but formed a highly active pair with CΔ-NitraPylRS. With regard to the previously characterised pyl tRNAs,6,7most class C and class S PylRS enzymes proved highly active with B-InttRNAPyl, giving rise to GFP levels over 80% of the wtGFP control in some cases. In addition, several class C and class S PylRS enzymes also showed moderate to strong activity with N-MmtRNAPyland AΔ- AlvtRNAPyl. Of the previously characterised PylRS enzymes, N+-MmPylRS was by far the most promiscuous, giving rise to over 50% of wild-type GFP production levels in the presence of eleven out of 16 pyl tRNAs (including all but two class S pyl tRNAs). These included S-ClostRNAPyland S-DebtRNAPyl, which showed only modest activity with the most active class S PylRS enzymes. To a lesser extent, class A and class B PylRS enzymes also cross-reacted with certain class S and class C pyl tRNAs. Despite this, we were pleased to observe that C-SCGCtRNAPyland C-Therm1tRNAPylwere orthogonal to N+-MmPylRS, AΔ-1R26PylRS, and BΔ- Lum1PylRS; this demonstrated that naturally occurring tRNAPylcan be found that are orthogonal to PylRS enzymes taken from all other classes. Two wild-type class S PylRS enzymes, S+- Gemmatimonadetes bacterium (Gem)PylRS and S+-DebPylRS, were expressed and showed convincing activity. S+-GemPylRS exhibited similar tRNAPylspecificity to its SΔvariant. However, S+-DebPylRS, the most active S+system characterised, showed a markedly different activity profile to SΔ-DebPylRS, for instance having much higher activity with A-AlvtRNAPyl(72% versus 2% of wtGFP control, respectively), but much lower activity with C-TrontRNAPyl(10% vs 64%). This is consistent with reports that PylSn proteins modulate tRNAPylspecificity.34,42Example 5 - Mutually orthogonal PylRS / tRNAPyl pairs Next, we determined whether any of the pyrrolysine systems we had discovered formed naturally mutually orthogonal sets. We first defined the criteria for mutually orthogonal pairs by reference to the interactions between them. The network of interactions between multiple aaRS / tRNA pairs may be represented as a matrix where each element ^^^ is the activity of the aaRS protein of column ^ with the tRNA of row ^ (measured in this case by GFP(150AllocK)His6 production in the presence of aaRSj and tRNAi). When aaRSi denotes the cognate aaRS of tRNAi all diagonal elements ^^^ represent the paired activities we wish to maximise. All off-diagonal elements represent the cross-reactivity between a non-cognate aaRS and tRNA, which should be minimised. A diagonal interaction matrix of order ^ therefore represents a perfectly orthogonal set of ^ pairs. In order to exclude sets of pairs with unacceptably low activity, or unacceptably high cross-reactivity, we deemed that the activity of a cognate pair should be greater than 40% of wild-type GFP production, but that each cross- reactivity (between a tRNAPyland a PylRS enzyme belonging to different pairs) should be less than 20% of wild- type GFP production. Since pairs composed of a PylRS and tRNAPylfrom different organisms can have activity equal to, or exceeding, that of the corresponding homologous pairs, we included heterologous PylRS / tRNAPylcombinations as possible cognate pairs in our search. In addition, we defined a new metric, henceforth known as ‘orthogonality coefficient’ (o.c.), as the quotient of the lowest intra-pair activity over the highest inter-pair cross- reactivity. This metric provides a quantitative measure of mutual orthogonality between a set of aaRS / tRNA pairs. Previously characterised triply orthogonal pairs (N+-MmPylRS / N-SpetRNAPyl, AΔ-1R26PylRS / A-AlvtRNAPyl-8, BΔ-Lum1PylRS / B-InttRNAPyl-17C10) used for the incorporation of three distinct non-canonical amino acids have an o.c. of approximately 5.0,6however, we reasoned that since mutual orthogonality could be improved by further engineering, a lower cut-off (o.c. > 2.5) would be more useful in initial screens. In our initial search we considered an interaction matrix to be sufficiently orthogonal if: (i) all diagonal elements were greater than 40% of the wtGFP control, (ii) all off-diagonal elements were less than 20% of the wtGFP control, and (iii) the quotient of the smallest diagonal element over the largest off-diagonal element was greater than 2.5. We uncovered 46 doubly orthogonal pairs (henceforth referred to as ‘doublets’); the highest doublet o.c. is 15.7. Since many doublets involve the same two PylRS enzymes, and differ only in the pyl tRNAs used, we grouped these doublets into families that use the same two PylRS enzymes. We thus obtained fifteen doublet families (Fig. 2c); all but one family contains a new class C or class S PylRS enzyme. Similarly, we obtained two triply orthogonal pairs (or ‘triplets’), both from the same family; the highest triplet o.c. is 2.9 (Fig.2d). These families shed important insights on the PylRS / tRNAPylactivity profiles. As expected, the highly orthogonal class C pyl tRNAs – C-Therm1tRNAPyland C-SCGCtRNAPyl– appear in doublets when either tRNAPylis paired with CΔ-NitraPylRS. However, these pyl tRNAs also form a surprising inter-class doublet family when paired respectively with SΔ-I2PylRS and SΔ-ClosPylRS; this doublet forms part of a triplet family with an N+-MmPylRS pair (e.g. N+-MmPylRS / S-SpitRNAPyl). Further doublet families involving S+or SΔPylRS enzymes (e.g. with S+- DebPylRS and SΔ-DebPylRS, or SΔ-DebPylRS and SΔ-I2PylRS) illustrate not only divergence between S+PylRS enzymes and their SΔvariants, but between different SΔPylRS variants. As such, we do not consider SΔPylRS enzymes as a distinct class, but rather as synthetically derived PylRS variants that expand the ΔN group (Fig.2e). The relationship between the five PylRS classes may itself be described on the basis of the doubly orthogonal pairs formed by representative PylRS enzymes from each class and the appropriate pyl tRNAs (which may or may not belong to the same classes). For five of these ten inter-class relationships, we obtained mutually orthogonal representative PylRS / RNAPylpairs (Fig.2f). For the remaining five, no PylRS / tRNAPylpairs met our criteria for mutual orthogonality. Two cases for lack of mutual orthogonality between general PylRS classes R1 and R2 can be defined: (1) 'two-sided cross-reactivity’ – for any two pairs of the form R1-PylRS / Ti-tRNAPyland R2- PylRS / Tj-tRNAPyl(where Ti and Tj are arbitrary tRNA classes), both cross-reactivities R1-PylRS / Tj-tRNAPyland R2-PylRS / Ti-tRNAPylare too high (i.e. off-diagonal elements in the interaction matrix are greater than 20% wtGFP control or result in o.c. < 2.5); (2) ‘one-sided cross-reactivity’ – there exist pairs R1-PylRS / Ti-tRNAPyland R2-PylRS / Tj-tRNAPylsuch that only one cross-reactivity R1-PylRS / Tj-tRNAPylor R2-PylRS / Ti-tRNAPylis too high (i.e. only one off-diagonal element in the interaction matrix is greater than 20% wtGFP control or results in o.c. < 2.5). Strikingly, all five non-orthogonal inter-class relationships fall into the second class. Therefore, of the 25 pairwise specificities required to make a set of quintuply orthogonal pairs (using one PylRS from each of the five classes), our computational approach was able to resolve 20 of these – all five cognate interactions and 15 out of 20 non-cognate interactions. Example 6 - Eliminating inter-class cross-reactivities As a starting point for generating a quintuply orthogonal pair, we examined the inter-class interaction matrix for five specific PylRS / tRNAPylpairs (Fig.3a-b). For classes N, A, and B, we used pairs N+-MmPylRS / N- MmtRNAPyl, AΔ-1R26PylRS / A-AlvtRNAPyl, and BΔ-Lum1PylRS / B-InttRNAPyl, since these were the starting point for a previously reported triplet.6 For class C, a natural starting point is CΔ-NitraPylRS / C-Therm1tRNAPyl, the most active class C pair for which the tRNAPylis orthogonal to all other PylRS classes. For class S, a good starting point is harder to define given diverse PylRS activity profiles and greater cross-reactivity of N+-MmPylRS with class S pyl tRNAs; we simply chose the most active pairing of a wild-type class S PylRS enzyme, S+-DebRS / S- SpitRNAPyl. Of the twenty possible inter-class synthetase / tRNA interactions in the matrix (off-diagonal elements), only nine are sufficiently low to meet our initial criterion for cross-reactivity (less than 20% of wild-type GFP production levels). In order to bring the other eleven interactions under this threshold, we sought to replace the tRNAs involved in undesired cross-reactions (off diagonal interactions) with more orthogonal variants, i.e. substitute the rows of the interaction matrix such that the off-diagonal elements are progressively eliminated. To find a class N tRNAPylwith orthogonality to all other classes, we screened seven previously reported pyl tRNAs from homologous class N Pyl systems (Fig.3c-d).6 To our delight, N-Methanococcoides methylutens (Met)tRNAPyland N-Methanococcoides burtonii (Bur)tRNAPylgave rise to around 10% or less activity with class A, B, C, and S PylRS enzymes while retaining over 85% of the activity of N-MmtRNAPylwith N+-MmPylRS. To find a class A tRNAPylwith orthogonality to all other classes, we screened ten previously reported A- AlvtRNAPylengineered variants (Fig.3e-f).7Pleasingly, two pyl tRNAs (A-AlvtRNAPyl-17and A-AlvtRNAPyl-21) gave rise to less than 10% of wild-type GFP levels in the presence of class N, B, C, and S PylRS enzymes while retaining over 70% of activity with AΔ-1R26PylRS. To find a class B tRNAPylwith orthogonality to all other classes, we screened seven previously reported B- InttRNAPylvariants (Fig.3g-h).6 However, although orthogonality to class N, A, and S PylRS proteins was obtained, all tested pyl tRNAs gave rise to significant levels of GFP production (over 40%) in the presence of CΔ- NitraPylRS. Since the B-InttRNAPylvariants carry a range of mutations from their parent tRNAPylin both the acceptor stem and variable loop, we hypothesised that CΔ-NitraPylRS may recognise multiple identity elements in B-InttRNAPyland therefore that its interaction cannot easily be abrogated without also destroying recognition with BΔ-Lum1PylRS. Overall, the screen led to the abrogation of a further nine inter-class PylRS / tRNAPylinteractions (Fig.3i); this left only two undesired interactions – between the class C PylRS and class B tRNAPyl, and between the class N PylRS and class S tRNAPyl. Notably, three out of five tRNAs now fulfilled all orthogonality requirements. Our results demonstrate the substantial extent to which the evolutionary divergence of PylRS sequences can be exploited to generate orthogonal interactions. Example 7 - Quadruply orthogonal PylRS / tRNAPyl pairs We investigated additional approaches to eliminate inter-class (off-diagonal) interactions for a fourth PylRS / tRNAPylpair, and form a mutually orthogonal quadruplet. As noted above, the engineered SΔPylRS variants belong to the expanded ΔN group (Fig.2e). The activities of different SΔvariants resemble the activities of different ΔN classes; for instance SΔ-I2PylRS is active with C- Therm1tRNAPyl(Fig.2b; much like CΔ-NitraPylRS), and SΔ-ClosPylRS is active with engineered B-InttRNAPylvariants (Fig.3g; much like BΔ-Lum1PylRS). We therefore speculated that substitution of the class B or C pair with a pair containing a SΔPylRS supplying a desired B-like or C-like activity might resolve the issue of cross- reactivity between class C PylRS enzymes and class B pyl tRNAs (Fig.4a). We refer to these substituting PylRS variants as SΔBor SΔC, respectively. In terms of the interaction matrices, this entailed substitution of columns such that off-diagonal interactions were eliminated. After allowing the replacement of class B or C PylRS enzymes with SΔPylRS variants, we obtain a total of 946 doublets in 25 families, 1425 triplets in 16 families, and – crucially – 96 quadruplets in four families (Fig.4b-c). Notably, the highest triplet o.c. is 24.5 – approximately five times higher than a previously reported triplet used for the incorporation of three distinct non-canonical amino acids (N+-MmPylRS / N-SpetRNAPyl, AΔ-1R26PylRS / A- AlvtRNAPyl-8, BΔ-Lum1PylRS / B-InttRNAPyl-17C10), and approximately two times higher than the highest o.c. triplet from the previously reported N+-MmPylRS, AΔ-1R26PylRS, BΔ-Lum1PylRS family.6 Most triplet and all quadruplet families involve substitution of the class B and / or class C pair with pairs containing SΔPylRS variants. To understand how far the SΔPylRS substitution strategy had advanced the development of quintuply orthogonal pairs, we considered the new quadruplets in the context of our inter-class interaction network (Fig.4d-h). For the [A, SΔB, C, S] (o.c.3.9) and [A, B, SΔC, S] (o.c.2.5) quadruplet families formed with a single SΔPylRS variant substituting for a class B or class C PylRS respectively, the cross-reactivity between classes B and C (Fig.3h) had effectively been replaced by cross-reactivity between class N and SΔBor B (Fig.4d-f). This results in the generation of a diagonal submatrix of order 4 in the overall interaction matrix, and thus the quadruplets. However, including cross-reactivity between classes N (N+-MmPylRS) and S (any tRNAPylpaired with S+-DebPylRS), two cross-reactivities still remained to be eliminated. Meanwhile, for the [N, A, SΔB, SΔC] and [A, SΔB, SΔC, S] quadruplets (both o.c.2.9) formed via substitution of both class B and class C PylRS enzymes with SΔPylRS variants, one main cross-reactivity (between N+-MmPylRS and any tRNAPylpaired with S+-DebPylRS) persisted (Fig.4g-h). However, there also remained some residual cross-reaction between the class SΔBand SΔC22 pairs which would restrict the o.c. of any potential quintuplets. We hypothesised that a general solution to all of these cross-reactivity problems would be further engineering of the pyl tRNAs paired with class B / SΔBand class S PylRS enzymes. Example 8 - Quintuply orthogonal PylRS / tRNAPyl pairs We aimed to: (1) discover a tRNAPylthat functions with a class B PylRS enzyme (or SΔBPylRS variant) but is orthogonal to all other PylRS classes, (2) discover a tRNAPylthat functions with a class S PylRS but is orthogonal to all other PylRS classes, and (3) replace the pyl tRNAs that pair with SΔB-ClosPylRS and S+-DebPylRS with our new pyl tRNAs in the highest o.c. quadruplet. We anticipated that this would mitigate undesired cross-reactivity with a fifth pair and thereby enable the generation of a quintuply orthogonal set of pairs (Fig.5a). We noted that CΔ-NitraPylRS was active with engineered B-tRNAPylvariants (Fig.3g), and postulated that a different parent tRNAPyl, such as one from a bacterial class S system, might provide a better starting point for the discovery of a class B- or SΔB-specific tRNAPylvariant with orthogonality towards CΔ-NitraPylRS (and the other PylRS enzymes, as necessary), via directed evolution. We chose the bacterial tRNAPylS-I2tRNAPylas a starting point for directed evolution, given its exceptional activity with SΔB-ClosPylRS (giving rise to 93% of wild-type GFP levels), yet fairly modest cross-reactivity with CΔ-NitraPylRS (23% of wild-type GFP levels). In fact, S-I2tRNAPylis most cross-reactive with N+-MmPylRS (68% of wild-type GFP levels); accordingly, we focussed our efforts on abrogating S-I2tRNAPylrecognition by N+-MmPylRS. Previous efforts demonstrated that N+-MmPylRS can be much more sensitive to expansions in the short variable loop of tRNAPylthan ΔN PylRS enzymes from classes A7 and B6, consistent with structural and biochemical studies.34,42Indeed, A-AlvtRNAPyland B-InttRNAPylvariants (Fig.3e and Fig.3g), which (unlike their parent pyl tRNAs)–are orthogonal to N+-MmPylRS, were obtained from libraries of mutants in which the variable loop was expanded (from three bases) to four, five, or six randomized nucleotides.6,7For instance, B-InttRNAPyl-B03, which differs from its parent tRNAPylby the insertion of a single uracil in its variable loop, is over thirty-fold less active with N+-MmPylRS than the parent tRNAPyl. Since SΔB-ClosPylRS is an artificial ΔN class PylRS, we hypothesised that it may tolerate insertions into the variable loop of S-I2tRNAPyl, and synthesised a library of S-I2tRNAPylmutants in which the variable loop was expanded to four nucleotides (Fig.5b). Positions 8 and 22, which could make important tertiary contacts with the variable loop, were also randomised. In addition, to widen the diversity of activity profiles within the library, three bases thought to be generally important to the recognition of S-I2tRNAPylwere also randomised; namely, position 69 (the discriminator base, an important identity element for PylRS), and positions 5 and 64. The latter two form a wobble base pair in the S-I2tRNAPylacceptor stem; such pairs are known to distort RNA helices and thereby dictate aminoacyl-tRNA synthetase recognition.52We selected S-I2tRNAPylmutants that allowed cells also expressing SΔB-ClosPylRS to grow on 100 μg mL-1 chloramphenicol in the presence of AllocK 1, by enabling production of protein from a gene coding for chloramphenicol acetyl transferase containing an amber codon at position 111. We then performed successive negative screens on the selected S-I2tRNAPylvariants to identify pyl tRNAs that have minimal cross-reactivity with N+-MmPylRS, AΔ-1R26PylRS, CΔ-NitraPylRS, and S+-DebPylRS. Cells harbouring GFP(150TAG)His6, an S-I2tRNAPylvariant, and one of N+-MmPylRS, AΔ-1R26PylRS, CΔ-NitraPylRS, or S+-DebPylRS were provided with AllocK and screened for the absence of GFP expression. These screens revealed three library members, S- I2tRNAPyl-B8, S-I2tRNAPyl-B32, and S-I2tRNAPyl-B72, which remain highly active with SΔB-ClosPylRS but have little cross-reactivity with N+-MmPylRS, AΔ-1R26PylRS, CΔ-NitraPylRS, and S+-DebPylRS (Fig.5c-d). In particular, S-I2tRNAPyl-B32and S-I2tRNAPyl-B72both retain over 75% of the activity of their parent S-I2tRNAPylwith SΔB- ClosPylRS but have over 20-fold lower activity than their parent tRNA with N+-MmPylRS and around three-fold lower activity than their parent with CΔ-NitraPylRS. While performing negative screens on S-I2tRNAPylmutants, we observed that – despite their expanded variable loops – some S-I2tRNAPylmutants have increased activity with S+-DebPylRS. We hypothesised that the N- terminal domain protein in the split S+-DebPylRS enzyme may have different recognition of variable loop nucleotides from N+PylRS. Therefore, to discover a tRNAPylthat pairs with a class S PylRS that is orthogonal to all other PylRS classes used in a quintuplet, we selected S-I2tRNAPylexpanded variable loop mutant library members that are selectively aminoacylated by S+-DebPylRS. We performed a positive selection for S-I2tRNAPylmutants that are active with S+-DebPylRS, followed by negative screening to minimize cross-reactivity with N+-MmPylRS, AΔ-1R26PylRS, SΔB-ClosPylRS, and CΔ- NitraPylRS. These screens identified one library member, S-I2tRNAPyl-S52, which is active with S+-DebPylRS (43% of wild-type GFP production) but possesses very low activity with SΔ-ClosPylRS, N+-MmPylRS, AΔ- 1R26PylRS, and CΔ-NitraPylRS (all less than 2% of wild-type GFP production) (Fig.5e-f). When compared with the wild-type parent tRNA S-I2tRNAPyl, S-I2tRNAPyl-S52is over three-fold more active with S+-DebPylRS and 120-fold less active with SΔB-ClosPylRS. By substituting our evolved S-I2tRNAPylmutants into the quadruplet with the highest o.c. (from the [A, SΔB, C, S] family), we minimized cross-reactivities with N+-MmPylRS. This enabled us to combine the updated quadruplets with a fifth orthogonal pair from class N (for example N+-MmPylRS / N-BurtRNAPyl) to generate a family of mutually orthogonal quintuplets with o.c. values of up to 4.0 (Fig.5f). Thus, by mutation, selection and screening from a single tRNA scaffold we eliminated the two final cross-reactivities (Fig.5g). Despite their very different activities, S-I2tRNAPyl-B32and S-I2tRNAPyl-S52differ by only four nucleotides; this demonstrates the power of synthetically engineering identity elements to control tRNA recognition by aminoacyl-tRNA synthetases. Because S-I2tRNAPyl-S52is also orthogonal to both BΔ-Lum1PylRS and SΔC-I2PylRS, we obtained two additional quintuplet families, by substitution of SΔB-ClosPylRS with BΔ-Lum1PylRS, or CΔ-NitraPylRS with SΔC-I2PylRS. Overall, using our original o.c. threshold of 2.5, we obtained 1136 doublets in 27 families (highest o.c.72.0), 2359 triplets in 26 families (highest o.c.39.6), 919 quadruplets in 14 families (highest o.c.7.2), and 90 quintuplets in 3 families (highest o.c.5.4). Upon increasing the o.c. threshold to 5.0 – comparable to the o.c. value for PylRS / tRNAPylpairs previously used for incorporating three distinct non-canonical amino acids – we obtained 924 doublets in 22 families, 1324 triplets in 18 families, 128 quadruplets in 7 families, and 8 quintuplets in 1 family (Fig.5h-i). The quintuplets with the highest o.c. values have the following composition: N+-MmPylRS with a (non-cognate) natural class N tRNAPyl; AΔ-1R26PylRS with an engineered A-AlvtRNAPylvariant; BΔ-Lum1PylRS with an engineered S-I2tRNAPylvariant; CΔ-NitraPylRS with a (non-cognate) natural class C tRNAPyl; and S+- DebPylRS with an engineered S-I2tRNAPylvariant (Fig.5j). We characterized the amber suppression efficiency and accuracy of each quintuply orthogonal PylRS / tRNAPylpair in the most orthogonal quintuplet by producing GFP150AllocKHis6 and Ub11AllocKHis6 from GFP150TAGHis6 or Ub11TAGHis6 respectively, and measuring protein titres as well as MS spectra. Our results demonstrate the successful and unprecedented division of homologous PylRS / tRNAPylsystems into five classes that are mutually orthogonal in their aminoacylation specificity (Fig.5k). Example 9 - Discussion We have defined sequence identity threshold criteria to effectively search genomic data for PylRS and tRNAPylorthogonality. By applying these thresholds to generate sequence clusters we have computationally searched PylRS sequences for multiply orthogonal systems. Using this approach we have identified orthogonal systems from hundreds of PylRS systems spanning all PylRS groups. By combining our computational approach with directed evolution and engineering we have generated the first quadruply and quintuply orthogonal PylRS systems. These advances, along with strategies for generating codons that can be used to encode non-canonical monomers, will be central to the synthesis of proteins containing an increasing number of ncAAs, and the encoded cellular synthesis of more diverse polymers and macrocycles.2,3,18,19,21-26Moreover, mining the data generated through our approach may provide further insight into the sequence requirements for mutually orthogonal systems and enable the creation of more refined rules for predicting orthogonality. Example 10 - Methods Identification of PylRS sequences We identified PylRS sequences by performing a BLAST search against the NCBI non-redundant protein sequence database using the AΔ-AlvPylRS protein as the query sequence, filtering for expected values below 1 x 10-30. Partial protein sequences and sequences from synthetic constructs were removed by manual inspection, and we ultimately obtained 351 PylRS sequences. We collected these sequences into a database, where each PylRS sequence was assigned a unique identifier based on the organism name and the NCBI Accession ID. We aligned the obtained PylRS sequences using Clustal Omega53and extracted sequences corresponding to the C terminal domain (CTD) of the PylRS enzymes, with reference to the known annotation of the CTD in N+-MmPylRS6. Identification of tRNAPyl sequences Using the NCBI Nucleotide database, we obtained all available nucleotide sequences of the host genome or metagenomic read containing each of our identified PylRS sequences. We ran the tRNA detection program ARAGORN49(version 1.2.38) on each nucleotide sequence, allowing for introns of up to 100 nucleobases and scoring thresholds at 90% of default levels. Discovered pyl tRNAs were added to the PylRS sequence database. For the PylRS sequence with identifier Marc.6481 (isolated from candidate division MSBL1 archaeon SCGC- AAA382A20, subsequently referred to as CΔ-SCGCPylRS), no corresponding tRNA could be found by ARAGORN, but a putative sequence had been previously reported54and was therefore added to the database. Meanwhile, PylRS sequences with identifiers Mthe.7552 and Mthe.9096 were found to originate from the same organism (Candidatus Methanohalarchaeum thermophilum), but ARAGORN only found a corresponding tRNAPylproximate to one of the PylRS sequences (Mthe.7552, subsequently referred to as CΔ-Therm1PylRS). Manual searching of the nucleotide sequence revealed a second tRNAPylproximate to the other PylRS sequence (Mthe.9096, subsequently referred to as CΔ-Therm2PylRS); this was also added to the database. During preparation of this manuscript these two pyl tRNAs were independently reported.46After manual curation to remove pseudogenes, we obtained pyl tRNAs for 284 PylRS genes. Analysis of previously characterised PylRS / tRNAPylpairs From our database of PylRS and tRNAPylsequences, we obtained the sequences of class A and B PylRS and tRNAPylsequences that we had previously characterized experimentally.6A matrix of pairwise sequence percentage identities for all pairs of PylRS sequences and all pairs of tRNAPylwas then calculated using python (version 3.9.7).55For PylRS sequences, percentage identities were calculated from the multiple sequence alignment of C terminal domains. For tRNAPylsequences, percentage identities were calculated from a manually performed multiple sequence alignment that was based on secondary structure predictions from ARAGORN and RNAfold.56,57We considered the experimental activity data previously reported for these sequences, namely the level of GFP production from a GFP gene containing an in-frame amber codon at position 150 (GFP150TAGHis6) obtained in the presence of each combination of PylRS enzyme, and tRNAPylCUA, as well as 8 mM Nε-Boc-L- lysine (subtracted by the level of GFP production in the presence of only GFP gene and tRNAPylCUA, due to background aminoacylation of the tRNAPylCUA by endogenous aaRSs). For each combination of PylRS enzyme and tRNAPylCUA, we plotted this activity against the percentage sequence identity of the PylRS sequence with the sequence of the PylRS from the same organism as the tRNAPylCUA. Similarly, we plotted the activity for each combination of PylRS enzyme and tRNAPylPyl CUA against the percentage identity of the tRNA CUA with the sequence of the tRNAPylCUAfrom the same organism as the PylRS enzyme. Clustering of PylRS C terminal domain sequences Using python (version 3.9.7), we calculated a matrix of percentage identities from the multiple sequence alignment of C terminal domains for all pairs of PylRS sequences in our database. We then used this matrix to perform unweighted average linkage agglomerative hierarchical clustering (UPGMA) of aligned PylRS CTD sequences with a cluster merging threshold of 55% sequence identity, using the biopython (version 1.79) and the scikit-learn (version 1.0.1) python libraries.55,58Alignment and clustering of tRNAPylsequences For each PylRS cluster, we chose a representative PylRS sequence for which a corresponding tRNAPylsequence could be found. For two of the 37 PylRS clusters, no tRNAPylsequence was found; these were excluded from further analysis. For the 35 obtained pyl tRNAs, we performed a manual multiple sequence alignment that was based on secondary structure predictions from ARAGORN and RNAfold. This multiple sequence alignment was then used to calculate a matrix of percentage identities for all pairs of chosen tRNAPyl, using the biopython (version 1.79) and scikit-learn (version 1.0.1) python libraries. We then used this matrix to perform unweighted average linkage agglomerative hierarchical clustering (UPGMA) of aligned chosen tRNAPylsequences with a cluster merging threshold of 75% sequence identity. DNA constructs PylRS and tRNAPylgenes were synthesized by IDT as gBlock double-stranded DNA fragments. We cloned all new pyl tRNAs into a minimal pMB1 backbone under an lpp promoter. Previously reported pyl tRNAs were used in the same format. Certain tRNAs differed from the canonical sequence at the anticodon loop; these positions were mutated to the consensus bases found in E. coli to improve the efficiency of the tRNAs in E. coli translation as has been previously described.6 PylRS sequences were cloned into a p15A backbone under a glnS promoter. For each new PylRS sequence, a 5’ untranslated region was generated using the online tool De Novo DNA59predicted to maximise translation initiation efficiency and inserted between the +1 site of the glnS promoter and the start codon of the gene. For class S PylRS enzymes, polycistronic operons consisting of the separately expressed N- and C-terminal domains were constructed using intergenic regions predicted by De Novo DNA. The optimal arrangement of the two domains was chosen by maximizing predicted translation initiation rates. N+MmPylRS, AΔ-AlvPylRS, and BΔ-Lum1PylRS were used in similar p15A constructs containing C-terminal tags as previously described.6The p15A vectors also encoded a chloramphenicol acetyltransferase (CAT) gene with an amber codon at position 111 under a constitutive cat promoter and a GFP gene with an amber codon at position 150 under an L-arabinose inducible pBAD promoter. Measuring the activity and specificity of PylRS / tRNAPylCUA pairs To measure the activity of the PylRS / tRNAPylCUA pairs we transformed 0.4 μL of pMB1 plasmid encoding a tRNAPylCUA gene into 4-10 μL E.coli DH10B chemically competent cells bearing a p15A plasmid encoding a PylRS gene, a CAT111TAG gene, as well as a GFP150TAGHis6 gene. We recovered the transformed cells for approximately 1 h at 37°C and 750 r.p.m. in 180 μL of SOC medium (Super optimal broth with catabolite repression) in a 96 well Costar microtitre plate format. We then used 40 μL of the rescued cells to inoculate 760 μL of selective 2xYT-st (2xYT medium containing 75 μg mL−1 spectinomycin and 12.5 μg mL-1 tetracycline) medium in a 1.2 mL 96 well plate format and the cultures were grown overnight at 37°C and 750 r.p.m. After a minimum of 16 h, 40 μL of the overnight cultures were used to inoculate 760 μL of 2xYT-st medium, containing 0.05% L-arabinose and 4 mM Nε-Alloc-L-lysine (AllocK), in a 1.2 mL 96 well plate format. Cells were grown for 18-24 h at 37°C and 750 r.p.m. Ultimately, 100 μL of each culture was transferred into 96 well flat bottom Costar plates and fluorescence and optical density (OD) were measured using a PHERAstar FS plate reader. Measured GFP OD600-1values were normalised by the GFP OD600-1value of cells expressing GFP from a GFP150AsnHis6 gene (referred to as ‘wtGFP control’). Identification of sets of mutually orthogonal PylRS / tRNAPylpairs Using python (version 3.9.7), we identified sets of mutually orthogonal PylRS / tRNAPylpairs based on the GFP activity data. For any given set of PylRS / tRNAPylpairs, the quotient of the lowest intra-pair activity over the highest inter-pair cross reactivity was defined as the orthogonality coefficient, o.c.. Sets of pairs were considered mutually orthogonal if the lowest intra-pair activity was greater than 40% of the wtGFP control, the highest inter- pair cross-reactivity was less than 20% of the wtGFP control, and the o.c. was higher than 2.5. We grouped mutually orthogonal sets together into families if they involved the same PylRS enzymes. S-I2tRNAPylCUA library generation The library of S-I2tRNAPylCUA with randomized nucleotides was constructed by Golden Gate cloning into a pMB1 vector using PCR primers, a Q5 DNA polymerase, a Bbs1-HF restriction enzyme, and a T4 DNA ligase (all enzymes were purchased from New England Biolabs (NEB)). The library was transformed into electrocompetent E.coli DH10B cells with a transformation efficiency of more than 1x108colony forming units. Selection and screening to identify orthogonal S-I2tRNAPylCUA hits The S-I2tRNAPylCUA library was transformed into electrocompetent E.coli DH10B cells bearing a p15A plasmid encoding CAT(111TAG), GFP(150TAG)His6 and either SΔ-ClosPylRS or S+-DebPylRS. Cells were recovered for one hour in 1 mL SOC at 37°C 220 r.p.m. supplemented with AllocK and plated onto LB agar plates containing 4 mM AllocK, 75 μg mL-1 spectinomycin, 12.5 μg mL-1 tetracycline and 100 μg mL-1 chloramphenicol. The plates were incubated at 37°C for 18-24 h. After incubation a combined total of 576 colonies from either selection (colonies grown in presence of SΔ-ClosPylRS or S+-DebPylRS, respectively) were picked into 500 μL 2xYT-st and the colonies were grown over night at 750 r.p.m. and 37°C.40 μL of the overnight culture were then given into 760 μL 2xYT-st containing 0.05% L-arabinose in presence and absence of 4 mM AllocK. Plasmids from clones which were selectively fluorescent in presence of AllocK were extracted (DNA miniprep from Qiagen), digested with NcoI restriction enzyme and T5 exonuclease (both from NEB) and retransformed into chemically competent cells bearing a p15A plasmid encoding CAT(111TAG), GFP(150TAG)His6 and one of the following PylRS genes – N+-MmPylRS, SΔ-ClosPylRS, CΔ-NitraPylRS, AΔ-1R26PylRS, or S+-DebPylRS. Plasmids of clones which fulfilled the orthogonality requirements were isolated and sequenced. Quantifying GFP150AllocKHis6 and Ub11AllocKHis6 protein production yields with quintuply orthogonal PylRS / tRNAPylCUA pairs To measure the protein yield for single ncAA incorporations with the quintuply orthogonal PylRS / tRNAPylCUA pairs from the set with the highest o.c., we co-transformed 0.8 μL of pMB1 plasmid encoding a tRNAPylCUA gene and p15A 0.8 μL plasmid encoding a PylRS gene, a CAT111TAG gene, as well as a GFP150TAGHis6, or Ub11TAGHis6 gene into competent E.coli DH10B by electroporation. As controls we also co-transformed GFPHis or Ub11TCAHis6 together with AlvtRNAPyl-21CUA and MmPylRS in the same plasmid set-up. We recovered the transformed cells for approximately 1 h at 37°C and 220 r.p.m. in 600 μL of SOC medium (Super optimal broth with catabolite repression). We used 160 μL of the rescued cells to inoculate 5 mL of selective 2xYT-st (2xYT medium containing 75 μg mL−1spectinomycin and 12.5 μg mL-1tetracycline) medium in a 50 mL glass tubes and the cultures were grown overnight at 37°C and 220 r.p.m in a shaking incubator. After a minimum of 16 h, 140 μL of the overnight cultures were used to inoculate 5 mL of 2xYT-st medium, containing 0.05% L-arabinose and 4 mM Nε-Alloc-L-lysine (AllocK), in a 50 mL glass tube. Cells were grown for 16-18 h at 37°C and 220 r.p.m. Cells were spun down, aspirated and the cell pellets were frozen at -20 ˚C for a minimum of 1 h. The pellets were resuspended in 800 μL BugBuster® Protein Extraction Reagent containing cOmplete™ protease inhibitor and lysed for one hour with head-over-tail rotation. Lysed cells were spun down and the supernatant incubated for 1-16 h at 4 ˚C with 160 μL NiNTA agarose beads. The beads were washed five times with 800 μL 25 mM imidazole in PBS at pH 8.5 and the proteins were eluted five times with 160 μL 250 mM imidazole in PBS pH 8.5 (for GFP samples), or five times with 100 μL 250 mM imidazole in PBS pH 8.5 (for Ub samples). Protein concentrations for GFP were measured by quantifying the absorption at 280 nm. Protein concentrations for ubiquitin were measured using Pierce™ BCA Protein Assay Kit from Thermo Fisher following the manufacturers protocol. Electrospray ionization mass spectrometry Denatured protein samples (~10 μM) were subjected to liquid chromatography–mass spectrometry analysis. Briefly, proteins were separated on a C4 BEH 1.7 μm, 1.0 × 100 mm ultraperformance liquid chromatography column (Waters) using a modified nanoAcquity (Waters) to deliver a flow of approximately 50 μl min−1. The column was developed over 20 min with a gradient of acetonitrile (2–80% v / v) in 0.1% v / v formic acid. The analytic column outlet was directly interfaced via an electrospray ionization source, with a hybrid quadrupole time-of-flight mass spectrometer (Xevo G2, Waters). Data were acquired over a m / z range of 300–2,000, in positive-ion mode with a cone voltage of 30 V. Scans were summed together manually and deconvoluted using MaxEnt1 (Masslynx, Waters). The theoretical molecular weights of proteins with ncAAs was calculated by first computing the theoretical molecular weight of wild-type protein using an online tool (http: / / web.expasy.org / protparam / ) and then manually correcting for the theoretical molecular weight of ncAAs. Example 11 – Table of PylRS for clustering analysis Table 1 )ssalcre r p eet? u s e.sma v s NulitO sNDn al∆ Nficifre nCiIiacr foi5at5rneQ m e(sitnits ndiseDIets serE S o p s D u SaleiC c e x cPSdIa T c A S Rulp Cer64 Arch ∆N Thaumarchaeota archaeon Tarc.8235 2026795 RLF98235.1 0 65 Bact S Proteobacteria bacterium Pbac.9365 1977087 NIQ39365.1 0 66 Bact S Desulfitibacter sp. BRH_c19 Dsp.1752 1734395 KUO51752.1 0 67 Bact S Thermincola ferriacetica Tfer.8912 281456 WP_052218912.1 0 68 Bact S Thermincola potens Tpot.8946 863643 WP_013118946.1 0 69 Bact S Desulfosporosinus sp. HMP52 Dsp.1943 1487923 WP_034601943.1 0 70 Bact S Desulfosporosinus meridiei Dmer.2489 79209 WP_014902489.1 0 71 Bact S Desulfosporosinus hippei Dhip.5591 569859 WP_092335591.1 0 72 Bact S Clostridia bacterium Cbac.0019 2044939 MBS3970019.1 0 73 Bact S Desulfosporosinus orientis Dori.4084 1563 WP_014184084.1 0 74 Bact S Desulfosporosinus sp. Dsp.4044 157907 MBC2724044.1 0 75 Bact S Sporomusa malonica Smal.8520 112901 WP_084578520.1 0 76 Bact S Desulfosporosinus youngiae Dyou.9325 339862 WP_007779325.1 0 77 Bact S Desulfitibacter alkalitolerans Dalk.6679 264641 WP_028306679.1 0 78 Bact S Sporomusa acidovorans Saci.6836 112900 WP_169716836.1 0 79 Bact S Desulfosporosinus sp. Dsp.5601 157907 HBV85601.1 0 80 Bact S Desulfitobacterium sp. LBE Dsp.9116 884086 TWH59116.1 0 81 Bact S Sporomusa acidovorans Saci.5126 112900 WP_211285126.1 0 82 Bact S Desulfitobacterium hafniense Dhaf.5117 49338 CDX05117.1 0 83 Bact S Peptococcaceae bacterium DCMF Pbac.8107 1761012 ATW28107.1 0 84 Bact S Clostridia bacterium Cbac.5628 2044939 MBS4025628.1 0 85 Bact S Desulfitobacterium hafniense DCB-2 Dhaf.2853 272564 ACL22853.1 0 86 Bact S Desulfitobacterium hafniense Dhaf.3507 49338 AAU93507.1 0 87 Bact S Desulfosporosinus lacus Dlac.3377 329936 WP_073033377.1 0 88 Bact S Chloroflexi bacterium Cbac.7656 2026724 MBE9477656.1 0 89 Bact S Syntrophomonadaceae bacterium Sbac.0035 2093811 HHW30035.1 0 90 Bact S Clostridia bacterium Cbac.6435 2044939 MBS4026435.1 0 91 Bact S Desulfitobacterium sp. LBE Dsp.7688 884086 WP_144677688.1 0 92 Bact S Desulfitobacterium hafniense Dhaf.5345 49338 WP_005815345.1 0 93 Bact S Desulfitobacterium chlororespirans Dchl.2347 51616 WP_072772347.1 0 94 Bact S Desulfitobacterium hafniense Dhaf.7530 49338 WP_018307530.1 0 95 Bact S Sporomusa acidovorans Saci.2727 112900 WP_176772727.1 0 96 Bact S Sporomusa malonica Smal.5631 112901 WP_084575631.1 0 97 Bact S Desulfitobacterium hafniense Dhaf.2028 49338 WP_018212028.1 0 98 Bact S Desulfosporosinus sp. FKB Dsp.7269 1969835 WP_088227269.1 0 99 Bact S Desulfitobacterium hafniense Dhaf.2270 49338 WP_011462270.1 000 Bact S Syntrophomonadaceae bacterium Sbac.8244 2093811 NLH28244.1 001 Bact S Syntrophaceticus schinkii Ssch.6330 499207 WP_044666330.1 002 Bact S Desulfosporosinus sp. Dsp.5453 157907 HBV85453.1 003 Bact S Peptococcaceae bacterium BRH_c23 Pbac.6016 1629714 KJS46016.1 004 Bact S Syntrophomonadaceae bacterium Sbac.8785 2093811 HHW28785.1 0 33 Bact S Desulfosporosinus sp. I2 Dsp.6271 1617025 WP_045576271.1 0 Yes05 Bact S Syntrophaceticus sp. Ssp.9299 2699755 HHY29299.1 006 Bact S Sporomusa sp. KB1 Ssp.0417 943346 WP_145090417.1 007 Bact S Zhaonella formicivorans Zfor.1312 2528593 TDF71312.1 008 Bact S Desulfosporosinus sp. Dsp.3686 157907 MBC2723686.1 009 Bact S Firmicutes bacterium Fbac.9496 1879010 MBP1759496.1 010 Bact S Desulfitobacterium dehalogenans Ddeh.2517 36854 WP_014792517.1 011 Bact S Proteobacteria bacterium Pbac.7733 1977087 NIS67733.1 012 Bact S Desulfitobacterium sp. PCE1 Dsp.5432 146907 WP_028305432.1 013 Bact S Proteobacteria bacterium Pbac.8025 1977087 NIQ38025.1 014 Bact S Desulfosporosinus fructosivorans Dfru.2604 2018669 WP_135552604.1 015 Bact S Clostridia bacterium Cbac.9511 2044939 HGF89511.1 016 Bact S Spirochaetes bacterium Sbac.2369 2202144 HEB32369.1 017 Bact S Proteobacteria bacterium Pbac.5562 1977087 NIO05562.1 018 Bact S Peptococcaceae bacterium SCADC1_2_3 Pbac.1416 1487582 KFD41416.1 019 Bact S Desulfosporosinus sp. OL Dsp.6945 1888891 WP_075366945.1 020 Bact S Sporomusa sphaeroides Ssph.2930 47679 WP_181382930.1 021 Bact S Dehalobacterium formicoaceticum Dfor.7528 51515 WP_157677528.1 0 Bact S uncultured Sporomusa sp. Ssp.8604 307249 SCM78604.1 0 Bact S Candidatus Aminicenantes bacterium Abac.2198 2052149 NOR12198.1 0 Bact S Firmicutes bacterium Fbac.3124 1879010 NMD43124.1 0 Bact S Sporomusa ovata Sova.8468 2378 WP_021168468.1 0 Bact S Clostridia bacterium Cbac.4592 2044939 NMA14592.1 0 Bact S Desulfosporosinus sp. Sb-LF Dsp.9917 2560027 WP_135379917.1 0 Bact S Sporomusa sp. KB1 Ssp.4848 943346 WP_145094848.1 0 Bact S Sporomusa silvacetica Ssil.5429 55504 WP_094605429.1 0 Bact S uncultured Sporomusa sp. Ssp.8578 307249 SCM78578.1 0 Bact S Clostridia bacterium Cbac.2504 2044939 HHT62504.1 0 Bact S Sporomusa sp. GT1 Ssp.8863 1534747 WP_188398863.1 0 Bact S Firmicutes bacterium Fbac.4365 1879010 MBP2664365.1 0 Bact S Sporomusa sp. GT1 Ssp.8855 1534747 WP_188398855.1 0 Bact S Sporomusa sphaeroides Ssph.7443 47679 WP_075757443.1 0 Bact S Sporomusa acidovorans Saci.7115 112900 WP_093797115.1 0 Bact S Thermacetogenium phaeum Tpha.1510 85874 WP_015051510.1 0 Bact S Thermacetogenium phaeum Tpha.5737 85874 KUK35737.1 0 Bact S Desulfitobacterium sp. PLL0 Dsp.3094 2816476 WP_206813094.1 0 Bact S Candidatus Aminicenantes bacterium Abac.6859 2052149 MBP1766859.1 0 Bact S Desulfosporosinus sp. BG Dsp.5211 1633135 WP_068965211.1 0 Bact S Desulfofarcimen acetoxidans Dace.3443 58138 WP_012813443.1 0 Bact S Chloroflexi bacterium Cbac.3905 2026724 RLC63905.1 0 Bact S Methylomusa anaerophila Mana.3968 1930071 WP_197723968.1 0 Bact S Pseudobacteroides cellulosolvens Pcel.9018 35825 WP_036939018.1 0 Bact S Candidatus Aminicenantes bacterium Abac.1782 2052149 MBN1221782.1 0 Bact S Desulfallas gibsoniae Dgib.4409 102134 WP_006524409.1 0 Bact S Anaerovorax sp. IOR16 Asp.0741 2773458 WP_206460741.1 1 Bact S Candidatus Cryptoclostridium obscurum Cobs.8365 2720830 WP_191478365.1 1 Yes Bact S Firmicutes bacterium Fbac.1862 1879010 MBQ1251862.1 1 Bact S Clostridiales bacterium BAD-6 Cbac.8809 2818044 MBR0598809.1 1 Bact S Clostridiales bacterium Cbac.2339 1898207 QOX62339.1 1 Bact S Dethiosulfatibacter aminovorans Dami.8817 332095 WP_073048817.1 1 Bact S Firmicutes bacterium Fbac.3216 1879010 MBQ4093216.1 1 Bact S Firmicutes bacterium Fbac.9146 1879010 MBR5329146.1 1 Bact S Firmicutes bacterium Fbac.4044 1879010 MBQ2764044.1 1 Bact S Olavius algarvensis Delta 1 endosymbiont Oalg.8898 334747 CAB1078898.1 2 Bact S Olavius sp. associated proteobacterium Delta 1 Osp.5413 698986 CAB1055413.1 2 Bact S Desulfosarcina sp. Dsp.3142 2027861 MBC2713142.1 2 Bact S Deltaproteobacteria bacterium Dbac.2193 2026735 MBC8442193.1 2 Bact S Desulfobacterales bacterium Dbac.1174 2044940 NNL41174.1 2 Bact S Deltaproteobacteria bacterium Dbac.1215 2026735 MBF0531215.1 2 Bact S Syntrophobacterales bacterium Sbac.0705 2282155 TFG90705.1 2 Bact S Gemmatimonadetes bacterium Gbac.3691 2026742 NNM03691.1 2 Yes Bact S Desulfosarcina sp. Dsp.4317 2027861 MBR9984317.1 2 Bact S Desulfobacter sp. Dsp.4131 2294 MAF34131.1 2 Bact S Desulfobacterales bacterium Dbac.7919 2044940 HEA67919.1 2 Bact S Deltaproteobacteria bacterium Dbac.1461 2026735 RLB41461.1 2 Bact S Desulfobacteraceae bacterium 4572_123 Dbac.6774 1971629 OQY06774.1 2 Bact S Deltaproteobacteria bacterium Dbac.4121 2026735 RLC04121.1 2 Bact S Desulforhopalus singaporensis Dsin.9473 91360 WP_092219473.1 3 Bact S Desulfobacula sp. Dsp.3302 2593537 MBC2703302.1 3 Bact S Deltaproteobacteria bacterium Dbac.4140 2026735 RLB94140.1 3 Bact S Desulfamplus magnetovallimortis Dmag.0266 1246637 WP_080800266.1 3 Bact S Desulfospira joergensenii Djoe.7843 53329 WP_022667843.1 3 Yes Bact S Desulfobacterium vacuolatum Dvac.7923 2298 WP_212637923.1 3 Bact S Desulfobacteraceae bacterium Dbac.8400 2049433 THB78400.1 3 Bact S Deltaproteobacteria bacterium Dbac.7308 2026735 RLB97308.1 3 Bact S Desulfobulbaceae bacterium S5133MH15 Dbac.1118 1869306 OEU51118.1 3 Bact S Phycisphaeraceae bacterium Pbac.5788 2026777 NQV35788.1 3 Bact S Deltaproteobacteria bacterium Dbac.5137 2026735 NOX35137.1 3 Bact S Deltaproteobacteria bacterium Dbac.5463 2026735 RLC15463.1 3 Bact S Deltaproteobacteria bacterium Dbac.9093 2026735 RLB89093.1 3 Bact S Desulfobacteraceae bacterium Dbac.8279 2049433 HCY88279.1 3 Bact S Phycisphaerales bacterium Pbac.4254 2052180 HCO94254.1 3 Bact S Firmicutes bacterium Fbac.2428 1879010 HAP32428.1 4 Bact S Peptococcaceae bacterium Pbac.7692 2052179 MBS3937692.1 4 Bact S Dethiobacter sp. Dsp.4212 2093370 RJX24212.1 4 Yes Arch ∆N B Methanomassiliicoccales archaeon RumEn M1 Marc.1560 1713724 KQM11560.1 5 Arch ∆N B Euryarchaeota archaeon Earc.6113 2026739 NLK26113.1 5 Arch ∆N B Methanomassiliicoccus sp. Msp.4194 2528041 MBI0584194.1 5 Arch ∆N B Euryarchaeota archaeon Earc.4405 2026739 NLI74405.1 5 Arch ∆N B Methanomassiliicoccus sp. Msp.7007 2528041 NLT37007.1 5 Arch ∆N B Methanomassiliicoccales archaeon PtaU1.Bin030 Marc.7871 1811729 OPY27871.1 5 Yes - for class B Arch ∆N B Methanomassiliicoccus luminyensis Mlum.9105 1080712 WP_026069105.1 5 PylRS Yes - for class B Arch ∆N B Candidatus Methanomassiliicoccus intestinalis Mint.8777 1406512 WP_020448777.1 5 tRNA Arch ∆N B Methanomassiliicoccus luminyensis Mlum.4533 1080712 WP_147654533.1 5 Arch ∆N B Methanomassiliicoccus sp. Msp.8548 2528041 NLT38548.1 5 Arch ∆N B Methermicoccus shengliensis Mshe.6913 660064 WP_042686913.1 5 Bact S Halarsenatibacter silvermanii Hsil.7707 321763 WP_089757707.1 6 Bact S Acetohalobium arabaticum DSM 5501 Aara.1903 574087 ADL11903.1 6 Yes Bact S Acetohalobium arabaticum Aara.1389 28187 WP_083771389.1 6 Bact S Firmicutes bacterium ML8_F2 Fbac.0826 1775675 OPL10826.1 7 Yes Bact S Firmicutes bacterium Fbac.6304 1879010 NLA46304.1 7 Bact S Firmicutes bacterium Fbac.2045 1879010 NLZ32045.1 7 Yes - for class A Arch ∆N A Candidatus Methanomethylophilus alvus Malv.5008 1291540 WP_015505008.1 8 tRNA Yes - for class A Arch ∆N A Candidatus Methanomethylophilus sp.1R26 Msp.7239 1769296 WP_058747239.1 8 PylRS Arch ∆N A Thermoplasmatales archaeon BRNA1 Tarc.8081 1054217 AGI48081.1 8 Arch ∆N A Thermoplasmata archaeon Tarc.7271 1906666 MBE6527271.1 8 Arch ∆N A Candidatus Methanomethylophilus sp. Msp.3097 2774294 MBO4503097.1 8 Arch ∆N A Candidatus Methanomethylophilus sp. Msp.9260 2774294 MBQ4369260.1 8 Arch ∆N A Candidatus Methanomethylophilus sp. Msp.2247 2774294 MBQ4412247.1 8 Arch ∆N A Candidatus Methanomethylophilus sp. Msp.8418 2774294 MBR1888418.1 8 Arch ∆N A methanogenic archaeon ISO4-H5 IH5.5040 1495144 AMH95040.1 8 Arch ∆N A Candidatus Methanomethylophilus sp. Msp.8905 2774294 MBO5518905.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.7097 2517205 MBO4357097.1 8 Arch ∆N A Thermoplasmata archaeon Tarc.7836 1906666 MBE6527836.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.5804 2517205 MBQ3685804.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.5683 2517205 MBP3385683.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.3162 2517205 MBQ8643162.1 8 Arch ∆N A methanogenic archaeon mixed culture ISO4-G1 IG1.3702 1452364 AMK13702.1 8 Arch ∆N A Methanomassiliicoccaceae archaeon DOK Marc.7765 1535962 QHK17765.1 8 Arch ∆N A Thermoplasmata archaeon Tarc.3881 1906666 MBE6513881.1 8 Arch ∆N A Candidatus Methanoplasma termitum Mter.1907 1577791 WP_048111907.1 8 Arch ∆N A Thermoplasmata archaeon Tarc.8562 1906666 MBE6518562.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.5161 2517205 MBR4685161.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.3539 2517205 MBR6213539.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.9261 2517205 MBQ8179261.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.2031 2517205 MBQ7622031.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.2341 2517205 MBR4202341.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.2488 2517205 MBO7352488.1 8 Arch ∆N A Thermoplasmata archaeon Tarc.4844 1906666 MBE6524844.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.9610 2517205 MBQ7979610.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.7379 2517205 MBR4227379.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.4498 2517205 MBR4244498.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.7299 2517205 MBR3477299.1 8 Arch ∆N A Thermoplasmata archaeon Tarc.0817 1906666 MBE6520817.1 8 Arch ∆N A Methanomassiliicoccales archaeon Mx-06 Marc.5181 1820007 TQS85181.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.4851 2517205 MBP5734851.1 8 Arch ∆N A Euryarchaeota archaeon Earc.5781 2026739 NLU45781.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.0821 2517205 MBR6870821.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.7842 2517205 MBR6037842.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.4345 2517205 MBR4504345.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.3801 2517205 MBR6203801.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.8587 2517205 MBO4798587.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.1067 2517205 MBR6911067.1 8 Arch ∆N A Thermoplasmatales archaeon Tarc.3713 2268204 NLF33713.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.3605 2517205 MBO6083605.1 8 Arch ∆N A Thermoplasmatales archaeon Tarc.5321 2268204 NLL95321.1 8 Arch ∆N A Candidatus Methanomethylophilaceae archaeon Marc.4399 2517205 MBP5394399.1 8 Bact S Gammaproteobacteria bacterium Gbac.4047 1913989 NCA74047.1 8 Arch ∆N C Methanonatronarchaeia archaeon Marc.2326 2099682 RZN62326.1 9 Arch ∆N C Methanonatronarchaeum thermophilum Mthe.6791 1927129 WP_143406791.1 9 Yes Bact S uncultured Eubacterium sp. Esp.8714 165185 SCJ68714.1 10 Bact S Emergencia sp.1XD21-10 Esp.9202 2304569 WP_161909202.1 10 Bact S Anaerotruncus A.0595 244127 WP_160200595.1 10 Bact S Aminipila sp. JN-18 Asp.6151 2507160 WP_128746151.1 10 Bact S Peptostreptococcaceae bacterium Pbac.3944 1904861 MBK5253944.1 10 Bact S Aminipila butyrica Abut.7413 433296 WP_163067413.1 10 Bact S Emergencia timonensis Etim.4473 1776384 WP_148484473.1 10 Bact S Emergencia sp.1XD21-50 Esp.3739 2304570 WP_161873739.1 10 Bact S Emergencia timonensis Etim.7639 1776384 WP_067537639.1 10 Bact S Clostridiales bacterium Cbac.2529 1898207 NLY82529.1 10 Yes Bact S Firmicutes bacterium CAG:238 Fbac.2449 1263011 CDA92449.1 10 Bact S Peptostreptococcaceae bacterium pGA-8 Pbac.5427 1520829 SFE25427.1 10 Bact S Hornefia porci Hpor.3506 2652292 WP_075713506.1 10 Bact S Clostridiales bacterium Cbac.5559 1898207 MBE6025559.1 10 Bact S Firmicutes bacterium Fbac.4992 1879010 MBR3374992.1 10 Bact S Firmicutes bacterium Fbac.6416 1879010 MBR6956416.1 10 Bact S Firmicutes bacterium Fbac.4230 1879010 MBR4024230.1 10 Bact S Firmicutes bacterium Fbac.4875 1879010 MBQ5954875.1 10 Bact S Firmicutes bacterium Fbac.8182 1879010 MBQ6088182.1 10 Bact S Firmicutes bacterium Fbac.4550 1879010 MBR6224550.1 10 Bact S Firmicutes bacterium Fbac.3139 1879010 MBR6473139.1 10 Bact S Firmicutes bacterium Fbac.8496 1879010 MBQ4468496.1 10 Bact S Firmicutes bacterium Fbac.0708 1879010 MBQ2160708.1 10 Bact S Firmicutes bacterium Fbac.8472 1879010 MBP3758472.1 10 Bact S Aminipila sp. CBA3637 Asp.2719 2697030 WP_162362719.1 10 Bact S Spirochaetales bacterium Sbac.7250 2026792 MBN1837250.1 11 Yes Bact S Spirochaetes bacterium GWB1_66_5 Sbac.6259 1802178 OHD26259.1 11 Bact S Eubacterium limosum Elim.2114 1736 WP_038352114.1 12 Bact S Acetobacterium fimetarium Afim.2153 52691 WP_186842153.1 12 Bact S Eubacterium limosum Elim.8747 1736 WP_133968747.1 12 Bact S Alkalibaculum bacchi Abac.1451 645887 WP_113921451.1 12 Yes Bact S Parasporobacterium paucivorans Ppau.7396 115544 WP_094757396.1 13 Yes Bact S Lachnospiraceae bacterium Lbac.7498 1898203 HCE77498.1 13 Bact S Acetobacterium sp. MES1 Asp.5325 1899015 OXS25325.1 13 Bact S Desulfosporosinus fructosivorans Dfru.2206 2018669 WP_206752206.1 13 Bact S Desulfosporosinus sp. HMP52 Dsp.8345 1487923 KGK88345.1 13 Bact S Desulfosporosinus sp. HMP52 Dsp.3388 1487923 WP_205623388.1 13 Bact S Lachnospiraceae bacterium NSJ-29 Lbac.0076 2763667 QNM10076.1 13 Bact S Megamonas funiformis Mfun.4926 437897 WP_167624926.1 14 Yes Bact S Pectinatus brassicae Pbra.3372 862415 WP_183863372.1 14 Bact S Selenomonas montiformis Smon.0940 2652285 WP_154620940.1 14 Bact S Desulforhopalus vacuolatus Dvac.9529 40414 WP_205219529.1 15 Yes Bact S Clostridiales bacterium Cbac.0707 1898207 NLV70707.1 16 Bact S Clostridiales bacterium Cbac.7415 1898207 HCU07415.1 16 Bact S Clostridia bacterium Cbac.1785 2044939 NCB41785.1 16 Yes Bact S Clostridiales bacterium UBA9856 Cbac.9944 2060925 HAF59944.1 17 Bact S Clostridiales Family XIII bacterium CFam.7930 2137877 NLM07930.1 17 Bact S Firmicutes bacterium HGW-Firmicutes-11 Fbac.5844 2013772 PKM85844.1 17 Yes Arch N Methanohalophilus halophilus Mhal.0754 2177 WP_072560754.1 18 Arch N Methanohalophilus sp. T328-1 Msp.2195 1794907 KXS42195.1 18 Arch N Methanohalophilus mahii Mmah.6758 2176 WP_013036758.1 18 Arch N Methanohalophilus profundi Mpro.7454 2138083 WP_129597454.1 18 Arch N Methanohalophilus sp. RSK Msp.5591 2485783 WP_123135591.1 18 Arch N Methanohalophilus levihalophilus Mlev.0695 1431282 WP_209680695.1 18 Arch N Methanohalophilus portucalensis Mpor.8806 39664 WP_072358806.1 18 Arch N Methanohalophilus sp. WG1-DM Msp.4488 2491675 WP_128754488.1 18 Arch N Methanohalophilus euhalobius Meuh.2291 51203 WP_096712291.1 18 Arch N Methanohalophilus sp. DAL1 Msp.4501 1864608 OBZ34501.1 18 Arch N Methanolobus profundi Mpro.6779 487685 WP_091936779.1 18 Arch N Methanosarcina sp. MTP4 Msp.1983 1434100 WP_048181983.1 18 Arch N Methanococcoides burtonii Mbur.0100 29291 WP_011500100.1 18 Arch N Methanococcoides sp. SA1 Msp.7821 2735871 WP_172367821.1 18 Arch N Methanosarcina sp.1.H.A.2.2 Msp.8488 1483601 KKH48488.1 18 Arch N Methanosarcina thermophila Mthe.L6A3 2210 Q1L6A3.1 18 Arch N unclassified Methanosarcina M.0001 2644672 WP_048130001.1 18 Arch N Methanosarcina sp.2.H.A.1B.4 Msp.9762 1483600 WP_048169762.1 18 Arch N Methanosarcina flavescens Mfla.8905 1715806 WP_054298905.1 18 Arch N Methanococcoides sp. SA1 Msp.7558 2735871 WP_172367558.1 18 Arch N Methanolobus zinderi Mzin.4005 536044 WP_176964005.1 18 Arch N Methanolobus sp. T82-4 Msp.0259 1794908 KXS40259.1 18 Arch N Methanohalobium evestigatum Meve.3804 2322 WP_013193804.1 18 Arch N Methanosarcina lacustris Mlac.8743 170861 WP_048128743.1 18 Arch N Methanosarcinaceae archaeon Marc.0294 1945595 HII00294.1 18 Arch N Methanosarcina acetivorans C2A Mace.3608 188937 AAM03608.1 18 Arch N Methanolobus psychrotolerans Mpsy.7644 1874706 WP_094227644.1 18 Arch N Methanosarcina sp.2.H.T.1A.15 Msp.4887 1483596 KKG14887.1 18 Arch N Methanosarcina horonobensis Mhor.2694 418008 WP_048142694.1 18 Arch N Methanosarcina sp.2.H.T.1A.6 Msp.0059 1483599 WP_048160059.1 18 Arch N Methanosarcina acetivorans Mace.0213 2214 WP_011020213.1 18 Arch N Methanosarcina sp.1.H.T.1A.1 Msp.4873 1483602 WP_048134873.1 18 Arch N Methanococcoides sp. Msp.8807 1966350 NOQ48807.1 18 Arch N Methanosarcina sp. Ant1 Msp.3301 1882735 OEU43301.1 18 Arch N Methanosarcina thermophila Mthe.7695 2210 WP_048167695.1 18 Arch N Methanosalsum zhilinae Mzhi.8320 39669 WP_013898320.1 18 Yes - for class N PylRS and Arch N Methanosarcina mazei Mmaz.3391 2209 WP_011033391.1 18 tRNA Arch N Methanosarcina mazei Mmaz.7042 2209 WP_048047042.1 18 Arch N Methanosarcina mazei Mmaz.9940 2209 WP_048049940.1 18 Arch N Methanolobus tindarius Mtin.5214 2221 WP_023845214.1 18 Arch N Methanolobus bombayensis Mbom.9327 38023 WP_209619327.1 18 Arch N Methanosarcina soligelidi Msol.0733 1036677 WP_048050733.1 18 Arch N Methanosarcina mazei Mmaz.9837 2209 NLO29837.1 18 Arch N Methanosarcina siciliae Msic.4670 38027 WP_148704670.1 18 Arch N Methanosarcina siciliae Msic.8985 38027 WP_048178985.1 18 Arch N Methanolobus sp. SY-01 Msp.9826 2052935 WP_135389826.1 18 Arch N Methanosarcina siciliae Msic.9178 38027 WP_048169178.1 18 Arch N Methanolobus psychrophilus R15 Mpsy.3928 1094980 AFV23928.1 18 Arch N Methanosarcina sp. MSH10X1 Msp.3320 2507075 WP_128503320.1 18 Arch N Methanosarcina sp. Msp.1167 2213 HII81167.1 18 Arch N Methanosarcina sp. Msp.4777 2213 HHV24777.1 18 Arch N Methanolobus vulcani Mvul.8514 38026 WP_154808514.1 18 Arch N Methanolobus vulcani Mvul.7851 38026 WP_154717851.1 18 Arch N Methanosarcina sp. Kolksee Msp.7962 1434099 WP_048157962.1 18 Arch N Methanosarcina sp. Msp.4411 2213 HIH74411.1 18 Arch N Methanosarcinales archaeon Marc.8799 2250255 NYT18799.1 18 Arch N Methanosarcina M.0621 2207 WP_048120621.1 18 Arch N Methanolobus vulcani Mvul.7898 38026 WP_154717898.1 18 Arch N Methanosarcina sp. Msp.3161 2213 NLN43161.1 18 Arch N Methanosarcina barkeri Mbar.6887 2208 WP_048176887.1 18 Arch N Methanosarcinaceae archaeon Marc.0544 1945595 MBN2110544.1 18 Arch N Methanosarcina barkeri Mbar.0867 2208 AAL40867.1 18 Arch N Methanosarcina spelaei Mspe.5044 1036679 WP_095645044.1 18 Arch N Methanosarcina barkeri Mbar.8458 2208 WP_048108458.1 18 Arch N Methanococcoides sp. NM1 Msp.4033 1201013 WP_135604033.1 18 Arch N Methanococcoides vulcani Mvul.9487 1353158 WP_091689487.1 18 Arch N Methanococcoides methylutens Mmet.7228 2226 WP_135607228.1 18 Arch N Methanococcoides sp. AM1 Msp.0773 1201011 WP_135610773.1 18 Arch N Methanococcoides methylutens Mmet.3608 2226 WP_048193608.1 18 Arch N Methanosarcina vacuolata Mvac.3171 2215 WP_048123171.1 18 Arch N Methanosarcina barkeri Mbar.5865 2208 WP_011305865.1 18 Arch N Methanimicrococcus blatticola Mbla.6947 91560 WP_133516947.1 18 Arch N Methanosarcinaceae archaeon Marc.9084 1945595 MBN2489084.1 18 Arch N Methanococcoides methylutens Mmet.4558 2226 WP_048204558.1 18 Arch N Methanomethylovorans hollandica Mhol.3491 101192 WP_015323491.1 18 Arch N Methanomethylovorans sp. PtaU1.Bin093 Msp.1133 1811679 OPY21133.1 18 Arch N Methanosarcinaceae archaeon Marc.1971 1945595 MBO4301971.1 18 Arch N Methanosarcinaceae archaeon Marc.0406 1945595 MBQ3620406.1 18 Arch N Methanosarcinaceae archaeon Marc.1876 1945595 NLI61876.1 18 Bact S Alphaproteobacteria bacterium Abac.7547 1913988 RPJ77547.1 18 Arch ∆N B Methanomassiliicoccales archaeon PtaU1.Bin124 Marc.1052 1811730 OPY31052.1 19 Yes Bact S Desulfovermiculus sp. Dsp.9351 2821143 MBS3779351.1 20 Yes Arch S-Arch C Candidatus Bathyarchaeota archaeon Barc.2298 2026714 RLI42298.1 21 Yes Arch ∆N C Candidatus Methanohalarchaeum thermophilum Mthe.9096 1903181 OKY79096.1 22 Yes Bact S Firmicutes bacterium HGW-Firmicutes-14 Fbac.3183 2013775 PKM83183.1 23 Yes Bact S Deltaproteobacteria bacterium Dbac.7859 2026735 RTZ97859.1 24 Bact S Deltaproteobacteria bacterium Dbac.9416 2026735 RTZ99416.1 24 Yes Bact S Desulfobacterales bacterium Dbac.3934 2044940 HHC23934.1 24 Bact S Deltaproteobacteria bacterium Dbac.7531 2026735 RLC07531.1 24 Arch ∆N C Nitrososphaeria archaeon Narc.2415 2268198 HHP52415.1 25 Yes Arch ∆N C Candidatus Methanohalarchaeum thermophilum Mthe.7552 1903181 OKY77552.1 26 Yes Bact S Carboxydothermus ferrireducens Cfer.2237 54265 WP_028052237.1 27 Yes Bact S Spirochaetes bacterium Sbac.2174 2202144 MBN2322174.1 28 Yes Bact S Desulfacinum sp. Dsp.7434 2301220 MBC7357434.1 29 Bact S Desulfacinum infernum Dinf.6409 35837 WP_084076409.1 29 Yes Bact S Actinobacteria bacterium Abac.2909 1883427 NLT92909.1 30 Bact S Actinobacteria bacterium ADurb.BinA094 Abac.2170 1852790 OPZ42170.1 30 Bact S Thermoleophilia bacterium Tbac.8218 2026888 MBE0528218.1 30 Bact S Actinobacteria bacterium Abac.2280 1883427 NLE22280.1 30 Yes Bact S Thermoleophilia bacterium Tbac.4777 2026888 MBN2204777.1 30 Bact S Actinobacteria bacterium Abac.4456 1883427 HGE64456.1 30 Bact S Actinobacteria bacterium Abac.4159 1883427 NLG64159.1 30 Arch S-Arch B Methanosarcinales archaeon Marc.1509 2250255 RLG21509.1 31 Yes Bact S Clostridia bacterium Cbac.8451 2044939 HHY38451.1 32 Yes Bact S Desulfobulbus rhabdoformis Drha.8345 34032 WP_205228345.1 33 Yes Bact S Bilophila wadsworthia Bwad.6770 35833 WP_029436770.1 34 Bact S Desulfovibrionaceae bacterium Dbac.7202 2049043 MBS1377202.1 34 Bact S Bilophila wadsworthia Bwad.0989 35833 WP_016360989.1 34 Yes Bact S Bilophila sp.4_1_30 Bsp.8528 693988 WP_009368528.1 34 Arch ∆N B Methanomassiliicoccales archaeon Marc.6584 1906667 MBP7086584.1 35 Arch ∆N B Methanomassiliicoccus sp. Msp.5345 2528041 TFG55345.1 35 Yes candidate division MSBL1 archaeon SCGC- Arch ∆N C AAA382A20 Marc.6481 1698280 KXB06481.1 36 Yes

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Claims

CLAIMS 1. A cell comprising an exogenous Class C acyl-tRNA synthetase (aRS) and comprising one, two, three, or four members of the group: an exogenous Class A aRS, an exogenous Class B aRS, an exogenous Class N aRS, and an exogenous Class S aRS, wherein each aRS is a pyrrolysyl-tRNA synthetase (PylRS) or a variant engineered to alter the PylRS acylation specificity.

2. A cell comprising an exogenous Class S aRS and comprising one, two, three, or four members of the group: an exogenous Class A aRS, an exogenous Class B aRS, an exogenous Class C aRS, and an exogenous Class N aRS, wherein each aRS is a PylRS or is a variant engineered to alter the PylRS acylation specificity.

3. The cell of claim 1 or claim 2, wherein: i) a Class A aRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the cell, that clusters with any of SEQ ID NOs: 4, 35, 203-245 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; and / or ii) a Class B aRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the cell, that clusters with any of SEQ ID NOs: 6, 36, 188-196, 375, 392, 399, 400 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; iii) a Class C aRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the cell, that clusters with any of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 247 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs: 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; iv) a Class N aRS is a PylRS that clusters with any of SEQ ID NOs: 295-373 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs: 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; and / or v) a Class S aRS is a PylRS that clusters with any of SEQ ID NOs: 26, 29, 31, 33, 65-187, 197-202, 246, 248-294, 374, 376-391, 393-398 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs: 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%.

4. The cell of any preceding claim, wherein a Class N aRS is a PylRS derived from an archaeal species, or variant engineered to alter the PylRS acylation specificity, and wherein the aRS comprises an aRS N-terminal domain as a part of the same polypeptide as an aRS C-terminal domain.

5. The cell of any preceding claim, wherein a Class S aRS is a PylRS derived from a bacterial species, or variant engineered to alter the PylRS acylation specificity, and wherein the cell expresses a separately encoded N- terminal domain associated with said Class S aRS.

6. The cell of any preceding claim, wherein a Class A aRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said aRS, expresses a tRNA according to SEQ ID NO: 37, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 38, ii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 39, iii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 40, and iv) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

7. The cell of any preceding claim, wherein a Class B aRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said aRS, expresses a tRNA according to SEQ ID NO: 38, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 39, iii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 40, and iv) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

8. The cell of any preceding claim, wherein a Class C aRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said aRS, expresses a tRNA according to SEQ ID NO: 39, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 38, iii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 40, and iv) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

9. The cell of claim 7 or claim 8 wherein a Class B aRS and / or a Class C aRS is a bacterial PylRS, or variant engineered to alter the PylRS acylation specificity, and wherein the cell does not express an associated N- terminal domain.

10. The cell of any preceding claim, wherein a Class N aRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said aRS, expresses a tRNA according to SEQ ID NO: 40, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA ischarged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 38, iii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 39, and iv) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

11. The cell of any preceding claim, wherein a Class S aRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said aRS, expresses a tRNA according to SEQ ID NO: 41, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 38, iii) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 39, and iv) in a cell that expresses said aRS and a tRNA according to SEQ ID NO: 40, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

12. The cell of any preceding claim, wherein13. The cell of claim 12, wherein: the 1R26PylRS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35, the Lum1PylRS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 36, the SΔ-ClosPylRS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 26, 27, or 28, the NitraPylRS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity any one of SEQ ID NOs: 11, 12, or 13, the SΔ-I2PylRS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 33 or 34, the MmPylRS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1 or 3, and / or the DebPylS is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 29 or 30 and an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 62.

14. The cell of claim 12 or claim 13, wherein the cell expresses one, two, three, four, or five tRNAs according to any combination of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO:

59.

15. The cell of any preceding claim, wherein each exogenous aRS is a part of an exogenous aRS-tRNA pair.

16. The cell of claim 15, wherein each exogenous aRS-tRNA pair is mutually orthogonal.

17. The cell of claim 16, wherein, in an experimental assay, each aRS-tRNA pair would lead to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein expression is measured in a cell that comprises a marker gene that is only capable of being decoded if said aRS-tRNA pair is charged, and the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and wherein every other pairing of the exogenous aRSs and exogenous tRNAs would lead to expression of said marker gene at less than 20% of expression for said control gene in said assay, and wherein the quotient of the lowest intra-pair activity over the highest inter-pair cross-reactivity is greater than 2.5 18. The cell of any preceding claim, wherein any one, two, three, four, or all five of the Class A, B, C, N, and / or S aRS enzymes are engineered to alter the acylation specificity.

19. The cell of any preceding claim, wherein the cell is a prokaryotic cell, a bacterial cell, or an Escherichia coli cell.

20. A method of producing a cell comprising at least two exogenous acyl-tRNA synthetases, wherein the method comprises: i) screening one or more PylRS in order to identify a first PylRS belonging to Class C or S; ii) screening one or more PylRS in order to identify a second PylRS belonging to Class A, B, C, N, or S; optionally iii) modifying the first and / or second PylRS in order to alter the acylation specificity; and iv) generating a cell expressing the first PylRS the second PylRS, wherein the first and the second PylRS are not of the same Class.

21. The method of claim 20, wherein: a Class A PylRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the cell, that clusters with any of SEQ ID NOs: 4, 35, 203-245 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs: 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; a Class B PylRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the cell, that clusters with any of SEQ ID NOs: 6, 36, 188-196, 375, 392, 399, 400 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs: 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; a Class C PylRS is a PylRS with no N terminal domain, or where the N terminal domain is not expressed in the cell, that clusters with any of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 247 after an average linkage clustering of the aligned sequence of its C-terminal domain with the aligned C-terminal domain sequences of SEQ ID NOs: 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 31, 33, 35, 36, 64-400, with a sequence identity lower threshold of 55%; a Class N PylRS is an PylRS derived from an archaeal species and wherein the PylRS comprises an PylRS N-terminal domain as a part of the same polypeptide as an PylRS C-terminal domain; and / or a Class S PylRS is an PylRS derived from a bacterial species and wherein the cell expresses a separately encoded N-terminal domain associated with said Class S PylRS.

22. The method of claim 20 or claim 21, wherein a Class A PylRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said PylRS, expresses a tRNA according to SEQ ID NO: 37, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 38, ii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 39, iii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 40, and iv) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

23. The method of any of claims 20 to 22, wherein a Class B PylRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said PylRS, expresses a tRNA according to SEQ ID NO: 38, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 39, iii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 40, and iv) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

24. The method of any of claims 20 to 23, wherein a Class C PylRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said PylRS, expresses a tRNA according to SEQ ID NO: 39, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 38, iii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 40, and iv) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

25. The method of claim 23 or claim 24, wherein the Class B PylRS and / or the Class C PylRS is a bacterial PylRS, or a variant engineered to alter the PylRS acylation specificity, and wherein the generated cell does not express an associated N-terminal domain.

26. The method of any of claims 20 to 25, wherein a Class N PylRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said PylRS, expresses a tRNA according to SEQ ID NO: 40, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNAis charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 38, iii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 39, and iv) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 41, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

27. The method of any of claims 20 to 26, wherein a Class S PylRS: leads to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein the expression is measured in a cell that expresses said PylRS, expresses a tRNA according to SEQ ID NO: 41, and comprises the marker gene, wherein the marker gene is only capable of being decoded if said tRNA is charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and leads to expression of a marker gene at less than 20% of expression for a control gene, wherein the expression is measured i) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 37, ii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 38, iii) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 39, and iv) in a cell that expresses said PylRS and a tRNA according to SEQ ID NO: 40, wherein said cells also express the marker gene which is only capable of being decoded if said tRNAs are charged, and wherein the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs.

28. The method of any of claims 20 to 27, further comprising screening one or more PylRS in order to identify a third PylRS belonging to Class A, B, C, N, or S optionally modifying the third PylRS in order to alter the acylation specificity; and generating a cell expressing the first PylRS, second PylRS, and third PylRS, wherein each PylRS is of a different class.

29. The method of claim 28, further comprising screening one or more PylRS in order to identify a fourth PylRS belonging to Class A, B, C, N, or S optionally modifying the fourth PylRS in order to alter the acylation specificity; and generating a cell expressing the first PylRS, second PylRS, third PylRS, and fourth PylRS, wherein each PylRS is of a different class.

30. The method of claim 29, further comprising screening one or more PylRS in order to identify a fifth PylRS belonging to Class A, B, C, N, or S optionally modifying the fifth PylRS in order to alter the acylation specificity; and generating a cell expressing the first PylRS, second PylRS, third PylRS, fourth PylRS, and fifth PylRS, wherein each PylRS is of a different class.

31. The method of any one of claims 20 to 30, comprising a step of determining whether an identified PylRS is active within the cell type of the cell-to-be-produced and if the PylRS is inactive, discarding the PylRS and re- screening to identify a PylRS of the relevant Class.

32. The method of any one of claims 20 to 31, wherein the PylRS enzymes expressed by the cell are engineered to be capable of charging a tRNA with an unnatural amino acid or a monomer that is a not an alpha amino acid.

33. The method of any one of claims 20 to 32, wherein, in the generated cell, each exogenous aRS is a part of an exogenous aRS-tRNA pair.

34. The method of claim 33, wherein each exogenous aRS-tRNA pair is mutually orthogonal.

35. The method of claim 34, wherein, in an experimental assay, each aRS-tRNA pair would lead to expression of a marker gene at greater than or equal to 40% of expression for a control gene, wherein expression is measured in a cell that comprises a marker gene that is only capable of being decoded if said aRS-tRNA pair is charged, and the control gene is an otherwise identical gene capable of being decoded by endogenous tRNAs; and wherein every other pairing of the exogenous aRSs and exogenous tRNAs would lead to expression of said marker gene at less than 20% of expression for said control gene in said assay, and wherein the quotient of the lowest intra-pair activity over the highest inter-pair cross-reactivity is greater than 2.

5.

36. A cell obtained or obtainable by the method of any one of claims 20 to 35.

37. A cell comprising: nucleic acid sequence encoding an exogenous protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 29 or SEQ ID NO: 30 and a protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 62; or nucleic acid sequence encoding an exogenous protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31 or SEQ ID NO: 32 and a protein that is of an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:

63.

38. The cell of claim 36 or 37, wherein the cell is a prokaryotic cell, a bacterial cell, or an Escherichia coli cell.

39. Use of a cell according to any one of claims 1-19 or 36-38, for the production of a polymer comprising at least one unnatural amino acid or monomer that is not an alpha amino acid.

40. The use of claim 39, wherein the monomer that is not an alpha amino acid is an alpha hydroxy acid or a beta amino acid.

41. The use of claim 39 or 40, wherein the polymer comprises at least one canonical amino acid.

42. A method for making a polymer comprising at least one unnatural amino acid or monomer that is not an alpha amino acid, the method comprising: culturing a cell according to any one of claims 1-19 or 36-38, providing the cell with a gene encoding the polymer, and obtaining the polymer.

43. The method of claim 42, wherein the monomer that is not an alpha amino acid is an alpha hydroxy acid or beta amino acid.

44. The method of claim 43, wherein the polymer comprises at least one canonical amino acid.

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