Orthogonal DNA replication system

Abstract

The invention relates to cells, such as bacterial cells, comprising orthogonal DNA replication machinery, linear plasmids that may be replicated by said machinery, uses of said cells and said linear plasmids, methods of maintaining linear plasmids in cells, methods of evolving sequences of interest, and methods of making polypeptides or nucleic acids. Orthogonal systems that are based on or derived from phage, such as phi29, that infect gram-positive bacteria are disclosed herein.

Description

[0001] ORTHOGONAL DNA REPLICATION SYSTEM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the priority and benefit of GB2416990.6, filed on 19 November 2024, and GB2508286.8, filed on 28 May 2025, the contents of which are incorporated herein in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to cells, such as bacterial cells, comprising orthogonal DNA replication machinery, linear plasmids that may be replicated by said machinery, uses of said cells and said linear plasmids, methods of maintaining linear plasmids in cells, methods of evolving sequences of interest, and methods of making polypeptides or nucleic acids. Orthogonal systems that are based on or derived from phage, such as phi29, that infect gram-positive bacteria are disclosed herein.

[0006] BACKGROUND OF THE INVENTION

[0007] The high-fidelity replication of an organism’s genome is essential for maintaining genetic integrity, and limits mutations that could be detrimental to survival or reproduction. The natural evolution of new gene function, resulting from the accumulation of mutations and selection within a population, is a slow process. Directed evolution enables the rapid diversification and selection of genes for a desired phenotype on laboratory timescales1. Classical directed evolution experiments rely on cycles of in vitro genetic diversification followed by transformation into an appropriate host for selection. These types of experiments are laborious and limited in terms of evolutionary depth and scale. Continuous evolution strategies, enabled by in vivo mutagenesis and selection of target genes, overcome these limitations and allow for the accelerated exploration of fitness landscapes at scale2.

[0008] Early strategies for continuous evolution included approaches that increased the genome -wide mutation rate3’4. However, these approaches were limited by: 1) the fitness burden associated with mutagenesis of essential genes5and, 2) the mutagenesis of the genes involved in the selection system that led to escape from the selection. These restrictions severely limited both the number of generations for which selection could be performed and the phenotypes that could be evolved.

[0009] Inserting target genes into viral genomes and iteratively infecting fresh mutagenic cells6,7sidesteps some of the limitations of the initial approaches. However, these approaches are limited to evolving small genes that can be packaged into viruses and are often run non-continuously for practical reasons8. Other strategies rely on the localization of a mutagenic moiety, such as an error-prone polymerase or a deaminase, to a target gene9-13. These approaches are generally restricted in terms of their mutagenic window and lack of orthogonality, leading to off- target mutations that can culminate in selection escape. By contrast, truly orthogonal replication systems, where a dedicated error-prone DNA polymerase maintains an episome harboring the target genes without interfering with genome replication, enable straightforward and robust continuous evolution experiments14-16.

[0010] Existing orthogonal replication systems rely on DNA polymerases that replicate linear replicons via a protein- primed mechanism of replication14-17. The first such system, OrthoRep, exploits natural linear plasmids from yeast and has enabled a range of continuous evolution experiments at high mutation rates18-20. Most genetic tools, however, are available in the workhorse of synthetic biology, E. coli, and are often not compatible with yeast21. Moreover, E. coli grows more rapidly and to higher densities than S. cerevisiae, enabling faster generation times - a key parameter governing the speed and scale of continuous evolution experiments. These advantages were realized by a recent orthogonal replication system, EcORep, developed based on components from the PRD1 bacteriophage which naturally infects E. coli15. However, the mutation rates achieved by this system were only modestly higher than the genomic mutation rates (2 x 10-7substitutions per base, per generation) and replicon transformation efficiencies were low. SUMMARY OF THE INVENTION

[0011] In an aspect, there is provided a cell comprising orthogonal DNA replication machinery, wherein the orthogonal DNA replication machinery is from or derived from a phage that infects gram-positive bacteria and / or a phage of the Salasmaviridae family.

[0012] The cell may be a bacterial cell, a gram-positive bacterium, a gram-negative bacterium, a Bacillus sp. cell, B. subtilis cell, or an E. coll cell.

[0013] In some embodiments, the cell comprises one or more non-orthogonal episomes that are capable of being replicated by the endogenous DNA polymerase, and the one or more non-orthogonal episomes encode at least one, a plurality, or all components of the orthogonal DNA replication machinery. In some embodiments, at least one, a plurality, or all components of the orthogonal DNA replication machinery are encoded by the cell genome. The orthogonal DNA replication machinery is capable of replicating an orthogonal episome that does not comprise at least one, a plurality, or all components of the orthogonal DNA replication machinery. The orthogonal DNA replication machinery may replicate DNA with a lower fidelity than the cell’s endogenous DNA replication machinery.

[0014] The orthogonal DNA replication machinery may comprise a gene encoding a Salasmaviridae terminal protein (TP) and a gene encoding an orthogonal DNA polymerase (ODNAP). The orthogonal DNA replication machinery may comprise a gene encoding a Salasmaviridae TP, a gene encoding an ODNAP, and a gene encoding a Salasmaviridae single-stranded DNA-binding protein (SSB). The orthogonal DNA replication machinery may comprise a gene encoding a Salasmaviridae TP, a gene encoding an ODNAP, and a gene encoding a Salasmaviridae double-stranded DNA-binding protein (DSB). The orthogonal DNA replication machinery may comprise a gene encoding a Salasmaviridae DSB.

[0015] The orthogonal DNA replication machinery may be from or derived from phi29 phage. The orthogonal DNA replication machinery may comprise a gene encoding a phi29 TP and a gene encoding an ODNAP. The orthogonal DNA replication machinery may comprise a gene encoding a phi29 TP, a gene encoding an ODNAP, and a gene encoding a phi29 SSB. The orthogonal DNA replication machinery may comprise a gene encoding a phi29 TP, a gene encoding an ODNAP, and a gene encoding a phi29 DSB. The orthogonal DNA replication machinery may comprise a gene encoding a phi29 DSB.

[0016] There is provided a viable cell comprising a gene encoding a Salasmaviridae TP and a gene encoding an ODNAP. The Salasmaviridae TP may be a phi29 TP. The cell may comprise a Salasmaviridae SSB and / or a Salasmaviridae DSB. The Salasmaviridae SSB and / or Salasmaviridae DSB may be a phi29 SSB and / or a phi29 DSB.

[0017] The ODNAP may be a Salasmaviridae or phi29 ODNAP or an engineered Salasmaviridae or phi29 ODNAP. In some embodiments, the cell comprises sequence encoding a protein that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to any one, two, three, or four of SEQ ID NOs: 1, 2, 3, and 4.

[0018] The ODNAP may replicate DNA with a lower fidelity than the cell’s endogenous DNA polymerase. The ODNAP may comprise any one the following mutations or sets of mutations described with reference to SEQ ID NO: 2: T15I, N62D, and / or F65S.

[0019] In some embodiments, at least one, a plurality, or all components of the orthogonal DNA replication machinery are encoded by a single operon.

[0020] In some embodiments, the cell comprises a gene encoding Gam protein. In some embodiments, the cell comprises an episome that is capable of being replicated by the orthogonal DNA replication machinery. The episome may be any as disclosed herein. The episome may comprise a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with the orthogonal DNA replication machinery. The 5’ ITR and 3’ ITR may be compatible with phi29 DNA replication machinery. The episome may comprise a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, or a truncation thereof, and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 or a truncation thereof. The episome may comprise a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

[0021] The episome may be a linear plasmid. The episome may comprise a sequence -of-interest. The cell may comprise a circular nucleic acid molecule that comprises the sequence of an episome that is capable of being replicated by the orthogonal DNA replication machinery. The episome that is capable of being replicated by the orthogonal DNA replication machinery may be any episome disclosed herein. The circular nucleic acid molecule is configured so that the episome that is capable of being replicated by the orthogonal DNA replication machinery may be excised from the circular episome within the cell.

[0022] In an aspect, there is provided a linear plasmid comprising a 5’ ITR and a 3’ ITR, each of which is compatible with DNA replication machinery from or derived from a phage that infects gram-positive bacteria and / or a phage of the Salasmaviridae family.

[0023] The 5’ ITR and 3’ ITR may be compatible with phi29 bacteriophage DNA replication machinery. The linear plasmid may comprise a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, or a truncation thereof and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, or a truncation thereof. The linear plasmid may comprise a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

[0024] In some embodiments, the plasmid does not encode one, two, three, or four of a TP, a DNA polymerase, an SSB, and a DSB. The plasmid may comprise a sequence-of-interest. In examples, the sequence -of-interest is not a sequence endogenous to the phage associated with the DNA replication machinery. In examples, the sequence-of- interest is not a sequence endogenous to phi29.

[0025] In an aspect, there is provided a circular nucleic acid molecule comprising the sequence of a linear plasmid disclosed herein.

[0026] The circular nucleic acid molecule may be a circular episome, a circular plasmid, or a bacterial artificial chromosome. The circular nucleic acid molecule may comprise one or more sites positioned such that the linear plasmid may be excised from the circular nucleic acid molecule.

[0027] In an aspect, there is provided a cell comprising first orthogonal DNA replication machinery and second orthogonal DNA replication machinery, wherein the first orthogonal DNA replication machinery is orthogonal to the endogenous DNA replication machinery of the cell and to the second orthogonal DNA replication machinery, and wherein the second orthogonal DNA replication machinery is orthogonal to the endogenous DNA replication machinery of the cell and to the first orthogonal DNA replication machinery.

[0028] The cell may be any cell as disclosed herein. The cell may comprise any a linear plasmid or circular nucleic acid molecule disclosed herein. The second orthogonal DNA replication machinery may comprise a Tectiviridae TP and a Tectiviridae ODNAP. The second orthogonal DNA replication machinery may comprise a Tectiviridae TP, a Tectiviridae ODNAP, and a Tectiviridae SSB. The second orthogonal DNA replication machinery may comprise a Tectiviridae TP, a Tectiviridae ODNAP, a Tectiviridae SSB, and a Tectiviridae DSB. The second orthogonal DNA replication machinery may comprise a PRD1 TP and a PRD1 ODNAP. The second orthogonal DNA replication machinery may comprise a PRD1 TP, a PRD1 ODNAP, and a PRD1 SSB. The second orthogonal DNA replication machinery may comprise a PRD1 TP, a PRD1 ODNAP, a PRD1 SSB, and a PRD1 DSB.

[0029] In an aspect, there is provided a method of maintaining a linear plasmid, wherein the method comprises: i) providing a cell as disclosed herein, transferring a linear plasmid as disclosed herein to a cell as disclosed herein, excising a linear plasmid from a circular nucleic acid molecule as disclosed herein within a cell as disclosed herein, transferring a first and / or a second linear plasmid to a cell as disclosed herein, or generating a first and / or a second linear plasmid within a cell as disclosed herein; and ii) incubating the cell under conditions conducive to growth.

[0030] In an aspect, there is provided use of a cell as disclosed herein, a linear plasmid as disclosed herein, or a circular nucleic acid molecule as disclosed herein for evolving a sequence-of-interest.

[0031] In an aspect, there is provided a method of evolving a sequence-of-interest, wherein the method comprises: i) providing a cell as disclosed herein, transferring a linear plasmid as disclosed herein to a cell as disclosed herein, excising a linear plasmid from a circular nucleic acid molecule as disclosed herein within a cell as disclosed herein, transferring a first and / or a second linear plasmid to a cell as disclosed herein, or generating a first and / or a second linear plasmid within a cell as disclosed herein; wherein the episome or linear plasmid comprises a sequence-of-interest and wherein the orthogonal DNA replication machinery replicates DNA with a lower fidelity than the cell’s endogenous DNA replication machinery; ii) incubating the cell under conditions conducive to growth; and iii) exposing the cell to a selection condition.

[0032] The method may comprise: iv) identifying the sequence of a sequence-of-interest from a cell meeting the selection condition.

[0033] The method may comprise: v) making a polypeptide or nucleic acid encoded by the identified sequence-of-interest.

[0034] In an aspect, there is provided a method of making a polypeptide or nucleic acid, wherein the method comprises: i) providing a sequence-of-interest identified by a method disclosed herein, and ii) producing a polypeptide or nucleic acid according to said sequence.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1. (A) Schematics of the phi29 genome (top) with the genes labelled 2, 3, 5, and 6 used in the synthetic replication operon (middle), and the synthetic linear replicon flanked by the left and right origins of the genome (bottom). (B) A synthetic replication operon was designed based on genes 3, 2, 6, and 5, encoding for the terminal protein (TP), DNA polymerase (DNAP), double-stranded DNA binding protein (DSB), and the single-stranded DNA binding protein (SSB), respectively. The synthetic replicon was designed to encode for an antibiotic resistance gene and a gene of interest (GDI) and was flanked by the left and right origins of replication (oriL and oriR) derived from the 29 genome.

[0037] Figure 2. Extraction of the TetR-sfGFP replicon from cells. Proteinase K addition was needed to remove the terminal proteins. The control is a PCR product. The operon-harbouring plasmid is also visible, as indicated. Figure 3. Assessing the efficiency of establishing a phi29 replicon via electroporation, (a) <T>29 synthetic replicons could be established by electroporating a PCR-derived product into cells harboring the <T>29 synthetic replication operon on a single-copy plasmid, (b) Efficiency of establishing O29 synthetic replicons via electroporation. A helper plasmid was used to additionally express gam and the genes encoding for the O29 SSB and DSB. O29 synthetic replicons extracted from cells could be transformed with a higher efficiency (n = 3, error bars indicate ± SD).

[0038] Figure 4. Assessing the stability of a phi29 replicon in the presence or absence of tetracycline to select for the replicon. Stability of the TcR-GFP O29 synthetic replicon over 550 generations with or without tetracycline, as assessed by maintenance of GFP fluorescence using flow cytometry (n = 4, the lines indicate ± SD).

[0039] Figure 5. Determining the essentiality of the individual genes for replicon maintenance.

[0040] Figure 6. Engineering phi29 replicons using lambda red recombination. (Upper Panel) Established O29 synthetic replicons can be engineered using lambda Red recombination via the electroporation of a PCR amplicon flanked by homologies to the replicon. (Lower Panel) Efficiency of replacing the tetracycline resistance gene on the O29 synthetic replicon with a kanamycin resistance gene (M = 3, error bars indicate ± SD).

[0041] Figure 7. Optimising the phi29 synthetic replication operon. a, A O29 synthetic replication operon library was generated where, for each gene in the operon, the spacer sequence between the Shine -Dalgamo sequence and the start codon were randomized, the N-terminal codon following the start codon was saturated, and the following six codons were synonymized. b, The ©29-opt operon, integrated on a single-copy plasmid, supports a higher replicon copy number, as measured via GFP fluorescence (M = 6, error bars indicate ± SD). c, Extracted 29 synthetic replicons can be efficiently transformed into cells where the ©29-opt operon was genomically integrated (M = 3, error bars indicate ± SD). d, The ©29-opt operon enables improved growth when maintaining a TcR-GFP ©29 synthetic replicon (M = 8, the boundary lines indicate ± SD).

[0042] Figure 8. The >29-based replication system is an orthogonal replication system in E. coli. An orthogonal replication system would enable hypermutation of a target DNA sequence without interfering with the high- fidelity replication of genomic DNA. Error-prone mutants of the ©29 DNAP do not increase the genomic mutation rate, but do increase the replicon mutation rate. To determine this, we performed fluctuation tests that assessed the frequency of a TAG stop codon reversion in a genomically- or replicon-integrated chloramphenicol resistance gene (Q38TAG) (M = 12, error bars indicate ± upper or lower 95% bounds).

[0043] Figure 9. Continuous evolution of tigecycline resistance using the >29-based replication system, a, Summary of the mutations in TetA that had enriched in at least two of the three independent evolution replicates, b, Mutants obtained from passaging a tetA-GFP ©29 synthetic replicon maintained by the error-prone N62D / F65S DNAP mutant were cloned into colEl circular plasmids and transformed into DH10B cells to assess the level of tigecycline resistance conferred. Two previously reported mutants were tested as a comparison, c, As in (b) but for tetracycline resistance.

[0044] Figure 10. Genotyping establishment of the linear >29 synthetic replicon. Colony PCRs to genotype for the TcR-GFP ©29 synthetic replicon (left) and the backbone of the circular plasmid (right) from which the PCR product used for establishing the replicon in vivo was amplified, as analyzed by agarose gel electrophoresis. The negative control is a circular plasmid which gives a band corresponding to the plasmid backbone which is absent from the linear ©29 synthetic replicons.

[0045] Figure 11. Repressing the copy number of the >29 synthetic replicon. The copy number of a TcR-GFP ©29 synthetic replicon can be reduced by downregulating the expression from a genomically-integrated ©29 synthetic replication operon via arabinose-inducible expression of dCas9 targeted to the promoter that is driving the replication operon. This change in copy number can be measured by GFP fluorescence and qPCR, and the two measurements are well correlated (n = 3, error bars indicate ± SD).

[0046] Figure 12. Engineering an improved >29 synthetic replication operon. a, Fluorescence of cells harboring a GFP-encoding ©29 synthetic replicon as maintained by the initial (WT) ©29 synthetic replication operon or engineered variants. The data for the WT and ©29-opt operons is also shown in Figure 7 (n = 6, error bars indicate ± SD). b, Comparison of the WT ©29 synthetic replication operon sequence with that of ©29-opt. Sequences within genes are shown in bold. Nucleotide changes in ©29-opt are highlighted in red. The sequences depicted are ggaggtctacacatggccaggagccctcgcatacgcatc (SEQ ID NO: 12), ggaggtgacgtgcatggcgagaagcccgcggatccgtatc (SEQ ID NO: 13), ggaggaatatttatgcccaggaagatgtactcg (SEQ ID NO: 14), ggaggccagcatatgtctcatatgagaaaaatgtactcg (SEQ ID NO: 15), ggaacgaggacaatggctaaaatgatgcaaagagagata (SEQ ID NO: 16), ggaggtaatagtatgagtaagatgatgcaaagagagata (SEQ ID NO: 17), ggataaccccagatggagaacacgaatatcgtcaaagcc (SEQ ID NO: 18), and ggaggactgagtatgcttaatactaatatagtcaaagcc (SEQ ID NO: 19).

[0047] Figure 13. Mutational spectra of the error-prone C>29 DNA polymerases. Cells containing a KanR-

[0048] CmR(Q38TAG) replicon as maintained by the indicated DNAP mutants were passaged for 100 generations with intermediate timepoints taken at 0, 10, 20, and 50 generations. Analysis of next- generation sequencing data from these samples was used to calculate the relative frequencies of all possible types of mutations.

[0049] Figure 14. Concentrations of tigecycline used at each passage of the continuous evolution.

[0050] Figure 15. Tigecycline and tetracycline resistance of pools of cells harboring evolved replicon-encoded tetA variants. After passaging cells containing the N62D / F65S DNAP mutant and a te tA -encoding <T>29 synthetic replicon in increasingly higher concentrations of tigecycline, we obtained pools of mutants able to sustain growth at higher tigecycline and tetracycline concentrations.

[0051] Figure 16. Evolved tetA variants display a range of sensitivities to a combination of fusaric acid and ZnCli.

[0052] A combination of fusaric acid (24 mg / L) and ZnCE (110 mg / L) can be used to counter-select against cells expressing TetA. WT TetA or mutants obtained from the continuous evolution for tigecycline resistance and cloned into colEl circular plasmids were tested for their sensitivity to these compounds. Variants displaying enhanced sensitivity might be of use as improved dual positive-negative selection markers.

[0053] DETAILED DESCRIPTION

[0054] Provided herein is a self-contained orthogonal DNA replication system. The inventors have generated a system comprising replication machinery from a phage that infects gram-positive bacteria that can be used to maintain an episome in host cells, wherein the episome does not need to encode the proteins that form the replication machinery. Thus, the inventors demonstrate that one or more, or all, components of the orthogonal replication machinery may be encoded by the host cell, for instance the host cell’s genome, rendering the cell capable of maintaining orthogonal episomes. The inventors demonstrate that this system is functional in bacterial cells.

[0055] This finding provides a system that allows continuous directed evolution techniques to be applied to a sequence- of-interest without affecting the endogenous genes of the host.

[0056] As an illustrative example, herein is reported the development of a highly mutagenic orthogonal replication system in E. coli. The system relies only on two genes from bacteriophage 29 which naturally infects exclusively Gram-positive hosts and has served as a model to study protein-primed DNA replication. The inventors achieve high replicon transformation efficiencies, develop an efficient strategy to engineer replicons in vivo, and identify an error-prone DNA polymerase that can mutagenize the replicon at rates approaching IO-4substitutions per base, per generation.

[0057] Thus, in a first aspect, there is provided a cell comprising orthogonal DNA replication machinery, wherein the orthogonal DNA replication machinery is from or derived from a phage that infects gram-positive bacteria and / or a phage of the Salasmaviridae family.

[0058] Orthogonal DNA replication machinery is not capable of replicating DNA that is endogenous to the host cell or is less capable of replicating DNA that is endogenous to the host cell compared to the host DNA replication machinery, but is capable of replicating DNA that is compatible with the orthogonal DNA replication machinery. The reduced ability to replicate DNA that is endogenous to the host cell may mean that, when active in a host cell, error-prone orthogonal DNA replication machinery does not introduce mutations into the host cell genome at a significant rate. For instance, the host genome mutation rate may be less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of the mutation rate for DNA that is compatible with the orthogonal DNA replication machinery. The reduced ability to replicate DNA that is endogenous to the host cell may mean that, when active in a host cell, error-prone orthogonal DNA replication machinery does not introduce mutations into the host cell genome. The reduced ability to replicate DNA that is endogenous to the host cell may mean that, when active in a host cell, the orthogonal DNA replication machinery is responsible for less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% of replication of endogenous DNA.

[0059] The orthogonal DNA replication machinery may be referred to as synthetic machinery; the genes for the machinery are artificially introduced into the cell. The genes may be referred to as recombinant genes.

[0060] The cell of the first aspect may be a bacterial cell. The bacterial cell may be from the genus or family Acinetobacter, Enterobacteria, Pseudomonas, Vibrio, or Escherichia. The cell may be a Pseudomonas putida cell or Vibrio natriegens cell. In a particular embodiment, the host cell is an E. coli cell. Suitable E. coli cells include K-12, MG1655, BL21, BL21(DE3), AD494, Origami, HMS174, BLR(DE3), HMS174(DE3), Tuner(DE3), Ongami2(DE3), Rosetta2(DE3), Lemo21(DE3), NiCo21(DE3), T7 Express, Shuffle Express, C41(DE3), C43(DE3), and ml5 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.

[0061] The cell of the first aspect may be a gram-positive or a gram-negative bacterium. The cell of the first aspect may be from the Bacillus genus. The cell of the first aspect may be a B. subtilis cell. In some embodiments, the cell of the first aspect is not of the Bacillus genus. In some embodiments, the cell of the first aspect is not a B. subtilis cell.

[0062] The cell of the first aspect is a viable cell. The cell does not comprise genes that encode sufficient lytic protein components of the phage from which the orthogonal DNA replication machinery is derived to result in cell lysis. In some embodiments, the cell does not comprise any genes that encode lytic protein components of the phage from which the orthogonal DNA replication machinery is derived. In a particular embodiment, the cell does not comprise any genes that encode structural or lytic protein components of the phage from which the orthogonal DNA replication machinery is derived.

[0063] One or more components of the orthogonal DNA replication machinery may be encoded by a non-orthogonal episome within the cell (i.e. an episome that can be maintained by the endogenous DNA replication machinery of the host cell but not by the orthogonal DNA replication machinery). For instance, at least one, a plurality, or all components of the orthogonal DNA replication machinery may be encoded by the genome of the cell. This provides a cell that contains the necessary machinery to replicate compatible orthogonal DNA. The cell may comprise one or more non-orthogonal plasmid, or other non-genomic episome, that encodes at least one, a plurality, or all components of the orthogonal DNA replication machinery. Such plasmids may be referred to as “helper plasmids”. In some embodiments, the components of the orthogonal DNA replication machinery are split between the genome of the cell and one or more non-orthogonal non-genomic episomes. For instance, one or more components of the orthogonal DNA replication machinery may be encoded by the cell genome and one or more components of the orthogonal DNA replication machinery may be encoded by one or more helper plasmid.

[0064] The orthogonal DNA replication machinery may be encoded within a single operon. Thus, the gene or genes of the orthogonal DNA replication machinery may be operably linked to a single promoter. The genes encoding the orthogonal DNA replication machinery may be referred to as recombinant genes. These genes are derived from phage but are encoded on non-orthogonal episomes within the cell, i.e. episomes that cannot be replicated by the wild-type phage DNA polymerase. The term “episome” has its ordinary meaning in the art, for example any accessory extrachromosomal replicating genetic element that can exist either autonomously or can become integrated with the chromosome. The episomes disclosed herein may be referred to as “replicons”.

[0065] Orthogonal DNA replication systems are suitable for evolving sequences because they allow target sequences to be replicated with a lower fidelity than the genes endogenous to the host. Thus, the target sequences are mutated at a higher frequency than the endogenous genes. Therefore, in an embodiment, the orthogonal DNA replication machinery replicates DNA with a lower fidelity than the host cell’s endogenous DNA replication machinery. This means that the orthogonal DNA replication machinery is more error prone when compared to the endogenous DNA replication machinery. Techniques to measure and compare the fidelity of replication by DNA polymerases are known in the art.

[0066] As discussed herein, the inventors have identified that DNA replication machinery of phages that are capable of infecting gram-positive bacteria is suitable for the creation of orthogonal DNA replication systems. The orthogonal DNA replication machinery may be from or derived from a bacteriophage of the Salasmaviridae family. The orthogonal DNA replication machinery may be from or derived from a phi29-like phage within the Salasmaviridae bacteriophage family. The orthogonal DNA replication machinery may be from or derived from a phi29 phage.

[0067] The bacteriophages from which the orthogonal replication machinery of the invention is derived comprise two genes that together provide DNA replication machinery capable of maintaining an orthogonal episome within the cell. These two genes are a gene encoding a terminal protein (TP) and a gene encoding an DNA polymerase. As discussed further herein, embodiments where the cell comprises only these two genes are particularly relevant when the orthogonal episome is provided as a complex that already comprises the TP. The genes may be naturally occurring genes or may comprise one or more substitutions, insertions, or deletions. The proteins encoded by the genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring protein from a phage that can infect gram-positive bacteria or to a naturally occurring protein from a Salasmaviridae phage. The proteins encoded by the genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring protein from the phi29 phage or a phi29-like phage within the Salasmaviridae family. The genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene from a phage that can infect gram-positive bacteria or to a naturally occurring gene from a Salasmaviridae phage. The genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene from the phi29 phage.

[0068] The bacteriophages from which the orthogonal replication machinery of the invention is derived comprise three genes that together provide DNA replication machinery capable of establishing an orthogonal episome within a cell. These are a gene encoding a TP, a gene encoding an DNA polymerase, and a gene encoding a singlestranded DNA-binding protein (SSB). The bacteriophages also include an optional further component that may form part of the orthogonal replication machinery: a gene encoding double-stranded DNA-binding protein (DSB). The genes may be naturally occurring genes or may comprise one or more substitutions, insertions, or deletions. The proteins encoded by the genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring protein from a phage that can infect gram-positive bacteria or to a naturally occurring protein from a Salasmaviridae phage. The proteins encoded by the genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring protein from the phi29 phage or a phi29-like phage within the Salasmaviridae family. The genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene from a phage that can infect gram-positive bacteria or to a naturally occurring gene from a Salasmaviridae phage. The genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene from the phi29 phage. Thus, a phi29 TP, phi29 SSB, or phi29 DSB as referred to herein encompasses the natural proteins and variants of said natural proteins that retain the appropriate DNA replication function. In an embodiment of the first aspect, therefore, there is provided a cell comprising an ODNAP and a TP. There is provided a cell comprising an ODNAP, a TP, and an SSB. There is provided a cell comprising an ODNAP, a TP, and a DSB. There is provided a cell comprising an ODNAP, a TP, an SSB, and a DSB.

[0069] As used herein, the term “SSB” refers to a protein from the orthogonal DNA replication machinery of a phage, wherein the SSB corresponds to the phi29 phage protein p5. The SSB need not necessarily function as a singlestranded DNA-binding protein, for instance it may be capable of also serving as a double-stranded DNA-binding protein (e.g. it may have a dual function in the manner of some naturally occurring phage SSBs).

[0070] As used herein, the term “DSB” refers to a protein from the orthogonal DNA replication machinery of a phage, wherein the DSB corresponds to the phi29 phage protein p6. The DSB need not necessarily function as a doublestranded DNA-binding protein, for instance it may be capable of also serving as a single-stranded DNA-binding protein (e.g. it may have a dual function in the manner of some naturally occurring phage DSBs).

[0071] Thus, the SSB and the DSB need only have the property of binding to DNA. They are identifiable by the skilled person as two of the four components of DNA replication machinery from the aforementioned phages and due to their correspondence with p5 and p6 of phi29.

[0072] The SSB is optional, particularly for embodiments where the orthogonal episome is delivered as a complex with the TP. The DSB is optional and not strictly necessary for maintenance of an orthogonal episome.

[0073] The orthogonal DNA replication machinery may be from or derived from a Salasmaviridae bacteriophage. Therefore, in a particular example, the cell of the first aspect comprises a gene encoding a Salasmaviridae TP. In other examples, the cell of the first aspect comprises a gene encoding a Salasmaviridae TP, a gene encoding a Salasmaviridae SSB, and optionally a gene encoding a Salasmaviridae DSB. In a particular embodiment, the orthogonal DNA replication machinery is from or derived from phi29 bacteriophage. Therefore, in a particular example, the cell of the first aspect comprises a gene encoding a phi29 TP. The cell may additionally comprise a gene encoding a phi29 SSB and optionally a gene encoding a phi29 DSB. The genes may be naturally occurring genes or may comprise one or more substitutions, insertions, or deletions. The proteins encoded by the genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring protein from a phi29 bacteriophage or a phi29-like phage within the Salasmaviridae family. The genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene from a phi29 bacteriophage. Thus, a phi29 TP, phi29 SSB, or phi29 DSB as referred to herein encompasses the natural proteins and variants of said natural proteins that retain the appropriate DNA replication function. The bacterial cell also comprises an ODNAP, which may be a Salasmaviridae DNA polymerase or, in particular, a phi29 DNA polymerase. The DNA polymerase may be a naturally occurring or engineered polymerase. For instance, the orthogonal DNA polymerase may be modified so as to increase the error rate or to render the DNA polymerase error prone. The orthogonal DNA polymerase may have a mutation rate of at least 10'9, 10'8, 10'710'6, or 10'5s.p.b. The gene encoding the DNA polymerase may be a naturally occurring gene or may comprise one or more substitutions, insertions, or deletions. The protein encoded by the gene may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring phi29 ODNAP or an ODNAP from a phi29-like phage within the Salasmaviridae family. The gene may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring phi29 ODNAP gene. Thus, a phi29 DNA polymerase as referred to herein encompasses the natural polymerase and variants of said natural polymerase that retains the appropriate DNA replication function. In an example, the cell of the first aspect comprises a gene encoding a Salasmaviridae TP and a gene encoding a Salasmaviridae ODNAP. In a particular example, the cell of the first aspect comprises a gene encoding a phi29 TP and a gene encoding a phi29 ODNAP. In another example, the cell of the first aspect comprises a gene encoding a Salasmaviridae TP, a gene encoding a Salasmaviridae SSB, and a gene encoding a Salasmaviridae ODNAP. In another example, the cell of the first aspect comprises a gene encoding a Salasmaviridae TP, a gene encoding a Salasmaviridae SSB, a gene encoding a Salasmaviridae DSB, and a gene encoding a Salasmaviridae ODNAP. In another example, the cell of the first aspect comprises a gene encoding a phi29-like phage TP and a gene encoding a phi29-like phage ODNAP. The cell may comprise a gene encoding a phi29-like phage TP, a gene encoding a phi29-like phage ODNAP, and a gene encoding a phi29-like phage SSB. The cell may comprise a gene encoding a phi29-like phage TP, a gene encoding a phi29-like phage ODNAP, and a gene encoding a phi29-like phage DSB. The cell may comprise a gene encoding a phi29-like phage TP, a gene encoding a phi29-like phage ODNAP, a gene encoding a phi29-like phage SSB, and a gene encoding a phi29-like phage DSB. In another particular example, the cell of the first aspect comprises a gene encoding a phi29 TP, a gene encoding a phi29 SSB, and a gene encoding a phi29 ODNAP. In another particular example, the cell of the first aspect comprises a gene encoding a phi29 TP, a gene encoding a phi29 SSB, a gene encoding a phi29 DSB, and a gene encoding a phi29 ODNAP. In another particular example, the cell of the first aspect comprises a gene encoding a phi29 TP, a gene encoding a phi29 SSB, and a gene encoding a phi29 ODNAP. In another particular example, the cell of the first aspect comprises a gene encoding a phi29 TP, a gene encoding a phi29 SSB, a gene encoding a phi29 DSB, and a gene encoding a phi29 ODNAP.

[0074] The cell of the first aspect may comprise more than one ODNAP. For instance, the cell may comprise two ODNAPs with different mutation rates. The cell may comprise a wild-type ODNAP and a mutated error-prone ODNAP. The ODNAPs may be inducible under different conditions and so it is possible to alter the culture conditions to determine which ODNAP is active.

[0075] The SSB and optionally DSB may be encoded by a non-orthogonal episome. The SSB and optionally DSB may be encoded by a plasmid that can be maintained by the DNA polymerase endogenous to the cell. The SSB and optionally the DSB may be overexpressed. An overexpressed SSB or DSB may be under the control of an inducible promoter.

[0076] In particular examples, the cell of the first aspect does not comprise any of lytic or structural genes associated with the phage from which the orthogonal DNA replication machinery is derived. For instance, the cell of the first aspect may comprise no genes other than the TP and ODNAP genes and optionally inverted terminal repeats (ITRs) from the phage or phages from which said genes were derived. In another example, the cell of the first aspect may comprise no genes other than the TP, optionally SSB, optionally DSB, and ODNAP genes and optionally inverted terminal repeats (ITRs) from the phage or phages from which said genes were derived. As discussed, the TP, ODNAP, DSB, and SSB may be referred to as synthetic or recombinant genes.

[0077] In particular examples, the cell of the first aspect does not comprise any lytic or structural genes derived from phi29. phi29 has a linear double stranded genome and the ends of the linear genome are composed of inverted terminal repeats that form the binding site for the TP and function as origins of replication. A schematic of the genome is provided in Figure 1. In some embodiments, the cell of the first aspect comprises no phi29 genes other than the two, three, or four genes mentioned in the preceding paragraphs and optionally ITRs.

[0078] An example of a sequence of a phi29 TP is provided below.

[0079] MARSPRIRIKDNDKAEYARLVKNTKAKIARTKKKYGVDLTAEIDIPDLDSFETRAQFNKWKEQASSFTNR ANMRYQFEKNAYGVVASKAKIAEIERNTKEVQRLVDEKIKAMKDKEYYAGGKPQGTIEQRIAMTSPAH VTGINRPHDFDFSKVRSYSRLRTLEESMEMRTDPQYYEKKMIQLQLNFIKSVEGSFNSFDAADELIEELKK IPPDDFYELFLRISEISFEEFDSEGNTVENVEGNVYKILSYLEQYRRGDFDLSLKGF (SEQ ID NO: 1)

[0080] An example of a sequence of a phi29 ODNAP is provided below.

[0081] MPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFII NWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTV LKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPT LSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDY PLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKF KATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETK DPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAK YLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVP GGVVLVDDTFTIK (SEQ ID NO: 2)

[0082] The ODNAP may be an error-prone ODNAP. For instance, the ODNAP may be mutated to disrupt the proofreading exonuclease activity. The ODNAP may be an error-prone polymerase according to SEQ ID NO: 2 and comprising one or more mutations. Exemplary mutations that may be applied to SEQ ID NO: 2 are: T15I (PMID: 8605889), N62D (also PMID: 8605889), and / or F65S (PMID: 9786901). In a particular embodiment, the ODNAP comprises N62D and F65S. The ODNAP may be an error-prone polymerase with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to SEQ ID NO: 2 and comprising N62D and F65S.

[0083] An example of a sequence of a phi29 DSB is provided below.

[0084] MAKMMQREITKTTVNVAKMVMVDGEVQVEQLPSETFVGNLTMEQAQWRMKRKYKGEPVQVVSVEP NTEVYELPVEKFLEVATVRVEKDEDQEEQTEAPEEQVAE (SEQ ID NO: 3)

[0085] An example of a sequence of a phi29 SSB is provided below.

[0086] MENTNIVKATFDTETLEGQIKIFNAQTGGGQSFKNLPDGTIIEANAIAQYKQVSDTYGDAKEETVTTIFAA DGSLYSAISKTVAEAASDLIDLVTRHKLETFKVKVVQGTSSKGNVFFSLQLSL (SEQ ID NO: 4)

[0087] In a particular example, the bacterial cell of the first aspect comprises one or more genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to any one, two, or three of SEQ ID NOs: 1, 2, and 4. The cell may comprise a gene encoding a protein that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 2, and comprising any of the mutations disclosed herein (e.g. T15I, N62D, and / or F65S). The cell may also comprise a gene encoding a protein that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 3. In another example, the bacterial cell of the first aspect comprises genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to any one, two, three, or four of SEQ ID NOs: 1, 2, 3, and 4.

[0088] In another example, the bacterial cell of the first aspect comprises genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 1 and SEQ ID NO: 2. In another example, the bacterial cell of the first aspect comprises genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4. In another example, the bacterial cell of the first aspect comprises genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In another example, the bacterial cell of the first aspect comprises genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0089] In embodiments comprising only a TP and an ODNAP, the TP and / or the ODNAP may be encoded by a non- orthogonal episome within the cell. For instance, at least one or both of the TP and the ODNAP may be encoded by the genome of the cell. This provides a cell that contains the necessary machinery to replicate compatible orthogonal DNA. The cell may comprise one or more non-orthogonal plasmid, or other non-genomic episome, that encodes the TP and / or the ODNAP. Such plasmids may be referred to as “helper plasmids”. In some embodiments, the TP and the ODNAP are split between the genome of the cell and one or more non-orthogonal non-genomic episomes. The TP and the ODNAP may be encoded within a single operon. Thus, in an embodiment, the cell is a bacterial cell, which may be E. coli, wherein the bacterial cell genome comprises an operon encoding the TP and ODNAP. One, two, or all three of the TP, SSB, and ODNAP may be encoded on a non-orthogonal episome within the cell (i.e. an episome that can be maintained by the endogenous DNA replication machinery of the host cell). One, two, or all three of the TP, SSB, and ODNAP may be encoded by the genome of the cell. The cell may be a bacterial cell comprising a genome encoding one, two, or all three of the TP, SSB, and ODNAP. One, two, or all three of the TP, SSB, and ODNAP may be encoded by a single operon. Thus, in an embodiment, the cell is a bacterial cell, which may be E. coli, wherein the bacterial cell genome comprises an operon encoding the TP, SSB, and ODNAP. In a particular embodiment, the operon comprises, from 5’ to 3’, the genes for TP, ODNAP, and SSB.

[0090] One, two, three, or all four of the TP, SSB, DSB, and ODNAP may be encoded on a non-orthogonal episome within the cell (i.e. an episome that can be maintained by the endogenous DNA replication machinery of the host cell). One, two, three, or all four of the TP, SSB, DSB, and ODNAP may be encoded by the genome of the cell. The cell may be a bacterial cell comprising a genome encoding one, two, three, or all four of the TP, SSB, DSB, and ODNAP. One, two, three, or all four of the TP, SSB, DSB, and ODNAP may be encoded by a single operon. Thus, in an embodiment, the cell is a bacterial cell, which may be E. coli, wherein the bacterial cell genome comprises an operon encoding the TP, SSB, DSB, and ODNAP. In a particular embodiment, the operon comprises, from 5’ to 3’, the genes for TP, ODNAP, DSB, and SSB.

[0091] The genes may be codon optimised for the host cell. For instance, the genes may be codon optimised for E. coli. As an example, the cell may comprise a gene cluster encoding a TP, an ODNAP, optionally a DSB, and optionally an SSB, each of which has been optimised for E. coli. As a more specific example, the cell may comprise a gene cluster encoding a phi29 TP, a phi29 ODNAP, optionally a phi29 DSB, and optionally a phi29 SSB, each of which has been optimised for E. coli.

[0092] A purely illustrative example of a phi29 gene cluster encoding a TP, ODNAP, DSB, and an SSB is provided below. The coding sequences in are underlined and the components are in the order: promoter-TP-DNAP-DSB- SSB. ttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaattctagtagaaaaaaggaggtctacacatggccaggagccctcgcatacgcatcaa agacaacgacaaggcggagtacgcacggttagtgaaaaacacaaaggcgaagatagcgagaactaagaagaaatatggcgtcgatctgacggcggaaatcgaca ttcccgatttggacagcttcgagacgcgcgctcagttcaataagtggaaagaacaggcttcgtctttcacgaacagggctaatatgaggtatcaattcgaaaagaatgct tacggtgtggtagcttcaaaagcgaagatcgcagaaattgaacgaaatacgaaggaagtccagcgcctcgtggacgagaagataaaggccatgaaggacaagga gtattacgctggtgggaagccacaaggtactattgagcagcgaatcgctatgaccagccccgctcacgtcactggcataaaccgaccacatgatttcgatttctccaag gtgaggagctattctcgtctgcgaacgttagaagaaagtatggaaatgcgtactgatccccagtactacgaaaaaaagatgattcagcttcaattaaatttcataaagag cgtggaaggctcgtttaattcgttcgacgcagcggatgaattgatcgaggagttgaagaagattccaccagacgatttctatgagctgttcttaaggataagtgagatctc cttcgaagagttcgactccgaaggcaacaccgttgagaatgtggagggaaatgtctacaagatcctcagctacttagaacagtatcgtaggggtgatttcgacctatctt taaaagggttttgagatccagtatcgaggaggaatatttatgcccaggaagatgtactcgtgcgatttcgaaaccacgactaaggtggaagactgcagggtttgggcgt acggttatatgaatatcgaagaccattctgaatacaaaatcggcaatagcctcgacgaattcatggcttgggtgctgaaggttcaggcagatctctattttcataatctcaa gttcgatggcgcgttcattattaactggttggagaggaacggttttaagtggagcgcggacggattgcccaacacctataacacgataatcagtcggatgggccagtg gtatatgattgatatctgtcttggttataaggggaaaaggaagatccacacggttatttacgactcgcttaagaaactgcctttccctgtgaaaaaaattgctaaagacttca agctcacggttctcaaaggagacatagattaccacaaggagaggccagtggggtacaaaatcacacctgaagagtacgcatacatcaaaaacgacatacagatcat cgcggaagcactcttaatccaatttaagcagggtttagatcgaatgaccgccggttcggattcgttaaaggggttcaaagacataatcacgacgaaaaagttcaagaag gtctttccaaccctgagcttaggtttggataaagaagttcgatacgcctatcgaggcggtttcacgtggctcaatgacaggtttaaggaaaaggaaattggagaaggcat ggtctttgacgtgaacagtctgtaccctgctcagatgtacagtagactcttgccgtacggagaacctatcgtatttgagggtaagtacgtatgggatgaggattacccact ccacatacagcacatacggtgcgaattcgagctgaaagagggttacatacccaccattcaaataaagaggtctcggttttataaagggaacgaatatttgaagagcagt gggggtgaaatcgcggatttatggttgtccaatgtggacttagagctgatgaaagaacactacgatttgtacaacgttgagtacataagcggacttaagttcaaagcgac caccgggctgtttaaggacttcattgataagtggacttacattaagaccactagcgaaggtgcgatcaagcaactcgccaagctgatgttgaacagtctgtacggtaaat ttgcgtcaaacccagatgtgacaggaaaagtcccatatctgaaagaaaacggagcgttagggtttcggcttggcgaagaggaaacgaaagatcctgtgtacacgccc atgggtgtcttcataacagcatgggctaggtacacgacaatcaccgcagcacaggcctgttacgaccggatcatctactgtgacacagattcaatacatcttactggga cggagatcccagacgtcataaaggacatcgtcgaccccaagaaactcggctattgggcccacgaatccactttcaagcgggctaaatacttacgacaaaagacttata tccaggacatctacatgaaagaagtggatggaaagctcgtagaaggaagccccgatgattacacggacattaaattcagcgtgaaatgtgccgggatgactgacaag attaagaaagaggtcacgtttgagaattttaaagtgggattcagtcgtaaaatgaagcccaagccagtgcaagtccctggcggtgtcgtcttggtcgacgacacctttac aataaagtgacacgctgcaataaggaacgaggacaatggctaaaatgatgcaaagagagataacgaaaactaccgtcaatgtggccaaaatggttatggtggatgg ggaagtacaggtagagcaacttccgtctgagacctttgttggcaacctcacgatggagcaggctcagtggcggatgaaacgcaaatacaaaggagagcccgtccag gtcgtgagtgtcgaaccaaacactgaggtgtacgaactacccgtagagaagttccttgaggtggctaccgtacgagtagagaaagatgaggaccaagaggagcag acagaagcgccggaggagcaggttgcagagtaagcaaactgtttaaggataaccccagatggagaacacgaatatcgtcaaagccacgttcgacactgagaccct ggaaggccagatcaagatattcaatgcacagacaggcggcggccaatcattcaagaatcttcctgatgggacaattatcgaggccaacgccatagcccaatacaaac aagtttcagacacatacggagacgcgaaagaagaaacagtcaccactatcttcgctgcggatggatctctatatagcgcaataagcaaaaccgtggcagaggctgcc agcgatctcattgacttagtaacacgccataagcttgaaacttttaaggtcaaggtggttcagggcaccagcagtaaaggcaacgttttcttctctcttcagctaagcctct ga (SEQ ID NO: 5)

[0093] Another illustrative example of a phi29 gene cluster encoding a TP, ODNAP, DSB, and an SSB is provided below. The open reading frames are underlined. ttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaattctagtagaaaaaaggaggtgacgtgcatggcgagaagcccgcggatccgtatca aagacaacgacaaggcggagtacgcacggttagtgaaaaacacaaaggcgaagatagcgagaactaagaagaaatatggcgtcgatctgacggcggaaatcgac attcccgatttggacagcttcgagacgcgcgctcagttcaataagtggaaagaacaggcttcgtctttcacgaacagggctaatatgaggtatcaattcgaaaagaatg cttacggtgtggtagcttcaaaagcgaagatcgcagaaattgaacgaaatacgaaggaagtccagcgcctcgtggacgagaagataaaggccatgaaggacaagg agtattacgctggtgggaagccacaaggtactattgagcagcgaatcgctatgaccagccccgctcacgtcactggcataaaccgaccacatgatttcgatttctccaa ggtgaggagctattctcgtctgcgaacgttagaagaaagtatggaaatgcgtactgatccccagtactacgaaaaaaagatgattcagcttcaattaaatttcataaaga gcgtggaaggctcgtttaattcgttcgacgcagcggatgaattgatcgaggagttgaagaagattccaccagacgatttctatgagctgttcttaaggataagtgagatct ccttcgaagagttcgactccgaaggcaacaccgttgagaatgtggagggaaatgtctacaagatcctcagctacttagaacagtatcgtaggggtgatttcgacctatct ttaaaagggttttgagatccagtatcgaggaggccagcatatgtctcatatgagaaaaatgtactcgtgcgatttcgaaaccacgactaaggtggaagactgcagggttt gggcgtacggttatatgaatatcgaagaccattctgaatacaaaatcggcaatagcctcgacgaattcatggcttgggtgctgaaggttcaggcagatctctattttcata atctcaagttcgatggcgcgttcattattaactggttggagaggaacggttttaagtggagcgcggacggattgcccaacacctataacacgataatcagtcggatggg ccagtggtatatgattgatatctgtcttggttataaggggaaaaggaagatccacacggttatttacgactcgcttaagaaactgcctttccctgtgaaaaaaattgctaaa gacttcaagctcacggttctcaaaggagacatagattaccacaaggagaggccagtggggtacaaaatcacacctgaagagtacgcatacatcaaaaacgacatac agatcatcgcggaagcactcttaatccaatttaagcagggtttagatcgaatgaccgccggttcggattcgttaaaggggttcaaagacataatcacgacgaaaaagtt caagaaggtctttccaaccctgagcttaggtttggataaagaagttcgatacgcctatcgaggcggtttcacgtggctcaatgacaggtttaaggaaaaggaaattgga gaaggcatggtctttgacgtgaacagtctgtaccctgctcagatgtacagtagactcttgccgtacggagaacctatcgtatttgagggtaagtacgtatgggatgagga ttacccactccacatacagcacatacggtgcgaattcgagctgaaagagggttacatacccaccattcaaataaagaggtctcggttttataaagggaacgaatatttga agagcagtgggggtgaaatcgcggatttatggttgtccaatgtggacttagagctgatgaaagaacactacgatttgtacaacgttgagtacataagcggacttaagttc aaagcgaccaccgggctgtttaaggacttcattgataagtggacttacattaagaccactagcgaaggtgcgatcaagcaactcgccaagctgatgttgaacagtctgt acggtaaatttgcgtcaaacccagatgtgacaggaaaagtcccatatctgaaagaaaacggagcgttagggtttcggcttggcgaagaggaaacgaaagatcctgtg tacacgcccatgggtgtcttcataacagcatgggctaggtacacgacaatcaccgcagcacaggcctgttacgaccggatcatctactgtgacacagattcaatacatc ttactgggacggagatcccagacgtcataaaggacatcgtcgaccccaagaaactcggctattgggcccacgaatccactttcaagcgggctaaatacttacgacaa aagacttatatccaggacatctacatgaaagaagtggatggaaagctcgtagaaggaagccccgatgattacacggacattaaattcagcgtgaaatgtgccgggatg actgataagattaagaaagaggtcacgtttgagaattttaaagtgggattcagtcgtaaaatgaagcccaagccagtgcaagtccctggcggtgtcgtcttggtcgacg acacctttacaataaagtgacacgctgcaataaggaggtaatagtatgagtaagatgatgcaaagagagataacgaaaactaccgtcaatgtggccaaaatggttatg gtggatggggaagtacaggtagagcaacttccgtctgagacctttgttggcaacctcacgatggagcaggctcagtggcggatgaaacgcaaatacaaaggagagc ccgtccaggtcgtgagtgtcgaaccaaacactgaggtgtacgaactacccgtagagaagttccttgaggtggctaccgtacgagtagagaaagatgaggaccaaga ggagcagacagaagcgccggaggagcaggttgcagagtaagcaaactgtttaaggaggactgagtatgcttaatactaatatagtcaaagccacgttcgacactga gaccctggaaggccagatcaagatattcaatgcacagacaggcggcggccaatcattcaagaatcttcctgatgggacaattatcgaggccaacgccatagcccaat acaaacaagtttcagacacatacggagacgcgaaagaagaaacagtcaccactatcttcgctgcggatggatctctatatagcgcaataagcaaaaccgtggcagag gctgccagcgatctcattgacttagtaacacgccataagcttgaaacttttaaggtcaaggtggttcagggcaccagcagtaaaggcaacgttttcttctctcttcagctaa gcctctga (SEO ID NO: 11)

[0094] In an example, a bacterial cell of the first aspect comprises an operon comprising a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5 or SEQ ID NO: 11. The operons with lower degrees of sequence identity may still encode proteins identical to those encoded by SEQ ID NO: 5 and SEQ ID NO: 11.

[0095] The operon may be designed according to the methods disclosed in WO2023285596 or Dunkelmann et al. (A 68- codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat. Chem. 13, 1110-1117 (2021). https: / / doi.org / 10.1038 / s41557-021-00764-5), each of which is herein incorporated by reference in their entirety. These documents disclose methods that enable the design of suitable synthetic ribosome binding sites. For instance, WO2023285596 discloses a method of designing an operon comprising at least two exogenous open reading frames for expression in a host cell. Thus, one, two, three, or all four of the TP, SSB, DSB, and ODNAP may be encoded by a single operon and the operon may be have been optimised according to the techniques disclosed in the aforementioned documents. The operon may comprise an inducible promoter operably linked to the genes; illustrative examples include the Parabinose promoter and the PIPTG promoter, or variants thereof. The operon may comprise a constitutive endogenous promoter operably linked to the genes; an illustrative example is the PdnaKJ promoter or a variant thereof. The operon may comprise a LexA-controlled promoter operably linked to the genes; illustrative examples include the PrecN promoter and the PuvrB promoter, or variants thereof. The operon may comprise a selfregulating promoter; an illustrative example is the PpN15 or a variant thereof. The operon may comprise an Anderson promoter; an illustrative example is the PJ23114 promoter or a variant thereof.

[0096] There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, comprising a gene encoding a Salasmaviridae TP and a gene encoding an ODNAP. There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, expressing a Salasmaviridae TP and an ODNAP. The genes may be any as described herein. In some embodiments, the genes are recombinant genes and are present on one or more non-orthogonal (i.e. endogenously replicated) episomes.

[0097] There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, comprising a gene encoding a phi29 TP and a gene encoding an ODNAP. There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, expressing a phi29 TP and an ODNAP. The genes may be any as described herein. In some embodiments, the genes are recombinant genes and are present on one or more non-orthogonal (i.e. endogenously replicated) episomes.

[0098] In an embodiment of the first aspect, there is provided a viable cell comprising a gene encoding a Salasmaviridae ODNAP and a gene encoding a Salasmaviridae TP. There is provided a viable cell comprising a gene encoding a Salasmaviridae ODNAP, a gene encoding a Salasmaviridae TP, and a gene encoding a Salasmaviridae SSB. There is provided a viable cell comprising a gene encoding a Salasmaviridae ODNAP, a gene encoding a Salasmaviridae TP, and a gene encoding a Salasmaviridae DSB. There is provided a viable cell comprising a gene encoding a Salasmaviridae ODNAP, a gene encoding a Salasmaviridae TP, a gene encoding a Salasmaviridae SSB, and a gene encoding a Salasmaviridae DSB. There is provided a viable cell expressing or capable of expressing (e.g. under an inducible promoter) a Salasmaviridae ODNAP and a Salasmaviridae TP. There is provided a viable cell expressing or capable of expressing a Salasmaviridae ODNAP, a Salasmaviridae TP, and a Salasmaviridae SSB. There is provided a viable cell expressing or capable of expressing a Salasmaviridae ODNAP, a Salasmaviridae TP, and a Salasmaviridae DSB. There is provided a viable cell expressing or capable of expressing a Salasmaviridae ODNAP, a Salasmaviridae TP, a Salasmaviridae SSB, and a Salasmaviridae DSB. The viable cell can be cultured and, under appropriate conditions, could maintain an orthogonal episome. The viable cell could be one of a population of viable cells that can be cultured and, under appropriate conditions, the population could maintain an orthogonal episome.

[0099] In an embodiment of the first aspect, there is provided a viable cell comprising a gene encoding a phi29-like phage ODNAP and a gene encoding a phi29-like phage TP. There is provided a viable cell comprising a gene encoding a phi29-like phage ODNAP, a gene encoding a phi29-like phage TP, and a gene encoding a phi29-like phage SSB. There is provided a viable cell comprising a gene encoding a phi29-like phage ODNAP, a gene encoding a phi29-like phage TP, and a gene encoding a phi29-like phage DSB. There is provided a viable cell comprising a gene encoding a phi29-like phage ODNAP, a gene encoding a phi29-like phage TP, a gene encoding a phi29-like phage SSB, and a gene encoding a phi29-like phage DSB. There is provided a viable cell expressing or capable of expressing (e.g. under an inducible promoter) a phi29-like phage ODNAP and a phi29-like phage TP. There is provided a viable cell expressing or capable of expressing a phi29-like phage ODNAP, a phi29-like phage TP, and a phi29-like phage SSB. There is provided a viable cell expressing or capable of expressing a phi29-like phage ODNAP, a phi29-like phage TP, and a phi29-like phage DSB. There is provided a viable cell expressing or capable of expressing a phi29-like phage ODNAP, a phi29-like phage TP, a phi29-like phage SSB, and a phi29-like phage DSB. The viable cell can be cultured and, under appropriate conditions, could maintain an orthogonal episome. The viable cell could be one of a population of viable cells that can be cultured and, under appropriate conditions, the population could maintain an orthogonal episome.

[0100] In an embodiment of the first aspect, there is provided a viable cell comprising a gene encoding a phi29 ODNAP and a gene encoding a phi29 TP. There is provided a viable cell comprising a gene encoding a phi29 ODNAP, a gene encoding a phi29 TP, and a gene encoding a phi29 SSB. There is provided a viable cell comprising a gene encoding a phi29 ODNAP, a gene encoding a phi29 TP, and a gene encoding a phi29 DSB. There is provided a viable cell comprising a gene encoding a phi29 ODNAP, a gene encoding a phi29 TP, a gene encoding a phi29 SSB, and a gene encoding a phi29 DSB. There is provided a viable cell expressing or capable of expressing (e.g. under an inducible promoter) a phi29 ODNAP and a phi29 TP. There is provided a viable cell expressing or capable of expressing a phi29 ODNAP, a phi29 TP, and a phi29 SSB. There is provided a viable cell expressing or capable of expressing a phi29 ODNAP, a phi29 TP, and a phi29 DSB. There is provided a viable cell expressing or capable of expressing a phi29 ODNAP, a phi29 TP, a phi29 SSB, and a phi29 DSB. The viable cell can be cultured and, under appropriate conditions, could maintain an orthogonal episome. The viable cell could be one of a population of viable cells that can be cultured and, under appropriate conditions, the population could maintain an orthogonal episome.

[0101] In some embodiments of the first aspect, there is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, comprising a gene encoding a Salasmaviridae TP, a gene encoding an ODNAP, a gene encoding a Salasmaviridae SSB, and optionally a gene encoding a Salasmaviridae DSB. There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, expressing a Salasmaviridae TP, an ODNAP, a Salasmaviridae SSB, and optionally a Salasmaviridae DSB. The genes may be any as described herein. In some embodiments, the genes are recombinant genes and are present on one or more non-orthogonal (i.e. endogenously replicated) episomes.

[0102] There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, comprising a gene encoding a phi29 TP, a gene encoding an ODNAP, a gene encoding a phi29 SSB, and optionally a gene encoding a phi29 DSB. There is provided herein a cell, such as a bacterial cell that is optionally an E. coli cell, expressing a phi29 TP, an ODNAP, a phi29 SSB, and optionally a phi29 DSB. The genes may be any as described herein. In some embodiments, the genes are recombinant genes and are present on one or more non-orthogonal (i.e. endogenously replicated) episomes.

[0103] An orthogonal episome, once established (for instance, in cells comprising a TP, ODNAP, SSB, and optionally a DSB) can be extracted in a manner that maintains the bound proteins. This complex can then be transferred to new cells. The new cells only require the TP and the ODNAP to maintain the orthogonal episome and they do not require the SSB or the DSB. Thus, in an embodiment the cell comprising a gene encoding a Salasmaviridae TP and a gene encoding an ODNAP and does not comprise (or does not express or is not required to express) a gene encoding a Salasmaviridae SSB or Salasmaviridae DSB. In another embodiment, the cell comprising a gene encoding a phi29 TP and a gene encoding an ODNAP does not comprise (or does not express or is not required to express) a gene encoding a phi29 SSB or phi29 DSB.

[0104] The TP and / or the ODNAP may be encoded by a non-orthogonal episome within the cell. For instance, at least one or both of the TP and the ODNAP may be encoded by the genome of the cell. This provides a cell that contains the necessary machinery to replicate compatible orthogonal DNA. The cell may comprise one or more non-orthogonal plasmid, or other non-genomic episome, that encodes the TP and / or the ODNAP. In some embodiments, the TP and the ODNAP are split between the genome of the cell and one or more non-orthogonal non-genomic episomes. The TP and the ODNAP may be encoded within a single operon. The genes encoding the TP and the ODNAP may be referred to as recombinant genes. These genes are derived from phage but are encoded on non-orthogonal episomes within the cell, i.e. episomes that cannot be replicated by the wild-type phage DNA polymerase. The TP and the ODNAP may be any as described herein. For example, in some embodiments, the TP and / or the ODNAP are from or derived from a Salasmaviridae family member. In a particular embodiment, the TP and the ODNAP are from phage phi29. The proteins encoded by the genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to a naturally occurring protein. The TP and / or ODNAP proteins may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similarity or identity to naturally occurring proteins from a phi29 bacteriophage. The genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene. The TP and / or ODNAP genes may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to a naturally occurring gene from a phi29 bacteriophage. The DNA polymerase may be a naturally occurring or engineered polymerase, as discussed for the first aspect. For instance, the orthogonal DNA polymerase may be modified to increase the error rate or to render the DNA polymerase error prone. The orthogonal DNA polymerase may have a mutation rate of at least 10'9, 10'8, 10'710'6, or 10'5s.p.b. The cell may comprise one or more genes encoding proteins that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 1 and / or SEQ ID NO: 2. The cell may comprise a gene encoding a protein that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to SEQ ID NO: 2, and comprising any of the mutations disclosed herein.

[0105] In particular examples, the cells do not comprise any of lytic or structural genes associated with a member of the Salasmaviridae family, as discussed above. For instance, the cell of these embodiments may comprise no Salasmaviridae family genes other than the TP and ODNAP genes and optionally inverted terminal repeats (ITRs). In particular examples, the cells of these embodiments do not comprise any lytic or structural genes derived from phi29. In some embodiments, the cell of these embodiments comprise no phi29 genes other than the TP and ODNAP and optionally ITRs.

[0106] Any of the cells of the first aspect may comprise a gene encoding Gam protein from the lambda phage. The cell of the first aspect may be a bacterial cell, such as an E. coli cell, that comprises a gene encoding Gam protein from the lambda phage. The cell of the first aspect may be a bacterial cell, such as an E. coli cell, that expresses Gam protein from the lambda phage. The Gam protein may be a wild-type protein or derivative that protects linear double-stranded DNA from degradation. The Gam protein may be a wild-type protein or derivative that inhibits host nucleases RecBCD and / or sbcCD.

[0107] The Gam protein may be encoded on a non-orthogonal episome, such as the cell’s genome or a plasmid. The Gam protein may be overexpressed. An overexpressed Gam protein may be under the control of an inducible promoter.

[0108] The cell of the first aspect may overexpress Gam, SSB, and DSB. The cell of the first aspect may overexpress Gam, Salasmaviridae SSB, and Salasmaviridae DSB. The cell of the first aspect may overexpress Gam, phi29 SSB, and phi29 DSB. The cell of the first aspect may express SSB from a first SSB gene and DSB from a first DSB gene, and the expression of SSB and / or DSB may be increased (e.g. when establishing an orthogonal replicon) by inducing expression from a second SSB gene and / or a second DSB gene.

[0109] The cell of the first aspect is capable of replicating orthogonal DNA that is compatible with the orthogonal DNA replication machinery. As such, one or more episomes compatible with the orthogonal DNA replication machinery may be present within the cell. Such episomes may be referred to as “orthogonal episomes” or “orthogonal replicons”.

[0110] The cell of the first aspect may comprise at least one, at least two, or at least three orthogonal episomes. The cell of the first aspect may comprise only one, only two, or only three orthogonal episomes. Thus, in an embodiment, the cell of the first aspect comprises an episome that is capable of being replicated by the orthogonal replication machinery. In embodiments where the cell comprises DNA replication machinery from a bacteriophage capable of infecting gram-positive bacteria, the episome comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with said phage. In embodiments where the cell comprises DNA replication machinery from a Salasmaviridae phage, the episome comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with said phage. In embodiments where the cell comprises DNA replication machinery from phi29, the episome comprises a 5’ ITR and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with phi29 DNA replication machinery. The episome may be a linear plasmid.

[0111] The orthogonal episome may not encode one, two, three, or all four of a TP, a DNA polymerase, an SSB, and a DSB. In particular embodiments, the episome does not comprise sequences of the bacteriophage genome other than the ITRs.

[0112] The orthogonal episome may comprise a sequence-of-interest. For instance, a gene to which it is desirable to apply continuous directed evolution techniques. The gene may encode a protein or polypeptide for which particular functionality, or an increase or decrease in functionality, is desired. The sequence-of-interest may have a function in its own right. For instance, the sequence-of-interest may be a promoter.

[0113] Purely illustrative examples include an antibiotic resistance gene, a fluorescent protein, and a promoter. However, the invention is not limited to these illustrations and, as will be apparent to the skilled person, the invention is compatible with any sequence-of-interest for which a change in functionality, efficacy, or similar may be brought about by mutations.

[0114] In a particular embodiment, the episome is a linear plasmid comprising: a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with phi29 DNA replication machinery.

[0115] The linear plasmid may lack 5’ phosphorylation. For instance, the linear plasmid may have been generated using non-phosphorylated primers.

[0116] The orthogonal episome may comprise a marker. For instance, a selectable marker. The marker may be a positive marker such that cells comprising the orthogonal episome may be identified or selected, or so that selection pressure can be applied to a population of cells to increase the retention of the orthogonal episome.

[0117] In an example, a 5’ ITR compatible with phi29 DNA replication machinery is as below, aaagtaagcccccaccctcacatgataccattctcctaatatcgacataatccgtcgatcctcggcataccatgatcagggagggaaactactacttaatatatcaatctat agacctactagataggtttgtcaatgaacaacataaaacgacacagaatcccacgttttagcgcttcgtctgtgtcgcatgtaaaaggttatcaaagtagcgtgcacttttg ccatgattgacaaccaatcaacaaagtatgtgggctgaac (SEQ ID NO: 6)

[0118] The 5’ ITR may be truncated. For instance, in vitro work has shown that 12 bp is sufficient for replication (PMID: 3399382). Thus, in an example, the 5’ ITR may comprise at least the first 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 100, 150, 200, or 250 bases of SEQ ID NO: 6.

[0119] In an example, a truncated 5’ ITR compatible with phi29 DNA replication machinery is as below, aaagtaagcccc (SEQ ID NO: 9)

[0120] The 5’ ITR may be or may comprise SEQ ID NO: 9. The 5’ ITR may comprise from 12 to 264 base pairs of SEQ ID NO: 6 and comprise SEQ ID NO: 9. The 5’ ITR may be a truncation of SEQ ID NO: 6 that is from 12 to 260, 12 to 200, 12 to 100, 12 to 50, or 12 to 30 base pairs in length and is truncated from the 3’ end of SEQ ID NO: 6 i.e. includes SEQ ID NO: 9). In an example, a 3 ’ ITR compatible with phi29 DNA replication machinery is as below. cccttttcgacaaattgatgataataaatagtataggtatatagtcgtgatttagttgttagattcttgtcgaagatagtcggtcaatggggaaatggtgtatgttgtcgctgta ccctacttt (SEQ ID NO: 7)

[0121] The 3’ ITR may be truncated. For instance, in vitro work has shown that 12 bp is sufficient for replication

[0122] (PMID: 3399382). In an example, the 3’ ITR may comprise at least the last 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 100, 120 bases of SEQ ID NO: 7.

[0123] In an example, a truncated 3 ’ ITR compatible with phi29 DNA replication machinery is as below. gtaccctacttt (SEQ ID NO: 10)

[0124] The 3’ ITR may be or may comprise SEQ ID NO: 10. The 3’ ITR may comprise from 12 to 124 base pairs of SEQ ID NO: 7 and comprise SEQ ID NO: 10. The 3’ ITR may be a truncation of SEQ ID NO: 7 that is from 12 to 120, 12 to 100, 12 to 50, or 12 to 30 base pairs in length and is truncated from the 5’ end of SEQ ID NO: 7 (i.e. includes SEQ ID NO: 10).

[0125] Thus, ITRs according to SEQ ID NOs: 9 and 10 may be used in conjunction with non-phi29 ODNAPs that are derived from members of the Salasmaviridae family.

[0126] In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 or 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 or 10. In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. The linear plasmid may comprise a truncation of SEQ ID NO: 6 and / or SEQ ID NO: 7 that is capable of being replicated by the orthogonal DNA replication machinery. In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a truncation of SEQ ID NO: 6 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a truncation of SEQ ID NO: 7, and wherein the linear plasmid is capable of being replicated by the orthogonal DNA replication machinery.

[0127] A purely illustrative example of linear plasmid is provided below. This linear plasmid comprises the 5’ and 3’ ITRs of SEQ ID NOs: 6 and 7. Upper case letters illustrate coding sequences and the order of the components is: oriL-tetracycline_resistance-sfGFP-oriR. As the skilled person would appreciate, these features are exemplary and not limiting. aaagtaagcccccaccctcacatgataccattctcctaatatcgacataatccgtcgatcctcggcataccatgatcagggagggaaactactacttaatatatcaatctat agacctactagataggtttgtcaatgaacaacataaaacgacacagaatcccacgttttagcgcttcgtctgtgtcgcatgtaaaaggttatcaaagtagcgtgcacttttg ccatgattgacaaccaatcaacaaagtatgtgggctgaactcatcatttcatttgtaagtttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCC TGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTC CGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCA CCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGC CACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTG GCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAA GATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGG CCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCT CAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTG AGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGA CTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGA CCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTC GCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCA TGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCAT TATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTA GATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTG GACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGT AGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCG ACCTGAtcagaattggttaattggttgctgcagtgggttgatgataccgctgccttactgggtgcattagccagtctgaatgacctgtcacgggataatccgaagtc aaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcTT AAT GGT GAT GAT GAT GG TGGCTGCCTTTATACAGTTCATCCATACCGTGGGTAATGCCCGCGGCGGTAACAAATTCCAGCAGCA CCATATGATCACGTTTTTCATTCGGATCTTTGCTCAGAACGCTCTGGGTGCTCAGATAATGATTATCC GGCAGCAGCACCGGGCCATCACCAATCGGGGTATTCTGCTGataATGATCCGCCAGCTGCACGCTACC ATCTTCCACGTTGTGACGGATTTTAAAGTTCGCTTTGATGCCATTTTTCTGTTTATCGGCGGTAATAT ACACGTTATGGCTGTTGAAATTATATTCCAGTTTATGACCCAGAATGTTGCCatcTTCTTTAAAATCAA TACCTTTCAGTTCAATGCGGTTCACCAGGGTATCGCCTTCAAATTTAACTTCCGCACGGGTTTTATAG GTGCCATCATCTTTGAAGCTAATGGTACGTTCCTGCACATAGCCTTCCGGCATCGCGCTTTTAAAGAA ATCATGGCGTTTCATATGATCCGGATAGCGGCTAAAGCACTGAACGCCATAggtCAGGGTGGTCACCA GGGTCGGCCACGGAACCGGCAGTTTACCGGTGGTGCAAATAAATTTCAGGGTCAGTTTACCgttGGTC GCATCACCTTCGCCTTCGCCACGAACGCTAAATTTATGGCCATTCACATCACCATCCAGTTCCACCAG AATCGGCACAACGCCGGTAAACAGTTCTTCACCTTTgctAACCATGGTTAATTCCTCCTGTTAGCCCAA

[0128] AAAACGGGTATGGAGAAAGGTCTGATCACATTATACGAGCCGATGATTAATTGTCAAgcgcaacgcaattaa tgcgtgcaatagttatggtgacccttttcgacaaattgatgataataaatagtataggtatatagtcgtgatttagttgttagattcttgtcgaagatagtcggtcaatgggga aatggtgtatgttgtcgctgtaccctacttt (SEQ ID NO: 8)

[0129] The cell of the first aspect, which may be a bacterial cell such as an E. coli cell, may comprise at least 2.5 copies, at least 10 copies, at least 50 copies, at least 100 copies, at least 250 copies, at least 500 copies, at least 1000 copies, or at least 1100 copies of the orthogonal episome per cell.

[0130] In a particular embodiment, the cell of first aspect is a bacterial cell that is optionally an E. coli cell, that comprises: a gene encoding a Salasmaviridae TP and a gene encoding an error-prone compatible ODNAP; and a linear plasmid comprising: a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with the error-prone compatible ODNAP.

[0131] In a particular embodiment, the cell of first aspect is a bacterial cell that is optionally an E. coli cell, that comprises: a gene encoding a phi29-like phage TP and a gene encoding an error-prone compatible ODNAP; and a linear plasmid comprising: a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with the error-prone compatible ODNAP.

[0132] In a particular embodiment, the cell of first aspect is a bacterial cell that is optionally an E. coli cell, that comprises: a gene encoding a phi29 bacteriophage TP and a gene encoding an error-prone phi29-compatible ODNAP; and a linear plasmid comprising: a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with the error-prone phi29-compatible ODNAP.

[0133] The orthogonal episome may be delivered to the cell of the first aspect in a circular nucleic acid molecule, such as a circular episome. Thus, the cell of the first aspect may comprise a circular nucleic acid molecule comprising sequences that are capable of being replicated by the orthogonal DNA replication machinery. The sequences that are capable of being replicated by the orthogonal DNA replication machinery may be excised from the circular nucleic acid molecule, for instance the excision may take place within the cell. This may be referred to as in vivo excision or in vivo cutting. And so, the cell of the first aspect may comprise a circular nucleic acid molecule, wherein the circular nucleic acid molecule comprises the sequence of a linear plasmid as disclosed herein. The circular nucleic acid molecule may be a circular episome, and the episome may be any suitable for use with the cell of the first aspect. For instance, the circular episome may be an episome that is compatible with E. coli. The circular episome may be a bacterial artificial chromosome (BAC). The circular episome may be a circular plasmid. In an embodiment, the cell of the first aspect comprises a circular plasmid or a BAC comprising sequence that, once excised, is any of the linear plasmids disclosed herein.

[0134] The circular nucleic acid molecule may comprise at least one site that can be cleaved to excise the orthogonal sequence. For instance, the linear plasmid may be circularised at a single point that can be cleaved to linearise the orthogonal episome. The circular nucleic acid molecule may comprise two sites that can be cleaved to excise the orthogonal sequence. For instance, the circular nucleic acid molecule, such as a circular episome, may contain the sequence of a linear plasmid that is flanked by sites that can be cleaved to excise the linear plasmid.

[0135] In embodiments with one site for cleavage, the site may be positioned between a 5’ ITR and a 3’ ITR as defined herein. Thus, cleavage of the site can result in a linear plasmid comprising, in order, a 5’ ITR, a sequence-of- interest, and a 3 ’ ITR.

[0136] In embodiments with two sites for cleavage, the sites may be positioned adjacent to a 5’ ITR and a 3’ ITR as defined herein. Thus, cleavage of the site can result in a linear plasmid comprising, in order, a 5’ ITR, a sequence-of-interest, and a 3 ’ ITR.

[0137] The cleavage may be inducible. Thus, the cell may comprise the circular nucleic acid molecule and cleavage may be induced under specific conditions.

[0138] In some embodiments, the sites that can be cleaved are target sites for an endonuclease or endonucleases. For instance, the sites may be targeted by RNA-guided endonucleases, such as CRISPR-Cas. However, the sites for cleavage need not be particularly limited, as long as they can be specifically cut.

[0139] The cell may comprise sequence encoding an endonuclease or endonucleases suitable for excising the orthogonal sequences from the circular nucleic acid molecule. The cell may comprise sequence encoding molecules suitable for guiding an endonuclease to a cleavage site, such as guide RNA. The endonuclease and / or guide molecules may be inducibly expressed, e.g. to allow for excision of the orthogonal sequences under particular conditions or at a desired time. The endonuclease and / or guide molecules may be encoded by the genome of the cell or by other episomes within the cell, such as a plasmid. Excision may be induced by the transfer of an episome encoding one or more components of the cleavage machinery to the cell.

[0140] Embodiments comprising circular nucleic acid molecules can be advantageous due to the easier manipulation of circular nucleic acid molecules compared to linear plasmids. For instance, a circular episome can be used to deliver a large linear plasmid, which may be hundreds of kilobases in size.

[0141] As discussed herein, the inventors provide herein an orthogonal DNA replication system suitable for use in cells such as bacterial cells (e.g. E. coif). In a second aspect, there is provided a linear plasmid comprising a 5’ ITR and a 3 ’ ITR, each of which is compatible with DNA replication machinery from or derived from a phage capable of infecting gram-positive bacteria and / or a phage of the Salasmaviridae family.

[0142] The linear plasmid may be as disclosed in connection with the first aspect. For instance, see illustrative sequences SEQ ID NOs: 6, 7, 8, 9, and 10.

[0143] The linear plasmid may be an artificial plasmid and so not naturally occurring. The linear plasmid may be isolated from the natural conditions. The linear plasmid may be an unnatural or engineered linear plasmid. The linear plasmid may comprise a sequence-of-interest. For instance, the sequence-of-interest may be a sequence to which it is desirable to apply continuous directed evolution techniques. The sequence-of-interest may be as discussed in relation to the first aspect. The sequence-of-interest may be a sequence that is not endogenous to the phage from which the ITRs are derived or are compatible. The sequence-of-interest may be a sequence that is not endogenous to a phi29 bacteriophage

[0144] The linear plasmid may comprise a marker. For instance, a selectable marker. The marker may be a positive marker such that cells comprising the linear plasmid may be identified or selected, or so that selection pressure can be applied to a population of cells to increase the retention of the linear plasmid.

[0145] The DNA replication machinery may be DNA replication machinery from any of the phages discussed herein. For instance, a phage of the Salasmaviridae family. The DNA replication machinery may be as discussed in relation to the first aspect of the invention. The linear plasmid may not encode one, two, three, or all four of a TP, a DNA polymerase, an SSB, and a DSB from said DNA replication machinery. In particular embodiments, the linear plasmid does not comprise sequences of the bacteriophage genome other than the ITRs.

[0146] Compatibility with DNA replication machinery may be compatibility with a Salasmaviridae TP, Salasmaviridae ODNAP, optionally Salasmaviridae SSB, and optionally Salasmaviridae DSB. Compatibility with DNA replication machinery may be compatibility with a phi29-like phage TP, phi29-like phage ODNAP, optionally phi29-like phage SSB, and optionally phi29-like phage DSB. Compatibility with DNA replication machinery may be compatibility with a phi29 TP, phi29 ODNAP, optionally phi29 SSB, and optionally phi29 DSB.

[0147] In a preferred embodiment, the 5’ ITR and 3’ ITR are compatible with phi29 DNA replication machinery. In particular, the ITRs may be compatible with replication machinery comprising phi29 proteins: TP, ODNAP, SSB, and optionally DSB, or variants thereof. The linear plasmid may not encode one, two, three, or all four of a TP, a DNA polymerase, an SSB, and a DSB. In particular embodiments, the linear plasmid does not comprise sequences of the bacteriophage genome other than the ITRs.

[0148] In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 or 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 or 10. The linear plasmid may comprise a truncation of SEQ ID NO: 6 and / or SEQ ID NO: 7 that is capable of being replicated by the orthogonal DNA replication machinery. In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a truncation of SEQ ID NO: 6 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a truncation of SEQ ID NO: 7, and wherein the linear plasmid is capable of being replicated by the orthogonal DNA replication machinery. The ITRs discussed in relation to cells of the first aspect may be applied to the linear plasmid of the second aspect.

[0149] In a particular embodiment, there is provided a linear plasmid comprising: a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with phi29 DNA replication machinery.

[0150] In a third aspect, there is provided a circular nucleic acid molecule comprising the sequence of a linear plasmid of the second aspect.

[0151] The circular nucleic acid molecule may be a circular episome. The circular episome may be any episome suitable for use with the cell of the first aspect. For instance, the circular episome may be an episome that is compatible with E. coli. The circular episome may be a bacterial artificial chromosome (BAC). The circular episome may be a circular plasmid. The sequence of the linear plasmid may be excised from the circular nucleic acid molecule and the excision may take place within a cell. Thus, the circular nucleic acid molecule may be configured so that the linear plasmid can be excised in vivo.

[0152] The circular nucleic acid molecule may comprise at least one site that can be cleaved to excise the linear plasmid. For instance, the linear plasmid may be circularised at a single point that can be cleaved to linearise the linear plasmid. The circular nucleic acid molecule may comprise two sites that can be cleaved to excise the orthogonal sequence. For instance, the circular nucleic acid molecule may contain the sequence of a linear plasmid that is flanked by sites that can be cleaved to excise the linear plasmid. In embodiments with one site for cleavage, the site may be positioned between a 5’ ITR and a 3’ ITR as defined herein. In embodiments with two sites for cleavage, the sites may be positioned adjacent to a 5’ ITR and a 3’ ITR as defined herein. Thus, cleavage of the site can result in a linear plasmid comprising, in order, a 5’ ITR, a sequence-of-interest, and a 3’ ITR.

[0153] In a particular embodiment, there is provided a circular episome suitable for use with E. coli, wherein the circular episome comprises the sequence of a linear plasmid of the second aspect, and wherein the circular episome is configured such that the linear plasmid may be excised by an endonuclease or endonucleases.

[0154] In some embodiments, the sites that can be cleaved are target sites for an endonuclease or endonucleases. For instance, the sites may be targeted by RNA-guided endonucleases, such as CRISPR-Cas. However, the sites for cleavage need not be particularly limited, as long as they can be specifically cut.

[0155] Given that the terminal origins of the phi29-based replication system differ substantially from those of PRD1 and other Tectiviridae family phages, the phi29 system is expected to be functionally orthogonal to EcORep (as described in Tian et al., Science, 25 Jan 2024, Vol 383, Issue 6681, pp. 421-426). This enables the creation of two independent orthogonal replicons for directed evolution in the same cellular context.

[0156] Thus, the inventors provide herein cells that comprise two separate orthogonal DNA replication systems. In fourth aspect, provided herein is a cell comprising first orthogonal DNA replication machinery and second orthogonal DNA replication machinery, wherein the first orthogonal DNA replication machinery is orthogonal to the endogenous DNA replication machinery of the cell and to the second orthogonal DNA replication machinery, and wherein the second orthogonal DNA replication machinery is orthogonal to the endogenous DNA replication machinery of the cell and to the first orthogonal DNA replication machinery.

[0157] As discussed in relation to the first aspect, orthogonality means that the first orthogonal DNA replication machinery is not capable or less capable of replicating endogenous episomes and not capable or less capable of replicating episomes compatible with the second orthogonal DNA replication machinery. It also means that the second orthogonal DNA replication machinery is not capable or less capable of replicating endogenous episomes and not capable or less capable of replicating episomes compatible with the first orthogonal DNA replication machinery. Further details of the meaning of orthogonality are provided in relation to the first aspect and are applicable to the fourth aspect.

[0158] The first orthogonal DNA replication machinery may be as described in relation to the first aspect. Thus, the cell of the fourth aspect may comprise any of the features described for the first aspect. For example, the first orthogonal replication machinery may be an orthogonal replication system based on genes from a Salasmaviridae family phage. In particular, the first orthogonal replication machinery may be an orthogonal replication system based on genes from phi29. For instance, the first orthogonal replication machinery may comprise a phi29 TP, an ODNAP compatible with phi29, optionally a phi29 SSB, and optionally a phi29 DSB. The cell of the fourth aspect may comprise a linear plasmid according to the second aspect or a circular nucleic acid molecule according to the third aspect. For instance, the cell may comprise a linear plasmid comprising Salasmaviridae -compatible ITRs or a circular nucleic acid molecule that may be cleaved to release a linear plasmid comprising Salasmaviridae-compatible ITRs. In an example, the cell comprises a linear plasmid comprising phi29-compatible ITRs or a circular nucleic acid molecule that may be cleaved to release a linear plasmid comprising phi29-compatible ITRs.

[0159] The second orthogonal DNA replication machinery may be based on genes from a lytic phage of the Tectiviridae family. The second orthogonal replication machinery may comprise a Tectiviridae TP, a compatible ODNAP, optionally a Tectiviridae SSB, and optionally a Tectiviridae DSB. The second orthogonal DNA replication machinery may be based on genes from any of phage PRD1, phage PR3, phage PR4, phage PR5, phage L17, phage PR772, and phage BCE1. In particular, the second orthogonal replication machinery may be an orthogonal replication system based on genes from PRD1. For instance, the second orthogonal replication machinery may comprise a PRD1 TP, an ODNAP compatible with PRD1, optionally a PRD1 SSB, and optionally a PRD1 DSB.

[0160] The cell of the fourth aspect may comprise a linear plasmid compatible with the second orthogonal DNA replication machinery. The cell may comprise a linear plasmid comprising ITRs compatible with the relevant Tectiviridae phage or a circular nucleic acid molecule that may be cleaved to release a linear plasmid comprising ITRs compatible with the relevant Tectiviridae phage. The ITRs may be compatible with any of phage PRD1, phage PR3, phage PR4, phage PR5, phage LI 7, phage PR772, and phage BCE1. In a particular embodiment, the cell may comprise a linear plasmid comprising PRD 1 -compatible ITRs or a circular nucleic acid molecule that may be cleaved to release a linear plasmid comprising PRD 1 -compatible ITRs.

[0161] The PRD 1 -based orthogonal DNA replication system may be as disclosed in Tian et al. (Science, 25 Jan 2024, Vol 383, Issue 6681, pp. 421-426 - herein incorporated by reference in its entirety). The PRDl-based orthogonal DNA replication system may be EcORep.

[0162] The second orthogonal DNA replication machinery may be any as disclosed or defined in PCT / EP2024 / 064301, filed on 23 May 2024, herein incorporated by reference in its entirety. In particular the second orthogonal DNA replication machinery may be a PRDl-based orthogonal DNA replication system as disclosed or defined in this document. In particular, claims 10-18 of PCT / EP2024 / 064301are explicitly incorporated by reference as an illustrative disclosure of PRDl-based orthogonal DNA replication machinery. Claim 33 of PCT / EP2024 / 064301 is explicitly incorporated by reference as an illustrative disclosure of a PRD 1 -compatible linear plasmid.

[0163] In an embodiment, there is provided a cell comprising a first orthogonal DNA replication system based on a Salasmaviridae bacteriophage and a second orthogonal DNA replication system based on a Tectiviridae bacteriophage.

[0164] In an embodiment, there is provided a cell comprising a first orthogonal DNA replication system based on bacteriophage phi29 and a second orthogonal DNA replication system based on bacteriophage PRD1.

[0165] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a Salasmaviridae TP and a Salasmaviridae ODNAP, wherein the Salasmaviridae ODNAP is mutated to be error- prone; and a second orthogonal DNA replication system comprising a Tectiviridae TP and a Tectiviridae ODNAP, wherein the Tectiviridae ODNAP is mutated to be error-prone.

[0166] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a Salasmaviridae TP, a Salasmaviridae ODNAP, a Salasmaviridae SSB, and optionally a Salasmaviridae DSB, wherein the Salasmaviridae ODNAP is mutated to be error-prone; and a second orthogonal DNA replication system comprising a Tectiviridae TP, a Tectiviridae ODNAP, a Tectiviridae SSB, and optionally a Tectiviridae DSB, wherein the Tectiviridae ODNAP is mutated to be error-prone.

[0167] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a phi29 TP and a phi29 ODNAP, wherein the phi29 ODNAP is mutated to be error-prone; and a second orthogonal DNA replication system comprising a Tectiviridae TP and a Tectiviridae ODNAP, wherein the Tectiviridae ODNAP is mutated to be error-prone.

[0168] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a phi29 TP and a phi29 ODNAP, wherein the phi29 ODNAP is mutated to be error-prone; and a second orthogonal DNA replication system comprising a PRD1 TP and a PRD1 ODNAP, wherein the PRD1 ODNAP is mutated to be error-prone.

[0169] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a phi29 TP, a phi29 ODNAP, a phi29 SSB, and optionally a phi29 DSB, wherein the phi29 ODNAP is mutated to be error-prone; and a second orthogonal DNA replication system comprising a Tectiviridae TP, a Tectiviridae ODNAP, a Tectiviridae SSB, and optionally a Tectiviridae DSB, wherein the Tectiviridae ODNAP is mutated to be error-prone.

[0170] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a phi29 TP, a phi29 ODNAP, a phi29 SSB, and optionally a phi29 DSB, wherein the phi29 ODNAP is mutated to be error-prone; and a second orthogonal DNA replication system comprising a PRD1 TP, a PRD1 ODNAP, a PRD1 SSB, and optionally a PRD1 DSB, wherein the PRD1 ODNAP is mutated to be error-prone.

[0171] In an embodiment, there is provided a cell comprising: a first orthogonal DNA replication system comprising a phi29 TP, a phi29 ODNAP, a phi29 SSB, and a phi29 DSB, wherein the phi29 ODNAP is mutated to be error- prone; and a second orthogonal DNA replication system comprising a PRD1 TP, a PRD1 ODNAP, a PRD1 SSB, and a PRD1 DSB, wherein the PRD1 ODNAP is mutated to be error-prone.

[0172] The cell of the first aspect, the linear plasmid of the second aspect, the circular nucleic acid molecule of the third aspect, or the cell of the fourth aspect may be used in methods of maintaining a linear plasmid in a cell. Thus, in a fifth aspect, there is provided a method of maintaining a linear plasmid, wherein the method comprises: i) providing a cell according to the first aspect or the fourth aspect that comprises an episome capable of being replicated by the orthogonal replication machinery, or transferring an episome capable of being replicated by the orthogonal replication machinery, or that comprises sequences that can be excised and are capable of being replicated by the orthogonal replication machinery, to a cell according to the first aspect or the fourth aspect; and ii) incubating the cell under conditions conducive to growth.

[0173] Step i) may comprise: transferring a linear plasmid according to the second aspect to a cell according to the first aspect, transferring a linear plasmid according to the second aspect to a cell according to the fourth aspect, transferring a circular nucleic acid molecule of the third aspect to a cell according to the first aspect, or transferring a circular nucleic acid molecule of the third aspect to a cell according to the fourth aspect.

[0174] In particular, the cell may any cell as discussed in relation to the first aspect and may comprise any features as discussed in relation to the first aspect. For instance, the cell may be a bacterial cell such as an E. coli cell. Alternatively, the cell may any cell as discussed in relation to the fourth aspect and may comprise any features as discussed in relation to the fourth aspect. The linear plasmid may be as discussed in relation to the second aspect and may comprise any features as discussed in relation to the second aspect. The circular nucleic acid molecule may be as discussed in relation to the third aspect and may comprise any features as discussed in relation to the third aspect.

[0175] The linear plasmid may lack 5’ phosphorylation. For instance, the linear plasmid may have been generated using non-phosphorylated primers. Thus, the method may comprise the generation of the linear plasmid using nonphosphorylated primers.

[0176] The linear plasmid may be transferred to a bacterial cell by electroporation. Alternatively, the linear plasmid may be transferred to a bacterial cell as a part of a circular nucleic acid and the linear plasmid may be excised by in vivo cutting. For instance, the cell may express an endonuclease, such as CRISPR-Cas, capable of specifically excising the linear plasmid from the circular nucleic acid. The circular nucleic acid molecule and the nature of the excision may be any as defined herein, for instance as discussed for the first aspect or the third aspect. Particular examples include episomes such as plasmids or BACs that are suitable for use with E. coli.

[0177] In an embodiment, the cell comprises, or is the recipient of, a circular nucleic acid molecule comprising the sequence of the linear plasmid. The linear plasmid can be excised from the circular nucleic acid molecule within the cell. For instance, an endonuclease or endonucleases within the cell may specifically cut the circular nucleic acid molecule to excise the linear plasmid. This excision may be inducible, e.g. the endonuclease or associated components - such as guide RNAs - may be inducibly expressed. Excision may be induced by the transfer of an episome encoding one or more components of the cleavage machinery to the cell.

[0178] Thus, in an embodiment, the method of the fifth aspect may comprise obtaining a cell comprising a circular nucleic acid molecule that comprises the sequence of the linear plasmid, and inducing excision of said linear plasmid. This step may comprise the induction of expression of cleavage machinery capable of excising the linear plasmid or the transfer of one or more components of said cleavage machinery. In such embodiments, the step of transferring a linear plasmid according to the second aspect to a cell according to the first aspect may be referred to as generating a linear plasmid according to the second aspect within a cell according to the first aspect or excising a linear plasmid according to the second aspect from a circular nucleic acid molecule within a cell according to the first aspect.

[0179] In some embodiments, the episome capable of being replicated by the orthogonal replication machinery, or that comprises sequences that can be excised and are capable of being replicated by the orthogonal replication machinery, is transferred to the cell in a format that does not comprise bound TPs. Cells capable of expressing a TP, ODNAP, SSB, and a DSB are particularly relevant to such embodiments.

[0180] The linear plasmid may be transferred to the cell as part of a complex. The transfer may be by electroporation. The linear plasmid for transfer may have been extracted from a cell by using techniques that do not disrupt any bound proteins. For instance, the linear plasmid for transfer may have been extracted from a cell using a technique that does not remove the bound TPs. To avoid the removal of the TPs, proteinase treatments may be avoided. Standard miniprep protocols for the purification of plasmids are suitable (e.g. QIAprep Spin Miniprep Kit, QIAGEN). Thus, the transferring of a linear plasmid according to the second aspect to a cell according to the first aspect or fourth aspect may be the transfer of a complex comprising a TP and a linear plasmid according to the second aspect. This is particularly relevant to embodiments that involve a cell comprising a TP and an ODNAP but not comprising an SSB or DSB.

[0181] In embodiments that involve a bacterial cell comprising genes encoding a Salasmaviridae TP and a Salasmaviridae-compatible ODNAP, a complex comprising Salasmaviridae TPs bound to a linear plasmid may be transferred to the bacterial cell. In embodiments that involve a bacterial cell comprising genes encoding a phi29 TP and a phi29-compatible ODNAP, a complex comprising phi29 TPs bound to a linear plasmid may be transferred to the bacterial cell.

[0182] In an embodiment, the method of the third aspect may comprise: transferring a complex to a cell according to the first aspect or the fourth aspect, wherein the complex comprises TPs bound to a linear plasmid according to the second aspect; and incubating the cell under conditions conducive to growth.

[0183] The incubation of the cell under conditions conducive to growth is exposure of the cell to conditions that maintain viability of the cell and allow for DNA replication. Such conditions are known in the art. The conditions may be selective conditions, e.g. for bacterial cells an antibiotic may be present. The conditions may select for cells that comprise the linear plasmid. For instance, the linear plasmid may comprise a marker gene or a resistance gene and the incubation of the cell may include conditions relevant to the marker gene or resistance gene.

[0184] Alternatively, the linear plasmid may comprise a sequence-of-interest for which is it anticipated that mutations to said sequence could increase the fitness of the cell during incubation. The incubation conditions may be such to enable only sub-optimal growth of the cell when comprising an unmutated sequence-of-interest. Further examples of growth and selection are provided in relation to the sixth aspect and are also applicable to the fifth aspect.

[0185] In a particular embodiment, there is provided a method of maintaining a linear plasmid, wherein the method comprises: i) transferring a linear plasmid to a bacterial cell or generating a linear plasmid within a bacterial cell, and ii) incubating the bacterial cell under conditions conducive to growth, wherein the linear plasmid comprises a 5’ ITR and a 3’ ITR, each of which is compatible with a phage capable of infecting gram-positive bacteria, and wherein the bacterial cell comprises orthogonal DNA replication machinery from said phage. In particular, the bacterial cell may express a phi29 TP, a phi29 ODNAP, a phi29 SSB, and optionally a phi29 DSB, or variants thereof.

[0186] In another embodiment, there is provided a method of maintaining a linear plasmid, wherein the method comprises: i) transferring a linear plasmid to a bacterial cell or generating a linear plasmid within a bacterial cell, and ii) incubating the bacterial cell under conditions conducive to growth, wherein the linear plasmid comprises a 5’ ITR and a 3’ ITR, each of which is compatible with phi29 DNA replication machinery, and wherein the bacterial cell comprises orthogonal DNA replication machinery from or derived from phi29 bacteriophage. In particular the bacterial cell may express a phi29 TP, a phi29 ODNAP, a phi29 SSB, and optionally a phi29 DSB, or variants thereof.

[0187] In an example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6 or 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 or 10. The linear plasmid may comprise a truncation of SEQ ID NO: 6 and / or SEQ ID NO: 7 that is capable of being replicated by the orthogonal DNA replication machinery. In a particular example, the linear plasmid comprises a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a truncation of SEQ ID NO: 6 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a truncation of SEQ ID NO: 7, and wherein the linear plasmid is capable of being replicated by the orthogonal DNA replication machinery. The ITRs discussed in relation to cells of the first aspect may be applied to the linear plasmid of the fifth aspect.

[0188] In another embodiment, there is provided a method of maintaining a linear plasmid, wherein the method comprises: i) transferring a first and / or second linear plasmid to a bacterial cell of the fourth aspect or generating a first and / or second linear plasmid within a bacterial cell of the fourth aspect, and ii) incubating the bacterial cell under conditions conducive to growth, wherein the first linear plasmid is compatible with the first orthogonal replication machinery and the second linear plasmid is compatible with the second orthogonal replication machinery. In examples, the first orthogonal replication machinery is a Salasmaviridae -based orthogonal DNA replication system and the second orthogonal replication machinery is a Tectiviridae-based orthogonal DNA replication system. In further examples, the first orthogonal replication machinery is a phi29-based orthogonal DNA replication system and the second orthogonal replication machinery is a PRD1 -based orthogonal DNA replication system.

[0189] The inventors provide herein a method of evolving a sequence-of-interest that makes use of the orthogonal DNA replication systems disclosed herein. Thus, in a sixth aspect, there is provided use of a cell according to the first aspect, a linear plasmid according to the second aspect, a circular nucleic acid molecule of the third aspect, or a cell of the fourth aspect for evolving a sequence-of-interest.

[0190] In an embodiment, there is provided a method of evolving a sequence-of-interest, wherein the method comprises: i) providing a cell according to the first aspect or the fourth aspect that comprises an episome capable of being replicated by the orthogonal replication machinery, or transferring an episome capable of being replicated by the orthogonal replication machinery, or that comprises sequences that can be excised and are capable of being replicated by the orthogonal replication machinery, to a cell according to the first aspect or the fourth aspect, wherein the episome comprises a sequence-of-interest and wherein the orthogonal DNA replication machinery replicates DNA with a lower fidelity than the cell’s endogenous DNA replication machinery; ii) incubating the cell under conditions conducive to growth; and iii) exposing the cell to a selection condition.

[0191] Step i) may comprise: transferring a linear plasmid according to the second aspect to a cell according to the first aspect, transferring a linear plasmid according to the second aspect to a cell according to the fourth aspect, transferring a circular nucleic acid molecule of the third aspect to a cell according to the first aspect, or transferring a circular nucleic acid molecule of the third aspect to a cell according to the fourth aspect.

[0192] The cell may be any cell as discussed in relation to the first aspect and may comprise any features as discussed in relation to the first aspect. For instance, the cell may be a bacterial cell such as an E. coli cell. Alternatively, the cell may any cell as discussed in relation to the fourth aspect and may comprise any features as discussed in relation to the fourth aspect. The linear plasmid may comprise any features as discussed in relation to the first or second aspects. The linear plasmid may be transferred to the cell or generated in the cell as discussed for the fifth aspect. For instance, the linear plasmid may be transferred by electroporation or may be excised from a circular nucleic acid molecule. A complex comprising the linear plasmid and TPs may be transferred to the cell.

[0193] As discussed in relation to the first aspect, replication of the linear plasmid may have a mutation rate of at least 10’9, IO'8, IO'7IO'6, or IO'5s.p.b.

[0194] The incubation of the cell under conditions conducive to growth is exposure of the cell to conditions that maintain viability of the cell and allow for DNA replication. Such conditions are known in the art and are discussed in relation to the fifth aspect.

[0195] Exposing the cell to a selection condition may be exposure of the cell to conditions that prevent or reduce the growth of cells in the absence of certain characteristics. The selection condition may be present during the incubation of the cell under conditions conducive to growth, and hence these steps may be concurrent. In particular, the selection conditions may be such to enable only sub-optimal growth of the cell when comprising an unmutated sequence-of-interest. The selection conditions may be such that the fitness of the cell is increased as the sequence-of-interest is mutated in a manner that leads to the acquisition of a particular function or that leads to decrease or increase in a particular function. As a purely illustrative example, the sequence-of-interest may be a potential antibiotic resistance gene, and hence the selection condition may be an amount of antibiotic that allows sub-optimal growth of bacterial cells comprising an unmutated antibiotic resistance gene but would allow improved growth of bacterial cells comprising a more effective antibiotic resistance gene. The selection condition may be applied after incubation of the cells under conditions conducive to growth for a period of time. The cells may be incubated and selected iteratively, for instance the cells may be incubated, selected, incubated, and then selected.

[0196] Another purely illustrative example of a selection condition and a sequence-of-interest is where the sequence-of- interest encodes an acyl-tRNA synthetase. The selection condition may be that the cell comprises a gene that confers reduced fitness or slower growth upon the cell in the presence of an unmutated acyl-tRNA synthetase. The gene may allow improved fitness or growth of the cell if the acyl-tRNA synthetase is mutated to acquire a new characteristic or to improve a characteristic, such as the ability to charge a particular tRNA with a particular substrate.

[0197] Alternatively, the selection condition may comprise sorting based on the expression level of a marker gene. The selection may comprise identifying cells for selection or sorting. For instance, the sequence-of-interest may encode a promoter operably linked to GFP. Cells with an above threshold level of GFP, or with a high level of GFP within the culture, may be sorted and retained. Potentially such cells may be re-entered into the method of evolving a sequence-of-interest. Such techniques are also compatible with other sequences-of-interest, for instance GFP gene may be arranged to be decoded only if an enzyme, such as an acyl-tRNA, is mutated to acquire a new characteristic or to improve a characteristic. The sequence-of-interest may, itself, act as a marker gene and may acquire or increase marker function during the evolving process.

[0198] The above examples are purely illustrative, and the invention is compatible with a wide range of sequences-of- interest, selection conditions, and desired functionality. These functionalities include enzyme activity, ribozyme activity, binding properties of proteins or aptamers, promoter or enhancer activity, capability of a nucleic acid or polypeptide to be bound by a known binding-molecule, etc.

[0199] The method of the sixth aspect may further comprise identifying the sequence of a sequence-of-interest from a cell meeting the selection condition. For instance, a cell that has an above-threshold level of fitness in the presence of the selection condition, a cell for which the selection condition is an above -threshold expression of a marker, or a cell for which the selection condition is an above -threshold detectable property or functionality. Such cells may be isolated and sequence information for the sequence-of-interest may be obtained.

[0200] The method may then comprise the making of a nucleic acid or polypeptide according to said identified sequence.

[0201] In an embodiment, there is provided a method of evolving a sequence-of-interest, wherein the method comprises: i) providing a bacterial cell comprising a linear plasmid, wherein the linear plasmid comprises a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ and 3’ ITR are compatible with DNA replication machinery from or derived from a phage capable of infecting gram-positive bacteria or from a Salasmaviridae family phage, and wherein the bacterial cell comprises orthogonal DNA replication machinery from or derived from the phage; ii) incubating the bacterial cell under conditions conducive to growth; and iii) exposing the bacterial cell to a selection condition.

[0202] The bacterial cell may express a TP and an ODNAP. The bacterial cell may express a TP, an ODNAP, and an SSB. The bacterial cell may express a TP, an ODNAP, an SSB, and a DSB. The bacterial cell may express a Salasmaviridae TP and a Salasmaviridae ODNAP. The bacterial cell may express a Salasmaviridae TP, a Salasmaviridae ODNAP, and a Salasmaviridae SSB. The bacterial cell may express a Salasmaviridae TP, a Salasmaviridae ODNAP, a Salasmaviridae SSB, and a Salasmaviridae DSB. In particular, the bacterial cell may express a phi29 TP, a phi29 ODNAP, a phi29 SSB, and a phi29 DSB, or variants thereof. In an embodiment, there is provided a method of evolving a sequence-of-interest, wherein the method comprises: i) providing a bacterial cell comprising a linear plasmid, wherein the linear plasmid comprises a 5’ ITR, a sequence-of-interest, and a 3’ ITR, wherein the 5’ and 3’ ITR are compatible with phi29 bacteriophage DNA replication machinery, and wherein the bacterial cell comprises orthogonal DNA replication machinery from or derived from phi29 bacteriophage; ii) incubating the bacterial cell under conditions conducive to growth; and iii) exposing the bacterial cell to a selection condition.

[0203] The bacterial cell may express a phi29 bacteriophage TP and a phi29 bacteriophage ODNAP, or variants thereof. The bacterial cell may express a phi29 bacteriophage TP, a phi29 bacteriophage ODNAP, and a phi29 bacteriophage SSB, or variants thereof. In particular, the bacterial cell may express a phi29 bacteriophage TP, a phi29 bacteriophage ODNAP, a phi29 bacteriophage SSB, and a phi29 bacteriophage DSB, or variants thereof.

[0204] In an embodiment, there is provided a method of evolving a sequence-of-interest, wherein the method comprises: i) transferring a first and / or second linear plasmid to a bacterial cell of the fourth aspect or generating a first and / or second linear plasmid within a bacterial cell of the fourth aspect, wherein the first linear plasmid is compatible with the first orthogonal replication machinery and the second linear plasmid is compatible with the second orthogonal replication machinery; ii) incubating the bacterial cell under conditions conducive to growth; and iii) exposing the bacterial cell to a selection condition.

[0205] In a seventh aspect, there is provided a method of making a polypeptide or nucleic acid, wherein the method comprises: i) providing a sequence-of-interest identified by a method of the sixth aspect, and ii) producing a polypeptide or nucleic acid according to said sequence.

[0206] 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.

[0207] The skilled technician will appreciate how to calculate the percentage identity between two nucleic sequences or two amino acid sequences. In order to calculate the percentage identity, 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.

[0208] Elaving 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.

[0209] 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).

[0210] 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.

[0211] 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.

[0212] EXAMPLES

[0213] Example 1 - Summary

[0214] We establish an orthogonal replication system in E. coli based on the phage phi29, of the family Salasmaviridae , which strictly infects Gram-positive bacteria (Bacillus sp.). The phi29 replication system utilises four genes from its genome which have been rearranged into a synthetic replication operon on a single-copy plasmid. These genes encode for the terminal protein, DNA polymerase, single-strand and double-strand DNA binding proteins. E. coli cells harbouring this plasmid were transformed with a linear PCR product consisting of a tetracycline resistance gene and sfGFP flanked by the left and right origins of the phi29 genome via electroporation. Green colonies emerged on tetracycline-supplemented plates and could be grown in tetracycline-supplemented medium. Plasmid extracts required treatment with proteinase K to visualise the linear replicon via agarose gel electrophoresis, validating that the replicon ends are bound by the terminal protein in vivo.

[0215] Our system requires only two genes to operate, making it the most minimal orthogonal replication system to date. We develop a highly efficient strategy to engineer replicons by using recombination components from the lambda phage. Engineering of the 029 DNA polymerase yielded a highly error-prone variant capable of introducing mutations at a frequency approaching 10'4per base per generation, thus introducing one mutation in a 1 kb gene every 10 generations. Our system is stable for hundreds of generations and enables the continuous evolution of new gene functions. To benchmark the system against other orthogonal replication systems, we demonstrate the rapid evolution of a tetracycline resistance gene to confer resistance to tigecy cline.

[0216] Example 2 - The establishment of an orthogonal replication system in E. coli based on the phage phi29

[0217] We arranged phi29 genes 3, 2, 6, and 5, encoding for the terminal protein, DNA polymerase, double-strand DNA binding protein, and single-strand DNA binding protein into a single operon driven by a tac promoter followed by a lac operator (Fig. 1). We assembled this operon onto a single-copy plasmid and maintained it in E. coli. We then generated a PCR product of a synthetic linear replicon carrying a tetracycline resistance gene and sfGFP and electroporated this into the operon-harbouring cells. We subsequently observed green colonies on the plate and could grow these in medium supplemented with tetracycline. To validate that the linear replicon is maintained in cells in the expected state where terminal proteins are attached at the 5’ ends, we extracted the linear plasmids from cells and visualised them via agarose gel electrophoresis before and after treatment with proteinase K. The linear plasmid could only be visualised after proteinase K treatment, indicating that terminal proteins are linked at the termini in vivo (Fig. 2). Thus, an in vivo replication system based on phi29 has successfully been established in E. coli.

[0218] More detail in relation to the design and establishment of a 029-based replication system in vivo is provided below.

[0219] Bacteriophage ©29 is a lytic, double-stranded DNA bacteriophage belonging to the Salasmaviridae family31. First discovered in 196532, it remains the smallest known phage that infects Bacillus. The ©29 genome (19,282 bp) encodes 27 protein-coding genes and a prohead RNA essential for genome packaging (Figure I)31. The encoded proteins are involved in both replicating and packaging the genome and, lysing the host cell31,33,34. The ©29 genome is linear and contains origin of replication sequences (oriL and oriR) at each end. The genome is capped at its 5’ ends by covalently bound terminal proteins (TPs); these proteins prime replication by the ©29 DNA polymerase (DNAP)35’36. The ©29 genome is an important model system for studying protein-primed DNA replication.

[0220] We aimed to generate a synthetic system for the controlled replication of a linear ©29 replicon in E. coli. Toward this goal, we designed a synthetic replication operon consisting of the genes encoding for the TP, DNAP, DSB and SSB (Figure 1); a combination of these proteins has been used to amplify portions of the ©29 genome in vitro31. We arranged these genes into an operon under the control of an isopropyl-P-D-thiogalactopyranoside (IPTG)- inducible promoter, PtacIPTG, on a single-copy plasmid (Figure 1). All genes were computationally codon- optimized for use in E. coli and their expression was driven by synthetic ribosome binding site sequences38-40.

[0221] We designed a ©29 synthetic replicon, to be replicated by the synthetic replication operon; the replicon consisted of genes encoding for tetracycline resistance and GFP, flanked by the left and right ©29 origins of replication (oriL and oriR) (Figure 1). We attempted to establish the ©29 synthetic replicon in E. coli by electroporating 5 pg of the ©29 synthetic replicon, generated by polymerase -chain reaction (PCR) amplification, into E. coli cells harboring the synthetic replication operon plasmid (Figure 3a). However, this yielded no tetracycline -resistant colonies (Figure 3b). This indicated that our initial attempt to establish the replicon system was unsuccessful.

[0222] With hypothesized that the establishment of the replicon may be limited by degradation of the electroporated PCR product in E. coli. To maximize the chance of establishing the synthetic replicon, we generated helper plasmids that express the Gam protein (derived from the lambda phage) to inhibit host nucleases (RecBCD and SbcCD), and the ©29 DSB and SSB to further protect the electroporated replicon DNA.

[0223] Upon electroporation of the ©29 synthetic replicon into cells bearing the helper plasmids and the synthetic replication operon, we observed a small number of tetracycline -resistant colonies that exhibited green fluorescence. These experiments suggested that the ©29 synthetic replicon had been established in these cells (Figure 3b); this conclusion was supported by additional genotyping experiments (Figure 10), and by experiments where we could reduce the replicon copy number by downregulating expression from the replication operon by targeting the promoter driving the operon with dCas9 (Figure 11). Extracting the established replicons, from cells bearing the helper plasmids and the synthetic replication operon, and electroporating them into fresh cells enabled substantially improved transformation efficiencies, even when the helper plasmids providing Gam and ©29 DSB and SSB were not present in the recipient cells (Figure 3b).

[0224] Example 3 - Assessing an orthogonal replication system in E. coli based on the phage phi29

[0225] We assessed the efficiency of establishing a phi29 replicon in cells harbouring the replication operon via the electroporation (Fig. 3 a) of either a high concentration of PCR product or a phi29 replicon extracted from cells where it had previously been established (standard miniprep). We additionally over-expressed Gam from the lambda phage and the phi29 SSB and DSB. This revealed that Gam and the SSB / DSB marginally increased the transformation efficiency (Fig. 3b) and that transformation of extracted replicons was substantially more efficient than from PCR products.

[0226] Next, we assessed the stability of the phi29 replicon (harbouring a tetracycline resistance marker and an sfGFP gene) over 550 generations using flow cytometry to measure the proportion of GFP+ cells. This revealed the replicon to be very stably maintained in its entirety while under tetracycline -based selection but rapidly lost without selection (Fig. 4).

[0227] To elaborate: the stability of the ©29 synthetic replicon through was assessed through many rounds of cell division, and we measured the proportion of cells that maintained the replicon, as indicated by GFP-fluorescence, over 550 generations (Figure 4). Without tetracycline, the replicon was lost in less than 50 generations. In the presence of tetracycline, the replicon was stably maintained for the entire 550 generations tested. Therefore, the ©29 synthetic replicon can be stably maintained for many generations, as required for continuous directed evolution experiments.

[0228] Example 4 - Defining the genes required for maintenance of the <D29 synthetic replicon

[0229] To assess which of the four genes was required for maintenance of the replicon, we generated operons where the TP, DNAP, SSB or both the SSB and DSB were knocked out. We then electroporated a replicon extracted from cells where it was initially established. Extracted replicons would already carry terminal proteins at their 5’ ends. This revealed that only the TP and DNAP genes are essential for maintaining the replicon (Fig 5). Our results are in agreement with in vitro efforts to replicate the ©29 genome using only the TP and DNAP, and self-replication of TP- and DNAP-encoding replicons in synthetic protocells41'43.

[0230] Example 5 - Efficient engineering of the <D29 synthetic replicon

[0231] To efficiently engineer established phi29 replicons in vivo, we sought to utilize lambda red recombination. To assess the efficiency of this process, we performed a lambda red recombination that exchanged the tetracycline resistance marker (TcR) with a kanamycin resistance marker (KanR) (Fig. 6). This was done by transforming a plasmid expressing the lambda red operon into cells harbouring the phi29 replicon and phi29 replication operon. The expression of the lambda red operon was under the control of an arabinose-inducible promoter. We then electroporated a PCR product of KanR flanked by 60 bp homology arms into arabinose-induced electrocompetent cells and determined the proportion of kanamycin-resistant transformants by counting colonies. To elaborate, we electroporated 0.5 pg of a PCR product consisting of a kanamycin resistance gene flanked by 60 bp homologies into cells harboring a ©29 synthetic replication operon plasmid, a tetracycline resistance conferring ©29 synthetic replicon, and a plasmid encoding for the lambda Red recombination system components under arabinose-inducible control (Figure 6 - upper panel). These experiments gave rise to kanamycin resistant colonies, where the tetracycline resistance gene had been replaced, at a frequency approaching 10'4. And so the recombination efficiency approaching 10'4(Fig. 6). All modifications of the ©29 synthetic replicon performed in this work were conducted using this approach.

[0232] Example 6 - Optimization of the C>29 synthetic replication operon

[0233] Next, we sought to improve the performance of the phi29 synthetic replication operon. Because the phi29 phage natively infects only Bacillus species, we had computationally re-designed the operon. Thus, its performance had not been evolutionarily optimised. To address this, we generated a library of phi29 synthetic replication operons where we randomised the spacer region between the RBS and the start codon, as well as the first codon following the start codon (N-end), and then synonymised the next six codons (Fig. 7a). We passaged cells containing these operon variants and the phi29 TcR-GFP replicon to enrich for better growing variants. We then plated out on agar plates and picked brightly fluorescing single colonies for further validation. This ultimately identified an operon variant which we term phi29-opt that enables a higher replicon copy number (Fig. 7b) and substantially improved bacterial growth (Fig. 7d). To elaborate on the above: we generated a ©29 synthetic replication operon library in which we varied three regions of the designed operon: we randomized the spacer sequence between the Shine-Dalgamo sequence and the start codon of each gene (this sequence can modulate the efficiency of translation initiation, which is rate determining for translation in E. coli), we randomized the first codon following the start codon (to allow for stability tuning via the N-end rule which differs between the Bacillus species that ©29 phage infect and E. coli), and we randomized codons 2-7 to synonyms (as the sequence in this region has been shown to influence translational efficiency) (Figure 7a)46-49. We passaged cells transformed with the ©29 synthetic replication operon library and a ©29 synthetic replicon to enrich operons with favorable properties. To select for operons that supported increased replicon copy numbers, we picked colonies exhibiting stronger GFP fluorescence. Ultimately, we identified an operon, ©29-opt, with improved performance; this operon supported: a higher replicon copy number (as evidenced by a seven-fold increase in culture fluorescence) (Figure 7b), improved transformation of extracted replicons (>106transformants per pg of transformed replicon, Figure 7c), and faster growth (Figure 7d, Figure 12).

[0234] Example 7 - Highly mutagenic <&29-based orthogonal replication system

[0235] We generated ©29 synthetic replication operons, based on ©29-opt, with variants (N62D and F65S, alone or in combination) of the ©29 DNAP; in in vitro experiments these individual DNAP mutants are reported to mutate the DNA that they replicate50’51. We used these operon variants to demonstrate the creation of a highly mutagenic orthogonal replication system in E. coli (Figure 8).

[0236] To measure the extent to which these ©29 DNAP mutants mutagenize the ©29 synthetic replicon, we generated a replicon harboring a chloramphenicol resistance gene with an in-frame stop codon (Q38TAG). After ten generations of growth, with each DNAP variant and a WT control, we measured the fraction of chloramphenicol-resistant cells arising from a point mutation in the TAG stop codon to generate sense codons. These experiments allowed us to determine the apparent mutation rates of the replicon in the presence of each DNAP, via fluctuation analysis52’53. This analysis revealed that the DNAP mutants were highly mutagenic on the replicon (Figure 8). The N62D / F65S double mutant outperformed the individual mutants, with a mutation rate approaching IO-4substitutions per base, per generation on the replicon (Figure 8). To determine the mutational spectra of these error-prone DNAP mutants, we grew cells harboring a ©29 synthetic replicon for 100 generations and measured the accumulation of mutations using next-generation sequencing. We primarily observed C or G to A or T mutations, though all types of mutations could be detected (Figure 13).

[0237] Next, we integrated a chloramphenicol resistance gene containing an in-frame stop codon (Q38TAG) into the genome of E. coli. We introduced the ©29 DNAP mutants into these cells and measured the fraction of chloramphenicol-resistant cells, arising from point mutations in the genomic TAG stop codon that generates a sense codon, after 10 generations. These experiments allowed us to determine the apparent mutation rates of the genome in the presence of each DNAP, via fluctuation analysis52-53. This analysis revealed that expression of the WT or mutant ©29 DNAPs did not lead to changes in the apparent genomic mutation rate with respect to control cells without a ©29 DNAP (Figure 8). By contrast, expression of an error-prone mutant of T7 DNAP led to increased mutagenesis of the genome54.

[0238] Overall, our data demonstrate that mutagenic ©29 DNAPs lead to mutation rates approaching 10'4substitutions per base, per generation on the replicon, but do not measurably affect the mutation rate of the genome. We conclude that the ©29-based replication system constitutes an orthogonal replication system in E. coli.

[0239] Example 8 - Continuous evolution of tigecycline resistance

[0240] Next, we sought to utilize the ©29 orthogonal replication system to continuously evolve new phenotypes. We initially investigated evolving the tetracycline resistance conferring tetA gene into a gene that confers resistance to tigecycline. This evolution has been carried out using previously reported continuous evolution approaches, enabling us to benchmark the performance of the ©29-based replication system9,15. We passaged cells containing a tetA-GFP replicon and the N62D / F65S error-prone DNAP in increasingly higher concentrations of tigecycline, until a final concentration of 40 pg ml / 1was reached (12 passages, Figure 14). The resultant pools of cells grew on plates containing tigecy cline concentrations as high as 50 pg mL-1whereas the pools of cells prior to continuous evolution grew on 0.2 pg mL-1but not on 0.5 pg mL-1(Figure 15). We sequenced replicons from cells growing on high tigecycline concentrations and found that certain mutations had convergently emerged in our three independent replicates. Of note are W233 which was mutated to C or S in every clone, A393 which was mutated to S or D in every clone, Pl 93 which was mutated to S, T or N in 79% of clones, and G388 which was mutated to V in some clones from all replicates but in all clones from the third replicate (Figure 9a). We cloned select sequences from each replicate into a standard circular plasmid (colEl) to further validate the performance of these mutants. This revealed that all mutants tested could confer resistance to tigecycline concentrations between 10 and 30 pg mL-1whereas the WT tetA control was unable to grow on 1 pg mL-1tigecycline. The tigecycline resistance conferred by our evolved mutants surpassed that of previously reported variants (Figure 9b)9,15. We also tested the susceptibility of these evolved variants to counter-selection as mediated by a combination of fusaric acid and ZnCL. revealing a range of sensitivities (Figure 16). The most sensitive variant had 104-fold higher sensitivity compared to the parent TetA and thus constitutes an improved dual positive-negative selection marker55. Thus, the ©29 orthogonal replication system enables the rapid and reproducible evolution of new gene function.

[0241] Example 9 - Discussion of preceding Examples

[0242] We have established an orthogonal replication system in E. coli via a synthetically designed operon encoding for components from bacteriophage ©29 which naturally infects only Gram-positive bacteria. This phage has served as a long-standing model system to study protein-primed replication, but our work constitutes the first report of sustained in vivo replication using ©29 components. We engineered the operon for improved performance and found that only two of the genes are needed for orthogonal replication, constituting the most minimal orthogonal replication system to date. Moreover, we utilize the lambda phage-derived recombination system to precisely and efficiently engineer the replicons and show that extracted replicons can be efficiently transformed into fresh cells harboring the optimized synthetic replication operon. We developed an error-prone DNAP with a mutation rate approaching IO-4, exceeding the highest mutation rates reported for EcORep DNAPs15. Given the faster generation time of E. coli compared to yeast, this mutation rate also exceeds the mutagenesis achievable using the newest generation of OrthoRep polymerases on a per day basis20. Thus, our ©29-based orthogonal replication system constitutes a best-in-class system for accelerated continuous evolution in E. coli.

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Claims

CLAIMS1. A cell comprising orthogonal DNA replication machinery, wherein the orthogonal DNA replication machinery is from or derived from a phage that infects gram-positive bacteria and / or a phage of the Salasmaviridae family.

2. The cell of claim 1, wherein the cell comprises one or more non-orthogonal episomes that are capable of being replicated by the endogenous DNA polymerase, and the one or more non-orthogonal episomes encode at least one, a plurality, or all components of the orthogonal DNA replication machinery; and / or at least one, a plurality, or all components of the orthogonal DNA replication machinery are encoded by the cell genome.

3. The cell of claim 1 or claim 2, wherein the orthogonal DNA replication machinery is capable of replicating an orthogonal episome that does not comprise at least one, a plurality, or all components of the orthogonal DNA replication machinery.

4. The cell of any preceding claim, wherein the orthogonal DNA replication machinery replicates DNA with a lower fidelity than the cell’s endogenous DNA replication machinery.

5. The cell of any preceding claim, wherein the orthogonal DNA replication machinery is from or derived from phi29 phage or a phi-29-like phage.

6. The cell of any preceding claim, wherein the orthogonal DNA replication machinery comprises a gene encoding a Salasmaviridae terminal protein (TP) and a gene encoding an orthogonal DNA polymerase (ODNAP).

7. The cell of claim 6, wherein the orthogonal DNA replication machinery comprises a gene encoding a Salasmaviridae single-stranded DNA-binding protein (SSB) and / or a gene encoding a Salasmaviridae doublestranded DNA-binding protein (DSB).

8. A viable cell comprising a gene encoding a Salasmaviridae TP and a gene encoding an ODNAP.

9. The cell of claim 8, wherein the cell comprises a gene encoding a Salasmaviridae SSB and / or a gene encoding a Salasmaviridae DSB.

10. The cell of claim 7 or claim 9, wherein the Salasmaviridae SSB is a phi29 SSB and / or the Salasmaviridae DSB is a phi29 DSB.

11. The cell of any one of claims 6 to 10, wherein the TP is a phi29 TP.

12. The cell of any one of claims 6 to 11, wherein the ODNAP is a Salasmaviridae or phi29 ODNAP or an engineered Salasmaviridae or phi29 ODNAP.

13. The cell of any preceding claim, wherein the cell comprises sequence encoding a protein that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% similar or identical to any one, two, three, or four of SEQ ID NOs: 1, 2, 3, and 4.

14. The cell of any one of claims 6 to 13, wherein the ODNAP replicates DNA with a lower fidelity than the cell’s endogenous DNA polymerase.3815. The cell of any one of claims 6 to 14, wherein the ODNAP comprises any one the following mutations or sets of mutations described with reference to SEQ ID NO: 2: T15I, N62D, and / or F65S.

16. The cell of any preceding claim, wherein the cell is a bacterial cell, a gram-positive bacterium, a gramnegative bacterium, a Bacillus sp. cell, B. subtilis cell, or an E. coll cell.

17. The cell of any preceding claim, wherein at least one, a plurality, or all components of the orthogonal DNA replication machinery are encoded by a single operon and / or wherein at least one, a plurality, or all of the TP, ODNAP, SSB, and DSB are encoded by a single operon.

18. The cell of any preceding claim, wherein the cell comprises a gene encoding Gam protein.

19. The cell of any preceding claim, comprising an episome that is capable of being replicated by the orthogonal DNA replication machinery and / or wherein the cell comprises an orthogonal episome that can be replicated by the TP and ODNAP.

20. The cell of claim 19, wherein the episome comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR and the 3’ ITR are compatible with the orthogonal DNA replication machinery and / or with the TP and ODNAP.

21. The cell of claim 20, wherein the 5’ ITR and 3’ ITR are compatible with phi29 DNA replication machinery.

22. The cell of any one of claims 19 to 21, wherein the episome comprises: a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, or a truncation thereof, and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 or a truncation thereof; or a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

23. The cell of any one of claims 19 to 22, wherein the episome is a linear plasmid.

24. The cell of any one of claims 19 to 23, wherein the episome comprises a sequence-of-interest.

25. The cell of any one of claims 1 to 24, wherein the cell comprises a circular nucleic acid molecule that comprises the sequence of an episome that is capable of being replicated by the orthogonal DNA replication machinery and / or the TP and ODNAP.

26. The cell of claim 25, wherein the episome that is capable of being replicated by the orthogonal DNA replication machinery is an episome according to any one of claims 19 to 24.

27. The cell of claim 25 or claim 26, wherein the circular nucleic acid molecule is configured so that the episome that is capable of being replicated by the orthogonal DNA replication machinery may be excised from the circular episome within the cell.

28. A linear plasmid comprising a 5’ ITR and a 3’ ITR, each of which is compatible with DNA replication machinery from or derived from a phage that infects gram-positive bacteria and / or a phage of the Salasmaviridae family.

29. The linear plasmid of claim 28, wherein the 5’ ITR and 3’ ITR are compatible with phi29 bacteriophage DNA replication machinery.

30. The linear plasmid of claim 28 or claim 29, wherein the linear plasmid comprises: a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, or a truncation thereof and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, or a truncation thereof; or a 5’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 and / or a 3’ ITR that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

31. The linear plasmid of any one of claims 28 to 30, wherein the plasmid does not encode one, two, three, or four of a TP, a DNA polymerase, an SSB, and a DSB.

32. The linear plasmid of any one of claims 28 to 31, wherein the plasmid comprises a sequence-of-interest.

33. The linear plasmid of claim 32, wherein the sequence-of-interest is: not a sequence endogenous to the phage associated with the DNA replication machinery; and / or not a sequence endogenous to phi29.

34. A circular nucleic acid molecule comprising the sequence of a linear plasmid according of any one of claims 28 to 33.

35. The circular nucleic acid molecule of claim 34, wherein the circular nucleic acid molecule is a circular episome, a circular plasmid, or a bacterial artificial chromosome.

36. The circular nucleic acid molecule of claim 34 or claim 35, wherein the circular nucleic acid molecule comprises one or more sites positioned such that the linear plasmid may be excised from the circular nucleic acid molecule.

37. A cell comprising first orthogonal DNA replication machinery and second orthogonal DNA replication machinery, wherein the first orthogonal DNA replication machinery is orthogonal to the endogenous DNA replication machinery of the cell and to the second orthogonal DNA replication machinery, and wherein the second orthogonal DNA replication machinery is orthogonal to the endogenous DNA replication machinery of the cell and to the first orthogonal DNA replication machinery.

38. The cell of claim 37, wherein the cell is according to any one of claims 1 to 27.

39. The cell of claim 38, wherein the cell comprises a linear plasmid according to any of claims 28 to 33 or comprises a circular nucleic acid molecule according to any one of claims 34 to 36.

40. The cell of any one of claims 37 to 39, wherein the second orthogonal DNA replication machinery comprisesl a Tectiviridae TP and a Tectiviridae ODNAP; or a Tectiviridae TP, a Tectiviridae ODNAP, and a Tectiviridae SSB; or a Tectiviridae TP, a Tectiviridae ODNAP, and a Tectiviridae DSB; or a Tectiviridae TP, a Tectiviridae ODNAP, a Tectiviridae SSB, and a Tectiviridae DSB.

41. The cell of any one of claims 37 to 40, wherein the second orthogonal DNA replication machinery comprisesla PRD1 TP and a PRD1 ODNAP; or a PRD1 TP, a PRD1 ODNAP, and a PRD1 SSB; or a PRD1 TP, a PRD1 ODNAP, and a PRD1 DSB; or a PRD1 TP, a PRD1 ODNAP, a PRD1 SSB, and a PRD1 DSB.

42. A method of maintaining a linear plasmid, wherein the method comprises: i) providing a cell according to any one of claims 19 to 27, transferring a linear plasmid according to any one of claims 28 to 33 to a cell according to any one of claims 1 to 18, excising a linear plasmid from a circular nucleic acid molecule according to any one of claims 34 to 36 within a cell according to any one of claims 1 to 18, transferring a first and / or a second linear plasmid to a cell according to any one of claims 37 to 41, or generating a first and / or a second linear plasmid within a cell according to any one of claims 37 to 41 ; and ii) incubating the cell under conditions conducive to growth.

43. Use of a cell according to any one of claims 1 to 27 or 37 to 41, a linear plasmid according to any one of claims 28 to 33, or a circular nucleic acid molecule according to any one of claims 34 to 36 for evolving a sequence-of-interest.

44. A method of evolving a sequence-of-interest, wherein the method comprises: i) providing a cell according to any one of claims 19 to 27, transferring a linear plasmid according to any one of claims 28 to 33 to a cell according to any one of claims 1 to 18, excising a linear plasmid from a circular nucleic acid molecule according to any one of claims 34 to 36 within a cell according to any one of claims 1 to 18, transferring a first and / or a second linear plasmid to a cell according to any one of claims 37 to 41, or generating a first and / or a second linear plasmid within a cell according to any one of claims 37 to 41 ; wherein the episome or linear plasmid comprises a sequence-of-interest and wherein the orthogonal DNA replication machinery replicates DNA with a lower fidelity than the cell’s endogenous DNA replication machinery; ii) incubating the cell under conditions conducive to growth; and iii) exposing the cell to a selection condition.

45. The method of claim 44, wherein the method comprises: iv) identifying the sequence of a sequence-of-interest from a cell meeting the selection condition.

46. The method of claim 45, wherein the method comprises: v) making a polypeptide or nucleic acid encoded by the identified sequence-of-interest.

47. A method of making a polypeptide or nucleic acid, wherein the method comprises: i) providing a sequence-of-interest identified by the method of any one of claims 44 to 47, and ii) producing a polypeptide or nucleic acid according to said sequence.

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