Large vectors and methods for high yield production

Abstract

Provided herein is a method for producing a vector of at least 16 kb in size from a bacterial cell, comprising the steps of: a) obtaining bacterial cells containing a vector of at least 16 kb in size, which contains an inducible origin of replication; b) inoculating a culture medium with bacterial cells containing the vector; c) culturing said bacterial cells in said medium; d) Optical density at 600 nm (OD ) of the bacterial culture 600 ) reaches at least 20, adding one or more inducers of said inducible replication origin to the culture medium; e) further culturing the bacterial cells in the medium; f) optionally separating the bacterial cells from the culture medium; and g) recovering the plasmid from the bacterial cells Also provided herein is a vector of at least 16 kb in size comprising an inducible origin of replication for use in such a method.

Description

[Technical field]

[0001] The present invention relates to methods for the high-yield production of vectors and large vectors. [Background technology]

[0002] Gene therapy and DNA vaccines are promising tools for the prevention, treatment and cure of diseases such as cancer and acquired immune deficiency syndrome (AIDS). Gene therapy and DNA vaccines require large amounts of highly purified plasmid DNA (pDNA) that must be homogenous in structural morphology and DNA sequence. Furthermore, gene therapy and DNA vaccines such as DNA vaccines against complex pathogens require plasmids that can tolerate large genomic DNA insertions. In biopharmaceutical applications, yield and quality are the main drivers for the plasmid manufacturing process. In the search for increased yield, plasmids that result in high copy numbers have been developed, meaning multiple copies of the plasmid per cell (>500 copies per cell, e.g., pUC18, pUC19 vectors). Such high copy numbers of plasmids per cell can be achieved with small plasmids (<10 kb). However, larger plasmids impose a greater metabolic burden on the bacterial cell and therefore replicate slower. Bacterial cells respond to the metabolic burden by introducing mutations into the plasmid. As a result, the quality of the plasmid DNA decreases. This can be optimized by using low copy number vectors such as bacterial artificial chromosomes (BACs), which are the preferred plasmids for maintaining large genomic DNA fragments, as individual DNA fragments can be stably maintained within the host cell in single copy (SC) vectors, even after more than 100 generations of continuous bacterial propagation.

[0003] Recent advances are related to optimizing the copy number of BACs. For example, WO2014174078 describes BACs that contain an inducible bacterial origin of replication (ori) sequence to amplify BACs to more than 10 copies per bacterial cell. However, these BACs do not provide commercially viable high BAC yields. As a result, very large-scale cultures are required to obtain significant amounts of BACs.

[0004] It is essential to develop new vectors and protocols to obtain high yields of high quality plasmids.

[0005] Conditional amplification of vectors containing inducible bacterial origin of replication (ori) sequences in bacteria has been described in the art. Induction of amplification is usually performed in the early exponential growth phase of bacteria, more specifically, in the early logarithmic (arithmetic) phase of the bacterial growth curve (i.e., low OD) (Wild et al. 2002, Genome Research 12:1434-1444). Summary of the Invention

[0006] We have found that by using an inducible system that maintains a low number of plasmids per cell until optimal fermentation conditions are reached and then switches the system to a high copy state, the exposure of the bacterial cell to high metabolic stress is minimized, resulting in higher yields of large plasmids and of high quality.

[0007] According to a first aspect of the invention, there is provided a method for producing a vector of at least 16 kb in size from a bacterial cell using a vector that comprises an inducible origin of replication, whereby induction occurs when the bacterial cell reaches an OD of at least 20. According to a particular embodiment, the method for producing a vector of at least 16 kb in size from a bacterial cell comprises the steps of: a) obtaining a bacterial cell containing a vector of at least 16 kb in size, which vector contains an inducible origin of replication; b) inoculating the medium with bacterial cells containing the vector; c) Optical density (OD) at 600 nm 600 ) reaches at least 20; d) adding to the culture medium one or more inducers of an inducible origin of replication; e) optionally separating the bacterial cells from the culture medium; and f) Recovering the plasmid from the bacterial cells A method is provided that includes:

[0008] It will be appreciated that culturing of the cells continues after step (d) to allow for production of the plasmid.

[0009] More specifically, the present invention relates to a method for culturing bacterial cells in a medium and measuring the optical density at 600 nm (OD 600 ) reaches at least 20, adding one or more inducers of the inducible origin of replication to the medium, and then further culturing the bacterial cells in the medium. Optionally, the bacterial cells are then separated from the medium, and the plasmid is recovered from the bacterial cells.

[0010] In certain embodiments, the method further comprises adding an inhibitor of bacterial protein synthesis to the medium after the one or more inducers of the inducible origin of replication have been added, such as after step d) and before step e).

[0011] In certain embodiments, glucose and / or yeast extract are added to the medium during cultivation of the bacterial cells in the medium.

[0012] In certain embodiments, the medium containing glucose is replaced with medium containing 0.50-2.0% (v / v) glycerol at least 30 minutes prior to adding one or more inducers of plasmid replication to the medium.

[0013] In a particular embodiment, step c) of culturing the bacterial cells in the medium is carried out at a temperature of about 30° C., and the temperature is increased from about 30° C. to a temperature of 36.0-38.0° C. at least 2 hours before adding one or more inducers of plasmid replication to the medium.

[0014] In certain embodiments, the inducible origin of replication comprises at least one LacO1 operator and the inducer of the inducible origin of replication is isopropyl β-D-1-thiogalactopyranoside (IPTG) and / or alpha-D-lactose.

[0015] In a particular embodiment, the inhibitor of bacterial protein synthesis is chloramphenicol or spectinomycin, preferably chloramphenicol.

[0016] In certain embodiments, the inhibitor of bacterial protein synthesis is added to the medium at least 1 hour after the addition of the one or more inducers of the inducible origin of replication.

[0017] In certain embodiments, the bacterial cells are separated from the culture medium up to 6 hours after addition of one or more inducers of the inducible origin of replication.

[0018] According to a further embodiment, there is provided a vector of at least 16 kb in size which comprises an inducible origin of replication.

[0019] In certain embodiments, the inducible origin of replication is: - the RNAI promoter is deleted or mutated such that its function is abolished or greatly reduced, forming a truncated RNAII preprimer; and - the nucleic acid sequence encoding RNAI is deleted or mutated such that the function of RNAI is abolished or significantly reduced, forming a truncated RNAII preprimer; and - at least one LacO1 operator is introduced upstream of the nucleic acid sequence encoding the truncated RNAII preprimer, and at least one LacO1 operator is operably linked to the nucleic acid sequence encoding the truncated RNAII preprimer. Contains the pMB1 origin of replication.

[0020] In certain embodiments, the endogenous RNAII promoter of the pMB1 origin of replication is replaced by a promoter that is not the endogenous promoter of the RNAII preprimer of the pMB1 origin of replication, and the non-endogenous promoter of the RNAII preprimer of the pMB1 origin of replication is operably linked to at least one LacO1 operator.

[0021] In a specific embodiment, the endogenous RNAII promoter of the pMB1 origin of replication is replaced by a promoter selected from the group consisting of the promoter of RNAI, the lac promoter, the trp promoter, the trc promoter and the T7 promoter, preferably the lac promoter, more preferably the L8 / UV5 lac promoter.

[0022] According to a further aspect, - the RNAI promoter is deleted or mutated such that its function is abolished or greatly reduced, forming a truncated RNAII preprimer; and - the nucleic acid sequence encoding RNAI is deleted or mutated such that the function of RNAI is abolished or significantly reduced, forming a truncated RNAII preprimer; and - at least one LacO1 operator is introduced upstream of the nucleic acid sequence encoding the truncated RNAII preprimer, and at least one LacO1 operator is operably linked to the nucleic acid sequence encoding the truncated RNAII preprimer; and - a nucleic acid corresponding to nucleic acid at position 322 of the wild-type pMB1 origin of replication is mutated from A to G and / or a nucleic acid corresponding to nucleic acid at position 543 of the wild-type pMB1 origin of replication is mutated from C to T Inducible origins of replication are provided, including the pMB1 origin of replication.

[0023] In a particular embodiment, the inducible origin of replication comprises a nucleic acid sequence having at least 95%, preferably 100%, sequence identity to the nucleic acid sequence shown in SEQ ID NO:9.

[0024] In a particular embodiment, the method is used to produce a vector at least 16 kb in size from a bacterial cell, hi a further embodiment, the vector at least 16 kb in size is a vector as set forth herein. [Brief description of the drawings]

[0025] [Figure 1] Figure 1 shows a schematic diagram of pViroVet-4-YFV17D plasmid and iOri. oriS: ori2 replicon of F factor; iOri: inducible origin of replication; repE: replication initiator gene; parB: split protein B gene; parC: split protein C gene; KanR: kanamycin resistance gene; HDrz: hepatitis virus delta ribozyme; UTR: untranslated region; YFV17D: yellow fever virus clone 17D; Lac-L8-UV: lac promoter with L8-UV5 modification; LacO1: lac operator; pMB1ori: origin of replication of modified pMB1(ColE1). [Diagram 2] FIG. 2 shows the nucleic acid sequences of the complete pMB1(ColE1)Ori and the complete iOri, which contain two point mutations in the RNAII sequence. [Diagram 3]Figure 3 (A) In pMB1(ColE1)Ori, RNAII DNA is transcribed from its cognate upstream promoter RNAIIp to generate RNAII transcripts. RNAII serves as a primer to initiate DNA synthesis at the Ori+1 start of the plasmid DNA. Expression of RNAII is post-transcriptionally regulated by binding of a complementary antisense RNAI. RNAI is expressed from an RNAI gene that resides in an inverted orientation within the RNAII gene from its RNAIp promoter. Plasmid-encoded ROP proteins stabilize the RNAI-RNAII duplex, which further restricts replication of the plasmid DNA. (B) In iOri, expression of the 5'-truncated RNAII* is initiated from the heterologous LacUVp promoter. The entire RNAI gene is deleted and RNAI is not expressed. Expression of the 5'-truncated RNAII* (i.e., the truncated RNAII preprimer) is regulated at the transcriptional level by binding of the LACI protein to a LacO1 repressor binding sequence inserted between the heterologous promoter and the original wild-type RNAII* sequence. In iOri, RNAII*, a transcriptional fusion between LacO1 and RNAII DNA sequences, serves as a primer to initiate plasmid DNA synthesis. Ori+1 is the origin of DNA replication; genes are in normal letters, transcripts in italics, proteins in bold capital letters; negative regulators are in shaded letters; transcripts are indicated by wavy arrows showing relative orientation. [Figure 4] FIG. 4 shows the inducibility of the plasmids presented herein by both IPTG and α-D-lactose. [Diagram 5] Figure 5 shows the beneficial effect of mutant iOri on vector production compared to wild type iOri. pVV4* means the first clone of pViroVet-4-YFV17D plasmid shown in Figure 1, pVV4 means the second clone of pViroVet-4-YFV17D plasmid shown in Figure 1, pVV-wt iOri means the pViroVet-4-YFV17D plasmid in which mutant iOri is not replaced with wild type (i.e., non-mutated) iOri, NI means not induced; I means induced. [Figure 6] FIG. 6 shows the beneficial effect on vector production by adding glucose or glycerol as a supplement to the feed. [Figure 7] Figure 7 shows the effect of chloramphenicol on plasmid production in a 1-liter fermenter. Procedure (a) is inoculation of bacterial cells; procedure (b) is initiation of glycerol feeding; procedure (c) is induction of bacterial cells with IPTG; procedure (d) is addition of chloramphenicol to the medium; and procedure (e) is harvesting of bacterial cells. [Figure 8] Figure 8 shows the effect of temperature shift on plasmid production in a 1 liter fermenter: procedure (a) is inoculation of bacterial cells; procedure (b) is initiation of glucose feeding; procedure (c) is increase of temperature to 37°C and change from glucose feeding to glycerol feeding; procedure (d) is induction of bacterial cells with IPTG; (e) is addition of chloramphenicol to the medium; and procedure (f) is harvesting of bacterial cells. [Figure 9] FIG. 9 shows the nucleic acid sequences represented by the SEQ ID NOs. Detailed Description of the Invention

[0026] The term "about" when used in connection with a numerical value has the meaning commonly understood in the relevant art. In certain embodiments, the term "about" may be omitted or may be interpreted to mean the numerical value +10%; or +5%; or +2%; or +1%.

[0027] Whenever used herein with respect to percentages, w / w means weight / weight and w / v means weight / volume.

[0028] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0029] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" when referring to recited members, elements, or method steps also include embodiments that "consist of" the recited members, elements, or method steps.

[0030] Additionally, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements or steps, unless otherwise noted, and are not necessarily used to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described herein can be operated in orders other than those described or illustrated herein.

[0031] References throughout this specification to "one embodiment" or "embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment contemplated herein. Thus, the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Also, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Also, while some embodiments described herein include some features that are included in other embodiments and not others, it is meant that combinations of features from different embodiments are within the scope of the invention and form different embodiments, as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the embodiments described in the claims may be used in any combination.

[0032] All references cited herein are hereby incorporated by reference.

[0033] When increasing the yield of large vectors (e.g., plasmids, bacterial artificial chromosomes (BACs)) in bacteria, there is a legitimate concern that the activity of the replication system will be greatly increased, leading to an increased mutation frequency of the vector DNA. Thus, the production of large vectors requires a manufacturing process that provides high yields of vector, but where vector replication occurs without the introduction of intolerable mutations.

[0034] The inventors have identified tools and methods to ensure high-yield production of large vectors. This is important, for example, in the generation of vectors used in vaccination, where large inserts are often required. The inventors have identified that this can be ensured by strategically utilizing an inducible origin of replication (ori) during the culture of host cells containing the vector. More specifically, bacterial cells containing vectors of at least 16 kb in size, including an inducible ori, are cultured at high culture density (e.g., optical density at 600 nm (OD 600 We have found that large amounts of the vector can be obtained by culturing the vector until the OD reaches at least 20 and then inducing replication by adding one or more inducers of the inducible ori to the medium. More specifically, we limit the copy number of the vector per cell unit during the growth phase (e.g., optical density at 600 nm (OD 600 ) less than 20), high biomass (e.g., OD 600We have found that by inducing replication at a concentration of at least 20% by volume, high quality pharmaceutical grade (i.e., low mutation frequency) vectors can be obtained in high yields (e.g., at least 55 mg / L of vector) for use in, for example, DNA-based vaccines. The methods for producing vectors of at least 16 kb in size presented herein are scalable (e.g., to at least 1000 L, at least 1500 L, or at least 2000 L). We have also found that vectors of at least 16 kb in size comprising an inducible ori comprising an inducible promoter and a nucleic acid sequence encoding a truncated RNAII preprimer of the ColE1-type ori, the nucleic acid sequence encoding a truncated RNAII preprimer of the ColE1-type ori being operably linked to the inducible promoter, are particularly advantageous for obtaining such high yields of vectors. The inventors have found that yields can be further increased (e.g., to at least 120 mg / L of vector) by (i) introducing a temperature shift from approximately 30°C to 36-38°C before induction; (ii) adding inhibitors of bacterial protein synthesis after induction; and / or (iii) adding glucose and / or yeast extract, or glycerol as supplements to the medium.

[0035] Thus, according to a first aspect, there is provided a method for producing a vector of at least 16 kb in size from a bacterial cell, comprising the steps of: a) obtaining a bacterial cell containing a vector of at least 16 kb in size, comprising an inducible ori; b) inoculating the medium with bacterial cells containing the vector; c) Optical density (OD) at 600 nm 600 ) reaches at least 20; d) adding one or more inducers of an inducible ori to the medium; e) optionally separating the bacterial cells from the culture medium; and f) Recovering the vector from the bacterial cells A method is provided that includes:

[0036] It will be appreciated that culturing of the cells continues after step (d) to allow for production of the plasmid.

[0037] Thus, the method for producing vectors of at least 16 kb in size from bacterial cells also includes the steps of: - obtaining a bacterial cell containing a vector of at least 16 kb in size, comprising an inducible ori, - inoculating the medium with bacterial cells containing the vector; - culturing bacterial cells in a culture medium; - Optical density at 600 nm (OD) of bacterial cultures 600 ) reaches at least 20, adding one or more inducers of the inducible ori to the medium; - Further culturing the bacterial cells in the medium, - optionally separating the bacterial cells from the culture medium, and - recovering the vector from the bacterial cells It can also be described as including.

[0038] In certain embodiments, these steps are performed in sequential order.

[0039] The term "vector" as used herein means a circular double-stranded DNA molecule into which a nucleic acid fragment, preferably a recombinant nucleic acid molecule as defined herein, can be inserted and cloned, i.e., propagated. Thus, a vector typically contains one or more unique restriction sites and is capable of autonomous replication in a defined cell or medium organism such that the cloned sequence can be reproduced. In the present case, the skilled artisan will understand that the vector is a vector capable of replicating in a bacterial cell. The vector may also contain a selection marker, such as, for example, an antibiotic resistance gene, to allow for the selection of recipient cells containing the vector. Vectors include, but are not limited to, plasmids, cosmids, phagemids, bacteriophage-derived vectors, PACs, BACs, etc., as appropriate. The plasmid may be a plasmid intended for the construction of a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector, among others. An expression vector is generally constructed to allow and / or provide for the expression of a nucleic acid or an open reading frame introduced therein in a desired expression system, e.g., in vitro, in a cell, an organ, and / or an organism. For example, expression vectors may advantageously contain appropriate regulatory sequences.

[0040] In certain embodiments, the vector is a large vector, such as a vector at least 16 kb, at least 17 kb, at least 18 kb, at least 19 kb, at least 20 kb, at least 21 kb, or at least 22 kb in size, preferably at least 16 kb. As used herein, "kb," "kbp," or "kilobase pair" refers to 1000 base pairs (bp) of DNA.

[0041] The term "bacterial cell" or "bacteria" refers to bacteria suitable as host cells for vectors. The advantages of producing vectors in bacteria lie, inter alia, in the relatively safe and easy handling of bacterial cells and the rapid replication cycle of microorganisms.

[0042] In certain embodiments, for example when the inducible ori comprises a Lac O1 operator, the bacterial cell is a bacterial cell comprising a Lac repressor (Lacl). In more specific embodiments, the bacterial cell is a cell having a sufficient amount of Lac repressor (Lacl). The sufficient amount of Lac repressor is an amount of Lac repressor that prevents leaky replication of the vector in the bacterial cell in the absence of one or more inducers of the inducible ori. Preferably, the sufficient amount of Lac repressor is an amount of Lac repressor that is capable of suppressing replication of 0.5-4 mg / L, 0.5-3 mg / L, 0.5-2 mg / L, or 0.5-1 mg / L of the vector in the absence of one or more inducers of the inducible ori.

[0043] In a particular embodiment, the bacterial cell is E. coli, preferably BL21 competent E. coli or derivatives such as BL21(DE3) competent E. coli and DH5-α cells as described by William Studier and Barbara A. Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5;189(1):113-30.

[0044] Bacterial cells containing vectors of at least 16 kb in size can be obtained by bacterial transformation methods known in the art, for example heat shock.

[0045] As used herein, the term "inducible origin of replication", "inducible ori", "iori", "conditional origin of replication" or "conditional ori" refers to a vector ori sequence that functions in a bacterial host cell and responds to one or more inducers of the inducible origin of replication that are foreign to the host cell (i.e., bacterial cell). Preferably, the replication function of the inducible ori is significantly suppressed or absent in the absence of one or more inducers of the inducible origin of replication. In certain embodiments, the inducible ori propagates the vector to a high copy number in the presence of one or more inducers of the inducible origin of replication, preferably at least 20 copies, at least 30 copies, at least 40 copies, at least 50 copies, at least 60 copies, at least 70 copies, at least 80 copies, at least 90 copies, at least 100 copies, at least 110 copies, at least 120 copies, at least 130 copies, at least 140 copies, at least 150 copies, at least 200 copies or at least 300 copies per cell, more preferably more than 50 copies per cell, even more preferably more than 100 copies per cell. Preferably, the inducible ori is responsive to one inducer of the inducible replication origin.

[0046] The term "inducer of an inducible origin of replication", "inducer of vector replication" or "replication initiator" refers to an agent capable of inactivating (e.g., by removal) a repressor. A repressor inhibits or reduces expression of one or more transcripts, usually by binding to an operator sequence of DNA. Binding of the repressor to the operator prevents binding of RNA polymerase to the promoter, thereby inhibiting transcription. An inducer of an inducible origin of replication can prevent binding of the repressor to the operator, thereby allowing transcription.

[0047] Those skilled in the art will understand that an inducible ori is selected for its compatibility with known inducers of vector replication, its usual tight downregulation in the absence of a compatible inducer of vector replication in the selected host cell, and its strong inducibility in the presence of an inducer of vector replication. Non-limiting examples of inducible ori include the oriV / TrfA amplification system tightly regulated by an L-arabinose-inducible parapromoter (araC-PBAD) that can be induced by the addition of L-arabinose, for example, the amplification of shuttle BAC vectors as described in Wild J. et al., Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Res. 2002.12(9):1434-1444. In a further example, oriV / TrfA can be tightly regulated by the rhamnose-inducible Prha promoter (rhaS-Prha) as described in Wild J. et al., Copy-control tightly regulated expression vectors based on pBAC / oriV. 2004.267:155-167.

[0048] ColEl-type plasmids are known in the art and include naturally occurring plasmids (such as pMB1, pl5A, pJHCMW1) and many commonly used cloning vehicles (such as pBR322 and related vectors, pUC plasmids, pET plasmids, and pBluescript vectors). The initiation and regulation of replication of these plasmids is well known in the art. More specifically, ColEl-type plasmids replicate DNA using a common mechanism that involves the synthesis of two RNA molecules (i.e., RNAI and RNAII) involved in the regulation of replication, their interaction on the one hand, and their interaction with the template plasmid DNA on the other hand. The ColEl region contains two promoters, one for RNAI and one for RNAII. Replication from ColEl-type plasmids is initiated by transcription of the preprimer RNAII, located 555 bp upstream of the replication origin, by the host RNA polymerase. During elongation, RNAII folds into a specific hairpin structure and begins to form a hybrid with the template DNA after approximately 550 nucleotides of polymerization. Subsequently, the RNAII preprimer is cleaved by RNase H to form an active RNAII primer with a free 3'OH end accessible to DNA polymerase I. On the opposite side of the origin strand of the ColEl type, RNAI, a 108-nucleotide RNA complementary to the 5' end of antisense RNAII, is transcribed. Transcription of RNAI is initiated 445 bp upstream from the replication origin and continues almost to the start of RNAII transcription. RNAI inhibits primer formation by binding to the elongating RNAII molecule before the RNA / DNA hybrid is formed, thus inhibiting replication. Apart from the RNAI / RNAII interaction, the rom / rop transcripts of ColEl contribute to the regulation of plasmid copy number by increasing the rate of complex formation between RNAII and RNAI.

[0049] Derivatives of ColEl-type plasmids with deleted rom / rop have been developed. Such derivatives have increased copy numbers. Examples of such derivatives include derivatives of pBR322, such as pUC18 and pUC19. The pUC18 and pUC19 plasmids further contain a single point mutation in RNAII, more specifically, a GA point mutation at nucleotide 112 of the RNAII transcript in the segment directly adjacent to the region commentary to RNAI, enhancing the ability to initiate plasmid replication.

[0050] Propagation of a plasmid within a host cell requires replication of the plasmid by the host's replication machinery. The origin of replication (ori) is a DNA sequence carried by a plasmid that instructs the host cell to initiate plasmid replication and is therefore essential for plasmid propagation. This is achieved by various proteins (initiators and repressors) that bind to sequences that make up the ori.

[0051] The pMB1(ColE1)ori contains regions that promote the synthesis of RNAI and RNAII, as described above. Plasmid replication is usually initiated by RNAII, which is transcribed from the plasmid 550 nucleotides (nt) upstream of theori and hybridizes tightly to the plasmid. The formation of this hybrid at the origin is an important prerequisite for plasmid replication. The RNA portion of this hybrid becomes a substrate for RNaseH, which digests RNAII, generating a 550-nt molecule that can be used as a primer for DNA polymerase I to initiate replication of the entire plasmid.

[0052] Those skilled in the art will understand that vectors of at least 16 kb in size taught or described herein may contain, in addition to an inducible ori, an ori that utilizes a replication mechanism different from the inducible ori, creating an unstable and unpredictable environment as the two ori compete for the same structure. For example, if the inducible ori is derived from a ColE1-type ori or an ori derived therefrom, the second ori may be an F1 or P1 ori or an ori derived therefrom. Thus, in certain embodiments, the vector is a vector replicated by an ori different from a ColE1-type ori or an ori derived therefrom (preferably a pMB1 ori or an ori derived therefrom), into which an inducible ori derived from a ColE-1-type ori (preferably derived from a pMB1 ori) is introduced as described herein. Non-limiting examples of vectors replicated by an ori different from a ColE-1-type ori (preferably derived from a pMB1 ori) include vectors replicated by an F1 or P1 ori or an ori derived therefrom, or vectors derived therefrom known in the art. In a more specific embodiment, the vector into which the inducible ori described herein is introduced is not a ColE1-type plasmid, and preferably, the vector into which the inducible ori described herein is introduced is not a pBR322 vector or a pUC vector.

[0053] The medium can be inoculated with bacterial cells containing the vector by any method known in the art for inoculating bacterial cells into culture medium.For example, a single colony of bacterial cells containing the vector can be selected from an agar plate using a sterile pipette tip or toothpick, and then the tip or toothpick is placed in liquid medium and the liquid medium containing the tip or toothpick is spun.Alternatively, the medium can be inoculated with a glycerol stock of bacterial cells containing the vector.

[0054] The medium can be any culture medium known in the art for culturing bacterial cells. Non-limiting examples include minimal medium such as Luria-Bertani Broth (LB) (ATCC medium formulation 1065) or M9 minimal medium. Those skilled in the art will understand that when minimal medium is used, supplementation with amino acids, salts and / or nutrients may be required. The use of minimal medium has the advantage that the nutrients supplied to bacterial cells can be tightly regulated.

[0055] In certain embodiments, the medium is LB medium supplemented with at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, or at least 90 mM phosphate. For example, the medium is LB medium supplemented with 90 mM phosphate.

[0056] In certain embodiments, the culture medium is a liquid (broth) medium.

[0057] The biological oxidation of carbohydrates, such as glucose, by bacteria leads to the synthesis of ATP as a source of chemical energy to generate simpler organic compounds than are required by the bacteria for biosynthetic or anabolic reactions.

[0058] Thus, in certain embodiments, glycerol, glucose and / or yeast extract are added to the medium after the step of inoculating the medium (step b) and before the step of culturing the bacterial cells in the medium (step c). In more specific embodiments, glycerol, glucose and / or yeast extract are added to the culture medium during the step of culturing the bacterial cells in the medium (step c).

[0059] In certain embodiments, one or more of glycerol, glucose and yeast extract (e.g., glycerol; glucose; yeast extract; glycerol and glucose; glycerol and yeast extract; glucose and yeast extract; or glycerol, glucose and yeast extract) are added to the medium at a total concentration of 0.50 to 1.0% (w / v or v / v for glycerol), 0.50 to 5.0% (w / v or v / v for glycerol), 1.0 to 5.0% (w / v or v / v for glycerol), 1.0 to 4.0% (w / v or v / v for glycerol), 1.0 to 3.0% (w / v or v / v for glycerol), 1.0 to 2.0% (w / v or v / v for glycerol), preferably 0.50 to 2.0% (w / v or v / v for glycerol). For example, one or more of glycerol, glucose and yeast extract is added to the medium at 1.0% (w / v or v / v for glycerol).

[0060] In certain embodiments, glycerol is added to the medium at a concentration of 0.50-5.0% (v / v), 1.0-5.0% (v / v), 1.0-4.0% (v / v), 1.0-3.0% (v / v), or 1.0-2.0% (v / v), preferably 0.5-2.0% (v / v). For example, glycerol is added to the medium at a concentration of 1.0% (v / v).

[0061] In a specific embodiment, glucose is added to the medium at a concentration of 0.50-5.0% (w / v), 1.0-5.0% (w / v), 1.0-4.0% (w / v), 1.0-3.0% (w / v), 1.0-2.0% (w / v), preferably 0.5-2.0% (w / v). For example, glucose is added to the medium at a concentration of 1.0% (w / v).

[0062] In certain embodiments, yeast extract is added to the medium at a concentration of 0.50-10.0% (w / v), 0.50-5.0% (w / v), 1.0-5.0% (w / v), 1.0-4.0% (w / v), 1.0-3.0% (w / v), or 1.0-2.0% (w / v).

[0063] The consumption of glycerol, glucose and / or yeast extract depends on the metabolic state of the bacterial cells. Exposure of bacterial cells to high concentrations of glycerol, glucose and / or yeast extract for a short period of time can lead to osmotic lysis of the bacterial cells.

[0064] Thus, in certain embodiments, glucose and / or yeast extract are continuously added to the medium during cultivation of the bacterial cells in the medium, for example by fed-batch cultivation.

[0065] In certain embodiments, glycerol is continuously added to the culture medium at a feed rate of up to 0.750% (v / v) per hour, up to 0.70% (v / v) per hour, up to 0.650% (v / v) per hour, up to 0.60% (v / v) per hour, up to 0.550% (v / v) per hour, up to 0.50% (v / v) per hour, up to 0.40% (v / v) per hour, up to 0.30% (v / v) per hour, up to 0.20% (v / v) per hour, or up to 0.10% (v / v) per hour. In certain embodiments, glucose is added to the medium at a feed rate of up to 0.60% (w / v) per hour, up to 0.50% (w / v) per hour, up to 0.40% (w / v) per hour, up to 0.30% (w / v) per hour, up to 0.20% (w / v) per hour, or up to 0.10% (w / v) per hour.

[0066] In certain embodiments, yeast extract is added to the medium at a feed rate of up to 0.30% (w / v) per hour, up to 0.20% (w / v) per hour, up to 0.10% (w / v) per hour, or up to 0.050% (w / v) per hour.

[0067] Bacterial cells require an energy source, especially during the growth phase. Thus, in certain embodiments, one or more of glycerol, glucose and yeast extract are added to the medium during the growth phase of the bacterial cells.

[0068] The growth phase of bacterial cells can be determined by the optical density of the medium containing the bacterial cells. The higher the concentration of bacteria in the medium, the higher the optical density of the culture at the time of measurement. Organic materials show a high optical density at a wavelength of 600 nm, so culture density can be measured using a spectrophotometer at a wavelength of 600 nm. The optical density of a medium sample at 600 nm is called the "OD 600 Preferably, the medium sample is not diluted. Thus, in certain embodiments, the glucose and / or yeast extract are added to the culture medium to obtain a culture medium with a high optical density at 600 nm (OD 600 ) is added to the medium during cultivation at 0 to 20, 0 to 21, 0 to 22, 0 to 23, 0 to 24, 0 to 25, 0 to 30, 5 to 20, 5 to 25, 5 to 30, 10 to 20, 10 to 25 or 10 to 30, preferably 0 to 30.

[0069] Cultivation of bacterial cells in a medium can be carried out by any method known in the art for culturing bacterial cells in a medium. For example, the liquid medium containing the bacterial cells can be placed in a shaking incubator at 30°C.

[0070] In a particular embodiment, the bacterial cells are cultured in a medium of at least 500 ml, at least 600 ml, at least 700 ml, at least 800 ml, at least 900 ml, at least 1000 ml, at least 1250 ml, at least 1500 ml, at least 1750 ml, at least 2000 ml or at least 2500 ml, preferably at least 800 ml. In a more particular embodiment, the bacterial cells are cultured in 500-2500 ml, 500-2000 ml, 500-1500 ml or 500-1000 ml, preferably 500-2500 ml. Typically, the medium is up to 85%, preferably up to 80% of the volume of the fermenter in which the bacterial cells are cultured. Thus, in a particular embodiment, the bacterial cells are cultured in a vessel having a volume of 400-2000 ml, 400-1600 ml, 400-1200 ml or 400-800 ml.

[0071] Preferably, the bacterial culture is initiated at a low confluency. Bacterial growth and confluency can be determined by measuring the optical density of the culture medium containing the bacteria.

[0072] In certain embodiments, culturing the bacterial cells in the medium comprises culturing the bacterial cells at an optical density of 600 nm (OD 600 ) starts at max 3, max 2, max 1, max 0.5, max 0.4, max 0.3, max 0.2, max 0.1, preferably max 0.1.

[0073] In certain embodiments, culturing the bacterial cells in the medium comprises culturing the bacterial cells at an optical density of 600 nm (OD 600 ) is started at 0.10-3.0, 0.10-2.0, 0.40-3.0, 0.40-2.0 or 0.50-2.0. For example, bacterial cells are grown at a starting OD of 600 It can be diluted to 0.1, 0.4 or 2.

[0074] Ensure that the culture of bacterial cells reaches a high confluency (e.g., at least OD 600 20), one or more inducers of the inducible replication origin are added to the medium.

[0075] Bacterial growth can usually be divided into four distinct growth phases: lag phase, log phase, stationary phase and death phase. The progression of these phases provides the data that is compiled into a bacterial growth curve.

[0076] Generally, a bacterial cell culture reaches a high culture density in the exponential or late logarithmic (arithmetic) phase of the bacterial growth curve. Thus, in certain embodiments, one or more inducers of an inducible replication origin are added to the medium when the number of bacterial cells is 70-95%, 75-95%, 80-95%, 85-95% or 85-90%, preferably 85-90%, of the estimated maximum bacterial cell number in the logarithmic phase of the bacterial growth curve determined for the bacterial cell culture.

[0077] One of skill in the art will know how to determine the bacterial growth curve of a bacterium. For example, the bacterial growth curve can be determined as described in Todar's online textbook of bacteriology at http: / / textbookofbacteriology.net / growth_3.html.

[0078] Similarly, in certain embodiments, one or more inducers of an inducible origin of replication are 600 ) (or the OD reached by a bacterial culture or a culture medium containing bacterial cells) 600 ) are added to the medium in an amount of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39 or at least 40, preferably at least 25, more preferably at least 30.

[0079] In certain embodiments, one or more inducers of an inducible origin of replication are administered at an optical density of 600 nm (OD 600 ) (or the OD reached by a bacterial culture or a culture medium containing bacterial cells) 600 ) is added to the medium at 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-45, 20-40, 25-35 or 30-40, preferably 30-40, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39. For example, one or more inducers of an inducible replication origin are added to the medium at an optical density at 600 nm (OD 600 ) can be added to the medium at 30°C.

[0080] Those skilled in the art will appreciate that in the methods of the present invention, optical density at 600 nm (OD 600 It will be appreciated that prior to the step of adding one or more inducers of an inducible origin of replication to the medium (step d) in which the number of inducers is at least 20, no inducer of the inducible origin of replication is present in or added to the medium.

[0081] Similarly, in certain embodiments, the one or more inducers of the inducible origin of replication are added to the medium 20-30 hours, 20-27 hours, 23-27 hours or 25-27 hours, for example 25 hours, 26 hours or 27 hours, after the step of inoculating the medium with bacterial cells containing the vector (step b).

[0082] In certain embodiments, the one or more inducers of the inducible origin of replication are incubated with the bacterial cells for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours or at least 5 hours, preferably at least 2 hours, after adding the one or more inducers of the inducible origin of replication to the culture medium.

[0083] In certain embodiments, the one or more inducers of the inducible origin of replication are incubated with the bacterial cells for 1-7 hours, 2-6 hours, 2-5 hours, 2-4 hours, 3-6 hours or 3-5 hours, preferably 2-6 hours, after adding the one or more inducers of the inducible origin of replication to the culture medium.

[0084] One or more inducers of inducible replication origin can be added to the medium by methods known in the art.For example, when the inducer of inducible replication origin is a chemical, the chemical can be added directly to the medium.In another example, when the inducer of vector replication is encoded by a polynucleotide, the polynucleotide can be provided in an expression cassette under the transcriptional control of a promoter.

[0085] Glucose may interfere with one or more inducers of plasmid replication, therefore, it is preferred that glucose is removed from the medium and / or completely consumed by the bacterial cells before adding one or more inducers of plasmid replication to the bacterial culture.

[0086] In certain embodiments, when the medium in which the bacterial cells are cultured is supplemented with glucose, one or more inducers of plasmid replication are added to the medium when substantially all (e.g., at least 99.0%, preferably at least 99.90%), or all, of the glucose present in the medium is consumed by the bacterial cells.

[0087] In certain embodiments, when the medium in which the bacterial cells are cultured is supplemented with glucose, the medium is replaced or substituted with a medium containing 0.50-2.0% (v / v), 0.50-1.50% (v / v) or 0.50-1.0% (v / v) glycerol, preferably 0.50-1.0% (v / v) glycerol, and up to 0.010% (v / w), preferably up to 0.0010% (v / w), including % (v / w), glucose, when substantially all (e.g., at least 99.0%, preferably at least 99.90%) or all glucose present in the medium is consumed by the bacterial cells prior to adding one or more inducers of plasmid replication to the medium. The amount of glucose in the medium can be determined as known in the art, for example, using high performance liquid chromatography (HPLC) of a medium sample or the GlucCell® Glucose Monitoring System (Cesco Bioproducts).

[0088] For certain bacterial cultures, after exchanging or replacing the glucose-supplemented medium with medium containing glycerol but no glucose (e.g., up to 0.010% (v / w), preferably up to 0.0010% (v / w) glucose), it may take at least 30 minutes for the bacterial cells to consume any glucose remaining in the bacterial cells.

[0089] Similarly, in certain embodiments, if the medium in which the bacterial cells are cultured is supplemented with glucose, the medium is exchanged or replaced with a medium containing 0.50-2.0% (v / v), 0.50%-1.50% (v / v) or 0.50-1.0% (v / v), preferably 0.50-1.0% (v / v), glycerol at least 30 minutes before adding one or more inducers of plasmid replication to the medium. For example, the medium containing glucose and yeast extract is exchanged or replaced with a medium containing 1.0% (v / v) glycerol and yeast extract at least 30 minutes before adding one or more inducers of plasmid replication to the medium. For example, if the medium supplemented with glucose and yeast extract is added to the medium continuously, e.g., by fed-batch culture, during the cultivation of the bacterial cells in the medium, the supply of the medium is switched from the medium supplemented with glucose and yeast extract to the medium supplemented with glycerol and yeast extract.

[0090] In certain embodiments, the glucose-containing medium is at least 10% of the optical density at 600 nm (OD ) of the medium (or bacterial culture) containing the bacterial cells. 600 ) is 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, 19-20, 13-18, 13-18, preferably 13-18, and is replaced or substituted with a medium containing 0.50-2.0% (v / v) glycerol. For example, a medium containing glucose is replaced or substituted with a medium containing 0.50-2.0% (v / v) glycerol when the optical density at 600 nm (OD) of the medium containing bacterial cells (or bacterial culture) is 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, 19-20, 13-18, 13-18, or preferably 13-18. 600 ) is 15, the medium is exchanged or substituted with medium containing 0.50 to 2.0% (v / v) glycerol.

[0091] In certain embodiments, for example when the medium in which the bacterial cells are cultured is supplemented with glucose, the cultivation of the bacterial cells is carried out at a temperature of about 30°C, and when the glucose-supplemented medium is replaced with a medium containing glycerol but not glucose (e.g., up to 0.010% (v / w), preferably up to 0.0010% (v / w) glucose), the temperature is increased from a temperature of about 30°C to a temperature of 36.0-38.0°C.

[0092] In a particular embodiment, the cultivation of the bacterial cells is carried out at a temperature of about 30° C. and the temperature is increased from a temperature of about 30° C. to a temperature of 36.0-38.0° C. at least 3 hours, at least 2 hours, at least 1.5 hours, at least 1 hour, at least 0.5 hours, preferably at least 2 hours, before adding one or more inducers of plasmid replication to the medium.

[0093] In certain embodiments, the temperature is increased from a temperature of about 30°C to a temperature of 36.0-38.0°C, for example, 36.0°C, 36.50°C, 37.0°C, 37.50°C or 38.0°C, preferably 37.0°C.

[0094] In certain embodiments, the temperature is adjusted to OD before one or more inducers of plasmid replication are added to the medium. 600 However, the temperature is increased from approximately 30°C to 36.0-38.0°C by 15-35, 20-35, 25-35, or 30-35.

[0095] The decrease in bacterial growth rate is associated with an increase in plasmid copy number. Therefore, as taught herein, inhibition of bacterial protein synthesis can further increase the yield of the method for the production of vectors of at least 16 kb in size.

[0096] Thus, in certain embodiments, the method further comprises adding one or more inhibitors of bacterial protein synthesis to the medium after the step of adding one or more inducers of an inducible origin of replication to the medium (step d) and before the optional step of separating the bacterial cells from the medium (step e) and before the step of recovering the plasmid from the bacterial cells (step f).

[0097] In certain embodiments, the method further comprises adding one or more inhibitors of bacterial protein synthesis to the medium during further culturing of the bacterial cells in the medium.

[0098] The skilled artisan will understand that prior to the step of adding one or more inducers of the inducible origin of replication to the medium (step d), no inhibitors of bacterial protein synthesis are added to the medium.

[0099] As used herein, the term "inhibitor of bacterial protein synthesis" or "bacterial protein synthesis inhibitor" refers to an agent that inhibits or slows down the growth or proliferation of bacterial cells by inhibiting the processes that directly lead to the production of a polypeptide or protein. Inhibitors of bacterial protein synthesis suppress the internal machinery of the bacteria, causing the bacteria to focus entirely on plasmid production. In addition, inhibitors of bacterial protein synthesis stabilize the bacteria's need for oxygen.

[0100] In certain embodiments, one or more inhibitors of bacterial protein synthesis are added to the culture medium at a concentration of 1-10 mM, 1-9 mM, 1-8 mM, 1-7 mM, 1-6 mM, 1-5 mM, 1-4 mM, 1-3 mM or 1-2 mM.

[0101] In certain embodiments, the one or more inhibitors of bacterial protein synthesis are agents that bind to bacterial ribosomes.Non-limiting examples of such inhibitors include chloramphenicol, spectinomycin, streptomycin, aminoglycosides, tetracyclines, macrolides, lincosamides and oxazolidinones.

[0102] In certain embodiments, the one or more inhibitors of bacterial protein synthesis are agents that inhibit the peptidyl transferase activity of bacterial ribosome.Preferably, the inhibitors of bacterial protein synthesis are agents that bind to the 50S or 30S ribosomal subunit of bacterial ribosome.Even more preferably, the inhibitors of bacterial protein synthesis are agents that bind to the 2451st and 2452nd amino acid residues of the 23S rRNA of the 50S subunit of bacterial ribosome.

[0103] In certain embodiments, the inhibitor of bacterial protein synthesis is an inhibitor of bacterial protein synthesis selected from the group consisting of chloramphenicol, spectinomycin, aminoglycosides, tetracyclines, macrolides, lincosamides and oxazolidinones, preferably chloramphenicol or spectinomycin, more preferably chloramphenicol.

[0104] In certain embodiments, one or more inhibitors of bacterial protein synthesis are added to the medium when the amount of dissolved oxygen in the medium decreases by more than 5%, more than 10%, more than 15%, more than 20% or more than 25%, preferably more than 5%.

[0105] In more specific embodiments, for example, the OD of a bacterial culture 600 increases by more than 10%, more than 15%, more than 20% or more than 25% after induction, one or more inhibitors of bacterial protein synthesis are added to the medium before the amount of dissolved oxygen in the medium is less than 40%, less than 35% or less than 30%, preferably before the amount of dissolved oxygen in the medium is less than 30%.

[0106] In more specific embodiments, for example, the OD of a bacterial culture 600 If increases by 1-10% per hour or 5-10% per hour after induction, one or more inhibitors of bacterial protein synthesis are added to the medium 2 hours before harvesting the bacterial cells.

[0107] In more specific embodiments, for example, the OD of a bacterial culture 600 If increases by 10-25% per hour after induction and the dissolved oxygen in the medium is greater than 35% or greater than 30%, one or more inhibitors of bacterial protein synthesis are added to the medium 2 hours before harvesting the bacterial cells.

[0108] In certain embodiments, the one or more inhibitors of bacterial protein synthesis are incubated with the bacterial cells for a period of at least 1 hour, at least 2 hours or at least 3 hours, preferably at least 1 hour, after the one or more inhibitors of bacterial protein synthesis are added to the medium.

[0109] In certain embodiments, the one or more inhibitors of bacterial protein synthesis are incubated with the bacterial cells for 1-5 hours, 1-4 hours, 1-3 hours, 2-3 hours or 1-2 hours, preferably 1-4 hours, after the one or more inhibitors of bacterial protein synthesis are added to the culture medium.

[0110] In certain embodiments, the one or more inhibitors of bacterial protein synthesis are added to the medium at least 1 hour after the addition of the one or more inducers of the inducible origin of replication.

[0111] Those skilled in the art will understand that in order to prevent the production of inferior quality vectors from the bacteria, it is necessary to harvest the bacterial cells before the RNA polymerase in the bacteria is depleted and / or the dissolved oxygen concentration reaches less than 40%, less than 35% or less than 30%, preferably less than 30%. Preferably, the pH of the medium is 6.5-7.5, preferably 6.9-7.1.

[0112] Thus, in certain embodiments, the bacterial cells are further cultured (in step e) for up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, preferably up to 6 hours, such as 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour. In certain embodiments, the bacterial cells are separated from the medium up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, preferably up to 6 hours, such as 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour after adding one or more inducers of an inducible origin of replication.

[0113] In certain embodiments, the bacterial cells are separated from the culture medium before the dissolved oxygen concentration in the medium reaches less than 40%, less than 35% or less than 30%, preferably less than 30%.

[0114] In certain embodiments, the bacterial cells are cultured at an optical density at 600 nm (OD 600 ) is preferably at the OD of the bacterial culture 600It is separated from the medium when the concentration of

[0115] The bacterial cells can be separated from the medium by any method known in the art for separating cells from the medium, for example, the medium containing the bacterial cells can be centrifuged and the pellet collected.

[0116] The vector can be recovered from the bacterial cells or bacterial environment by any method known in the art for recovering vectors from bacteria. For example, the bacterial cells are first lysed by using a lysis buffer (e.g., an alkaline-containing buffer) and / or sonication, followed by purification of the vector DNA by ethanol precipitation, spin-column-based nucleic acid purification, or phenol-chloroform extraction, etc. Since bacterial proteins can be toxic to mammalian cells, the purified vector DNA can be extensively extracted with phenol, chloroform, and ether to ensure that bacterial proteins are not present in the plasmid DNA preparation.

[0117] The presence of an inducible origin of replication in the vector allows for induction of transcription within the cell, thus allowing for a regulated switch from low to high copy number at any time during bacterial culture.

[0118] Inducible origins of replication and their compatible inducers are well known in the art.

[0119] The origin of replication is made inducible by operably linking an inducible promoter / repressor system to the origin of replication.

[0120] For example, the inducible origin of replication is the oriV / TrfA amplification system tightly regulated by the l-arabinose inducible parapromoter (araC-PBAD) that can be induced by the addition of l-arabinose, as described for example for the amplification of shuttle BACs in Wild J. et al., Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Res. 2002.12(9):1434-1444. In a further example, the inducible origin of replication is tightly regulated by the rhamnose inducible Prha promoter (rhaS-Prha) as described in Wild J. et al., Copy-control tightly regulated expression vectors based on pBAC / oriV. 2004.267:155-167.

[0121] In a preferred embodiment, the inducible origin of replication is, as described elsewhere herein: - an inducible promoter (i.e., an inducible promoter / repressor system); preferably, the inducible promoter comprises, consists essentially of, or consists of a lac promoter and at least one LacO1 operator, wherein the at least one LacO1 operator is operably linked to the lac promoter; and - a nucleic acid sequence encoding a truncated RNA II preprimer of a ColE1-type replication origin, operably linked to an inducible promoter; preferably a nucleic acid sequence encoding a truncated RNA II preprimer of a ColE1-type replication origin, operably linked to a Lac promoter and a LacO1 operator Comprise, consist essentially of, or consist of.

[0122] In certain embodiments, the methods taught herein allow for the regulation of the copy number of a vector in a bacterial cell.

[0123] In certain embodiments, the methods taught herein reduce the mutation rate and / or the number of undesired mutations in the resulting vector. Mutation rates in bacterial populations can be determined by methods known in the art, such as those described in Rosche et al., Determining mutation rates in bacterial populations, Methods, 2000, 20(1):4-17.

[0124] The present inventors have developed large vectors containing inducible ori that result in particularly high plasmid production when the vectors are produced by the methods taught herein. Furthermore, the present inventors have developed improved inducible ori that result in even higher plasmid production than large vectors containing wild-type inducible ori.

[0125] Thus, according to a further aspect there is provided a vector of at least 16 kb comprising an inducible origin of replication.

[0126] In certain embodiments, the inducible ori is - an inducible promoter (i.e., a promoter / repressor system); preferably, the inducible promoter comprises, consists essentially of, or consists of a lac promoter and at least one LacO1 operator, wherein the at least one LacO1 operator is operably linked to the lac promoter; and - a nucleic acid sequence encoding a ColE1-type ori truncated RNAII preprimer, said nucleic acid sequence being operably linked to an inducible promoter, preferably a LacO1 operator and a Lac promoter; Comprise, consist essentially of, or consist of.

[0127] "Operable linkage" refers to a linkage in which the regulatory sequence and the sequence desired to be transcribed or expressed are connected in such a way that their transcription or expression is possible. For example, sequences such as a promoter and an ORF can be said to be operably linked if the nature of the linkage between those sequences: (1) does not result in the introduction of a frameshift mutation, (2) does not inhibit the ability of the promoter to direct the transcription of the ORF, and (3) does not inhibit the ability of the ORF to be transcribed from the promoter sequence. Thus, "operably linked" means that an expression regulatory sequence such as a promoter is incorporated into a genetic construct so as to effectively regulate the transcription / expression of the sequence of interest.

[0128] As used herein, the term "wild-type RNAII pre-primer" generally refers to a nucleic acid sequence that includes a sequence encoding RNAII, an antisense RNAI promoter, and an antisense sequence encoding RNAI.

[0129] The term "truncated RNAII pre-primer" as used herein generally refers to the fact that there is no sequence encoding a functional RNAI, i.e., there is no sequence capable of interacting with RNAII and / or there is no sequence encoding a functional RNAI promoter operably linked to the sequence encoding RNAI. RNAI is a reverse transcription product of a fragment of RNAII. Typically, the deletion of the sequence encoding RNAI and / or the RNAI promoter means that the RNAII pre-primer sequence is shorter than the wild-type RNAII pre-primer sequence that includes the RNAI sequence and / or the RNAI promoter. However, as further described herein, it will be clear to those skilled in the art that the deletion of a portion of the sequence encoding RNAI and / or the mutation of said sequence will also result in a non-functional RNAI sequence. Similarly, it will be clear to those skilled in the art that the deletion of a portion of the RNAI promoter and / or the mutation of said sequence will also result in a non-functional RNAI promoter. For example, the truncated RNAII pre-primer may comprise, consist essentially of, or consist of the sequence shown in SEQ ID NO: 6 or 7.

[0130] Thus, in certain embodiments, the inducible origin of replication does not contain a nucleic acid sequence encoding a functional (i.e., capable of interacting with RNAII) RNAI of a ColE1-type ori, and preferably, the inducible origin of replication does not contain a nucleic acid sequence encoding a functional (i.e., capable of interacting with RNAII) RNAI of pMB1(ColE1) ori.

[0131] In a more specific embodiment, the inducible ori does not comprise the nucleic acid sequence set forth in SEQ ID NO:1.

[0132] In a particular embodiment, the inducible ori does not comprise a functional RNAI promoter of a ColE1-type ori, preferably, the inducible ori does not comprise a functional RNAI promoter of pMB1(ColE1) ori. In a more specific embodiment, the inducible ori does not comprise the nucleic acid sequence shown in SEQ ID NO:2.

[0133] In certain embodiments, the inducible ori and / or inducible promoter does not comprise the endogenous promoter of the RNAII pre-primer. In more specific embodiments, the inducible ori and / or inducible promoter does not comprise the endogenous promoter of the RNAII pre-primer of the pMB1 ori. In even more specific embodiments, the inducible ori and / or inducible promoter / repressor system does not comprise the nucleic acid sequence shown in SEQ ID NO:3.

[0134] In certain embodiments, the vector does not contain a rop gene or contains a rop gene encoding an inactive rop.

[0135] As used herein, the term "promoter" refers to a DNA sequence that allows transcription of a gene. Promoters are recognized by, and transcription is initiated by, RNA polymerase. Thus, promoters include DNA sequences that directly bind to, or are involved in the recruitment of, RNA polymerase. Promoter sequences may include "enhancer regions," which are one or more DNA regions that can bind proteins (i.e., trans-acting factors) to increase the transcription level of genes in a gene cluster. Enhancers are typically at the 5' end of the coding region, but may also be separate from the promoter sequence, e.g., within an intron region of a gene or 3' of the coding region of a gene. When referring to an "inducible promoter" herein, an inducible promoter / repressor system is intended to include a promoter region and an operator region (i.e., operator) to which one or more regulatory proteins (repressors and / or activators) can bind. Binding of one or more regulatory proteins to the operator region can increase or decrease the affinity of the promoter for RNA polymerase. When referring to a "promoter" in this specification, the promoter itself is intended, i.e., it is meant not to include the operator region.

[0136] In certain embodiments, the inducible promoter comprises a promoter selected from the group consisting of the promoter of RNAI, the lac promoter, the trp promoter, the trc promoter and the T7 promoter, preferably the lac promoter.

[0137] In certain embodiments, the lac promoter is a wild-type lac promoter or a modified lac promoter. In more specific embodiments, the lac promoter comprises the lacL8 promoter, also known as the lacUV5 promoter or the L8-UV5 lac promoter. The lacL8 promoter comprises two base pair mutations in the -10 hexamer region compared to the wild-type lac promoter. The lacL8 promoter has been previously described in Magasanik, The lactose operon, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1972.

[0138] In certain embodiments, the lac promoter comprises, consists essentially of, or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, preferably 100%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO:4.

[0139] One of skill in the art will appreciate that final transcription initiation is determined by the promoter present in the inducible ori.

[0140] In certain embodiments, the inducible promoter comprises at least one (e.g., one, two, or three) LacO1 operator. Replication of the vector can be more tightly regulated by using multiple LacO1 operators upstream of the nucleic acid sequence encoding the truncated RNAII preprimer.

[0141] In certain embodiments, the LacO1 operator comprises, consists essentially of, or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, preferably 100%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5.

[0142] In a specific embodiment, the nucleic acid sequence encoding a truncated RNAII preprimer for a ColE1-type origin of replication encodes a truncated RNAII preprimer for a pMB1(ColE1) origin of replication.

[0143] One skilled in the art will understand that since RNAI is a reverse transcription product for a fragment of RNAII, the nucleic acid sequence encoding the RNAII of the ColE1-type ori is truncated so as not to include the reverse transcription product encoding the RNAI of the ColE1-type ori. Furthermore, the nucleic acid sequence encoding the RNAII of the ColE1-type ori can be further truncated so as not to include the antisense RNAI promoter of the ColE1-type ori. The truncated RNAII preprimer abolishes or at least reduces the negative RNAI-RNAII duplexing feedback loop.

[0144] In certain embodiments, the nucleic acid sequence encoding the truncated RNAII pre-primer of the pMB1 (ColE1) origin of replication comprises, consists essentially of, or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, preferably 100%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6. Preferably, the nucleic acid sequence encoding the truncated RNAII pre-primer has a nucleic acid sequence comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 6.

[0145] The inventors found that the presence of two mutations in the truncated RNAII pre-primer increased vector production compared to the wild-type truncated pMB1 origin of replication.

[0146] In certain embodiments, the truncated RNAII pre-primer comprises a C to T point mutation in the nucleic acid at a position corresponding to nucleic acid 543 of the complete (i.e., before cleavage) RNAII pre-primer transcript, and / or an A to G point mutation in the nucleic acid at a position corresponding to nucleic acid 322 of the complete (i.e., before cleavage) RNAII pre-primer transcript. In certain embodiments, the truncated RNAII pre-primer comprises a C to T point mutation in the nucleic acid at a position corresponding to nucleic acid 543 of SEQ ID NO:10, and / or an A to G point mutation in the nucleic acid at a position corresponding to nucleic acid 322 of SEQ ID NO:10.

[0147] In certain embodiments, the truncated RNAII pre-primer comprises a CT point mutation at the 306th nucleic acid of the truncated RNAII transcript and / or an AG point mutation at the 85th nucleic acid of the truncated RNAII transcript.

[0148] In certain embodiments, the nucleic acid sequence encoding the truncated RNAII pre-primer comprises or consists of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, preferably 100%, sequence identity to SEQ ID NO: 7. Preferably, the nucleic acid sequence encoding the truncated RNAII pre-primer has a nucleic acid sequence comprising or consisting of the nucleic acid sequence shown in SEQ ID NO: 7. The inducible ori disclosed herein is (i) deleting the nucleic acid sequence encoding RNAI from pMB1 ori or mutating the nucleic acid sequence encoding RNAI in pMB1 ori to disable or significantly reduce the function of RNAI; (ii) deleting the RNAI promoter from the pMB1 ori or mutating the RNAI promoter in the pMB1 ori to abolish or greatly reduce the function of the RNAI promoter; (iii) optionally, replacing the RNAII promoter of pMB1 ori with a promoter that is not the endogenous promoter of the RNAII preprimer of pMB1 ori, preferably, the promoter that is not the endogenous promoter of the RNAII preprimer of pMB1 ori is a promoter selected from the group consisting of an RNAI promoter, a lac promoter, a trp promoter, a trc promoter and a T7 promoter, more preferably, the promoter that is not the endogenous promoter of the RNAII preprimer of pMB1 ori is a lac promoter; the promoter that is not the endogenous promoter of the RNAII preprimer of pMB1 ori is operably linked to a nucleic acid sequence encoding a truncated RNAII preprimer; and (iv) by introducing upstream (5') of the nucleic acid sequence encoding the truncated RNA II preprimer at least one (e.g., one, two, three or more) operator that is operably linked to the nucleic acid sequence encoding the truncated RNA II preprimer and that allows for conditional transcription of the truncated RNA II preprimer in combination with the endogenous RNA II promoter or any replacement promoter for the endogenous RNA II promoter; It can be derived from a plasmid containing the pMB1 (ColE1 type) ori, such as the pUC18 plasmid.

[0149] As described above, RNAI is a reverse transcription product of a fragment of RNAII, so deleting the nucleic acid sequence encoding RNAI from pMB1 ori results in a nucleic acid sequence encoding a truncated RNAII preprimer.

[0150] In more specific embodiments, the inducible origin of replication disclosed herein can be derived from the pMB1 ori by modifying the pMB1 ori as described in European Patent EP0179786B1, which is incorporated herein by reference, and Panayotatos et al., DNA replication regulated by the priming promoter, Nucleic Acids Research, 1984, Volume 12(8):2641-2648.

[0151] In certain embodiments, the inducible origin of replication is: - the RNAI promoter is deleted or mutated such that its function is abolished or greatly reduced; this results in a truncated RNAII preprimer; - a mutation is introduced into the nucleic acid sequence of the RNAI / RNAII overlap that reduces the affinity of the RNAI / RNAII interaction; preferably, the nucleic acid sequence encoding RNAI is deleted or mutated such that the function of RNAI is abolished or greatly reduced; this allows obtaining a truncated RNAII preprimer; - at least one (such as one, two, three or more) operator is introduced upstream (5') of the nucleic acid sequence encoding the truncated RNA II preprimer, the at least one operator being operably linked to the nucleic acid sequence encoding the truncated RNA II preprimer, the at least one operator allowing conditional transcription of the truncated RNA II preprimer in combination with the endogenous RNA II promoter or any replacement promoter of the endogenous RNA II promoter; and / or - optionally, the RNAII promoter is replaced with a promoter that is not an endogenous promoter of the RNAII preprimer, preferably, the RNAII promoter is replaced with a promoter selected from the group consisting of an RNAI promoter, a lac promoter, a trp promoter, a trc promoter or a T7 promoter; the promoter that is not an endogenous promoter of the RNAII preprimer is operably linked to a nucleic acid sequence encoding a truncated RNAII preprimer. pMB1 origin of replication.

[0152] Mutations in the region of the RNAI / RNAII overlap that reduce the affinity of the RNAI / RNAII interaction are known in the art and are described, for example, in Table 2 of Camps et al., Modulation of ColE1-like plasmid replication for recombinant gene expression. Recent Pat DNA Gene Seq. 2010, 4(1):58-73.

[0153] In a preferred embodiment, the inducible origin of replication is - the nucleic acid sequences encoding the RNAI gene and the RNAI promoter have been deleted; - at least one (such as one, two, three or more) LacO1 operator is introduced upstream (5') of the nucleic acid sequence encoding the RNAII preprimer, and the at least one LacO1 operator is operably linked to the nucleic acid sequence encoding the (truncated) RNAII preprimer; and - the RNAII promoter is replaced with a promoter that is not the endogenous promoter of the RNAII preprimer, preferably the RNAII promoter is replaced with a promoter selected from the group consisting of the RNAI promoter, the lac promoter, the trp promoter, the trc promoter and the T7 promoter; and the promoter that is not the endogenous promoter of the RNAII preprimer is operably linked to a nucleic acid sequence encoding a truncated RNAII preprimer. pMB1 origin of replication.

[0154] In a particular embodiment, the inducible origin of replication comprises, consists essentially of, or consists of the sequence of SEQ ID NO:8.

[0155] As described above, the present inventors have found that introduction of one or two mutations into the truncated pMB1 origin of replication increases vector production compared to the wild-type truncated pMB1 origin of replication.

[0156] Thus, in certain embodiments, the induced ori comprises an A to G mutation at nucleic acid position 176 of the amino acid sequence set forth in SEQ ID NO:8, and / or a C to T mutation at nucleic acid position 397 of the amino acid sequence set forth in SEQ ID NO:8.

[0157] In a preferred embodiment, the inducible origin of replication comprises, consists essentially of, or consists of a sequence as a set having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, preferably 100%, sequence identity to SEQ ID NO: 9. Those skilled in the art will appreciate that the sequence shown in SEQ ID NO: 9 may additionally include mutations at nucleic acid positions 176 and / or 397.

[0158] In certain embodiments, when the inducible origin of replication described herein comprises a LacO1 operator operably linked to a lac promoter, the inducer is isopropyl β-D-1-thiogalactopyranoside (IPTG) and / or α-D-lactose. IPTG removes the endogenous repressor LacI from the LacO1 operator, causing the inducible origin of replication to disable other origins of replication present in the plasmid.

[0159] Those skilled in the art will appreciate that the vectors taught herein of at least 16 kb are - repE for plasmid replication and copy number regulation, parA and parB to partition the F plasmid DNA to daughter cells during division and / or ensure stable maintenance of the vector; and / or - a selection marker such as an antibiotic resistance gene or an antibiotic-independent selection marker (e.g., the Ccda / ccdb toxin / antitoxin system (Staby® Operating System; Delphi Genetics) or a metabolically dependent selection marker); It will be understood that the vector may further include other factors required for vector replication, cell division and / or selection, including, but not limited to,

[0160] In certain embodiments, the empty backbone vector into which the inducible ori described herein is cloned is a pShuttle vector and / or a BAC vector, or a combination thereof, such as the pShuttle vector from Addgene, product number #16402.

[0161] The vectors of the present invention can be used in next generation vaccine platform technologies such as plasmid launched live attenuated virus (PPLAV) vaccines. Thus, in certain embodiments, the vector comprises a viral expression cassette comprising the cDNA of a live attenuated (flavi) virus vaccine. In more specific embodiments, the vector comprises a viral expression cassette comprising the cDNA of a live attenuated Yellow Fever Virus (YFV)-17D vaccine into which heterologous DNA sequences have been inserted and / or into which native viral sequences have been deleted.

[0162] According to further aspects, there is provided a use of the vectors taught herein for vaccination, for the production of large polypeptides or proteins, or for the production of viral vectors.The following examples are intended to illustrate the invention and should not be construed as limiting its scope. EXAMPLES

[0163] Example 1: Comparison of pMB1(ColE1)Ori and iOri In pMB1(ColE1)Ori (see SEQ ID NO: 10), RNAII DNA was transcribed from its cognate upstream promoter RNAIIp to give rise to RNAII transcripts. RNAII served as a primer to initiate DNA synthesis at the Ori+1 start of the plasmid DNA. Expression of RNAII was posttranscriptionally regulated by binding of a complementary antisense RNAI. RNAI was expressed from the RNAI gene, which resides in an inverted orientation within the RNAII gene, from its RNAIp promoter. Plasmid-encoded ROP protein stabilized the RNAI-RNAII duplex, which further restricted replication of the plasmid DNA (Figures 2-3).

[0164] In iOri, 5'-truncated RNA II * Expression of was driven from the heterologous LacUVp promoter. The RNAII sequence was truncated by deleting the entire RNAI gene, so that RNAI was not expressed. * Expression of the gene was determined using both a heterologous promoter and the original RNAII. * iOri is regulated at the transcriptional level by binding of the LACI protein to the LacO1 repressor binding sequence inserted between the LacO1 and RNAII DNA sequences. * served as a primer to initiate plasmid DNA synthesis. * contained two point mutations, C→T at nucleotide position 397 and A→G at nucleotide position 176 (see SEQ ID NO: 9) (FIGS. 2-3).

[0165] Example 2: RNAII of iOri for plasmid production * Effect of point mutations on BL21 bacteria were transformed with a plasmid having the structure shown in FIG. 1 (pViroVet-4-YFV17D with mutated iOri) or a plasmid having the structure shown in FIG. 1 in which the mutated iOri was replaced with a wild-type iOri (i.e., an iOri that does not contain the point mutations C→T at 397th nucleotide position and A→G at 176th nucleotide position, as shown in FIG. 2) (unmutated iOri).

[0166] Two different bacterial clones (pVV) transformed with pViroVet-4-YFV17D containing the mutated iOri were grown in phosphate-buffered LB medium. * and pVV4), and one bacterial clone transformed with pViroVet-4-YFV17D containing the unmutated iOri (pVV-wt iOri)) were inoculated and incubated overnight in a shaker flask.

[0167] The next morning, the OD of the bacterial culture was 600 Measure the OD of the bacteria in fresh medium supplemented with 1% glycerol in the fermenter. 600 The cultures were diluted to a pH of 2. The bacteria were grown at 37°C for 2 hours and then induced with IPTG. Two hours after induction, chloramphenicol was added to a) repress the internal machinery of the bacteria and focus entirely on plasmid production, and b) stabilize the oxygen requirements of the bacteria. Two hours after the addition of chloramphenicol, the cells were harvested and the yield was measured.

[0168] Figure 5 shows the bacterial clone pVV4 * or induction of pVV4 (I) resulted in higher plasmid production compared to induction of the bacterial clone pVV-wt iOri. Thus, the two mutations, the point mutation C→T at 397th nucleotide position of iOri and the point mutation A→G at 176th nucleotide position of iOri, have beneficial effects on plasmid production.

[0169] Example 3: Effect of the level of biomass at the time of induction of replication on the yield of vectors taught herein Transform BL21 bacteria with a plasmid having the structure shown in FIG. 1 (pViroVet-4-YFV17D) and containing iOri, e.g. https: / / international.neb.com / Protocols / 0001 / 01 / 01 / transformation-protocol-for-bl21-de3-competent-cells-c2527 Transformation Protocol for BL21(DE3) competent cells from New England BioLabs Inc. and the Addgene website. https: / / www.addgene.org / protocols / create-glycerol-stock / Glycerol stocks were prepared using state-of-the-art techniques as described.

[0170] Table 1 shows the effect of the level of biomass at the time of induction of replication on the yield of the plasmid of the invention.

[0171] [Table 1] Abbreviation: ON: Overnight

[0172] LB medium was inoculated with a glycerol stock of bacteria transformed with pViroVet-4-YFV17D (Figure 1) and incubated overnight in a shaker flask. The following morning, OD 600 Measure the biomass of the bacteria, reflected in the OD value at the start 600 The OD of one culture was 0.1, 0.4, and 2. 600 The cells were seeded when the OD reached 0.2 and incubated overnight to obtain the OD at induction. 600 The densities were allowed to reach 10. After bacterial densities reached 1, 2, 3.78 and 10, the cultures were supplemented with 1 mM IPTG to induce plasmid replication. Two hours after induction, cells were harvested to determine the yield of plasmid production (expressed as mg / l). More specifically, alkali was added to lyse the cells, after which the mixture was neutralized and cell debris was removed by tangential flow filtration. The plasmids were then isolated and purified by ion exchange chromatography, ultrafiltration and tangential flow filtration (for possible buffer exchange), followed by a final purification column.

[0173] The temperature was constantly maintained at 37°C.

[0174] The yield increased with increasing biomass upon induction, including fermenter runs that achieved much higher biomass compared to the limits of shaker flasks (see Table 1). It was concluded that the higher the bacterial biomass, the higher the yield of plasmid production.

[0175] Example 4: Induction of a vector containing iOri as taught herein with both IPTG and α-D-lactose The medium was inoculated with a glycerol stock of bacteria transformed with pViroVet-4-YFV17D (Figure 1) and incubated overnight in a shaker flask. The following morning, OD 600 Measure the OD of the bacteria 600 The cultures were diluted to an A = 2 and grown for a further 2 hours. One cell culture was then grown in the absence of plasmid inducer (NI = not induced) while the other culture was induced with 1 mM IPTG or increasing amounts of α-D-lactose (i.e. 0.5%, 1%, 2% and 5%). All cultures were harvested after 4 hours and DNA was isolated, digested with restriction enzymes and applied to a gel for visualization. The temperature was always kept at 37°C.

[0176] All induced cultures showed the presence of the plasmid, with no obvious difference between IPTG and various concentrations of α-D-lactose (Figure 4). It was concluded that the plasmid could be induced by both IPTG and α-D-lactose.

[0177] Example 5. Enhancement of bacterial growth by addition of glycerol or glucose to the medium The medium was inoculated with a glycerol stock of bacteria transformed with pViroVet-4-YFV17D (Figure 1) and incubated overnight in a shaker flask. The following morning, OD 600 Measure the OD of the bacteria 600 The cells were diluted with fresh medium supplemented with or without 1% glycerol, 1% glucose, or both, to an OD of 0.1. Cells were grown for 8 h and then incubated at 37 °C for 1 h at 4 °C for 1 h. 600 was measured every hour. The cells were not induced. The temperature was constantly kept at 37°C.

[0178] The addition of glycerol or glucose increased bacterial growth by 2- to 2.5-fold (Figure 6).

[0179] Example 6. Benefits of plasmid production by addition of chloramphenicol, an inhibitor of bacterial protein synthesis The medium was inoculated with a glycerol stock of bacteria transformed with pViroVet-4-YFV17D (Figure 1) and incubated overnight in a shaker flask at 37 °C. The following morning, OD 600 Measure the OD of the bacteria in fresh medium supplemented with glucose in the fermenter. 600 The mixture was diluted to a concentration of 0.1 and incubated overnight at 37° C. (FIG. 7, procedure (a): seeding). In the morning, the feed was supplemented with glycerol (FIG. 7, procedure (b)).

[0180] The bacteria were grown under these conditions for approximately 1.5 hours and then induced with IPTG (Figure 7, procedure (c)). Approximately 2 hours after induction, chloramphenicol was added to i) inhibit the internal machinery of the bacteria and focus entirely on plasmid production, and ii) stabilize the oxygen requirement of the bacteria (Figure 7, procedure (d)). Approximately 4 hours after the addition of chloramphenicol, the cells were harvested (Figure 7, procedure (e)) and the yield was measured.

[0181] Soon after adding chloramphenicol, the bacterial growth reached a plateau, suggesting the cessation of the internal bacterial machinery (Figure 7). At the same time, the yield and DNA content increased exponentially, suggesting that the bacteria were mainly focused on plasmid production (Figure 7). This led us to conclude that adding chloramphenicol was beneficial for plasmid production.

[0182] Example 7. Benefits of switching from 30° C. to 37° C. prior to induction of an inducible origin of replication to a manufacturing process The medium was inoculated with a glycerol stock of bacteria transformed with pViroVet-4-YFV17D (Figure 1) and incubated overnight in a shaker flask at 37 °C. The following morning, OD 600 Measure the OD of the bacteria in fresh medium supplemented with glucose in the fermenter. 600 The mixture was diluted to a concentration of 0.1 and incubated at 30° C. overnight (FIG. 8, procedure (a): seeding and procedure (b)).

[0183] In the morning, the feed was supplemented with glycerol instead of glucose to avoid inhibiting induction, and the temperature was switched from 30°C to 37°C to create an optimal environment for plasmid production (Figure 8, step (c)).

[0184] The bacteria were grown under these conditions for approximately 1.5 hours and then induced with IPTG (Figure 5, procedure (d)). Approximately 2 hours after induction, chloramphenicol was added (Figure 5, procedure (e)), and approximately 4 hours later, the cells were harvested (Figure 5, procedure (f)) and the yield was measured.

[0185] The temperature switch resulted in higher yields compared to the run shown in Figure 7 (26 mg / l vs. 45 mg / l), leading to the conclusion that switching from 30°C to 37°C was beneficial to the production process (Figure 8).

Claims

1. 1. A method for producing a vector of at least 16 kb in size from a bacterial cell, comprising the steps of: a) obtaining a bacterial cell containing a vector of at least 16 kb in size, comprising an inducible origin of replication; b) inoculating a culture medium with bacterial cells containing the vector; c) culturing said bacterial cells in said medium to obtain a bacterial culture; d) Optical density at 600 nm (OD) of the bacterial culture ６００ adding one or more inducers of said inducible origin of replication to the medium when the number of the inducible origins of replication reaches at least 20; e) further culturing said bacterial cells in said medium; and g) recovering vectors of at least 16 kb in size from said bacterial cells. A method comprising:

2. The method described in claim 1, further comprising step f) of separating the bacterial cells from the culture medium, said step f) being performed between step e) and step g).

3. 3. The method of claim 2, further comprising adding an inhibitor of bacterial protein synthesis to the medium after step d) and before step f).

4. The method according to any one of claims 1 to 3, wherein glucose and / or yeast extract are added to the medium during culturing the bacterial cells in the medium.

5. 5. The method of claim 4, wherein the glucose and / or yeast extract containing medium is replaced with a medium containing 0.50 to 2.0% (v / v) glycerol at least 30 minutes prior to adding one or more inducers of the inducible origin of replication to the medium.

6. 6. The method according to any one of claims 1 to 5, wherein said step c) of culturing said bacterial cells in said medium is carried out at a temperature of 30°C, and said temperature is increased from 30°C to 36.0-38.0°C at least 2 hours before adding one or more inducers of said inducible origin of replication to the medium.

7. 7. The method of claim 1, wherein the inducible origin of replication comprises at least one LacO1 operator and the inducer of the inducible origin of replication is isopropyl β-D-1-thiogalactopyranoside (IPTG) and / or α-D-lactose.

8. 4. The method of claim 3, wherein the inhibitor of bacterial protein synthesis is chloramphenicol or spectinomycin.

9. 4. The method of claim 3, wherein the inhibitor of bacterial protein synthesis is added to the culture medium at least 1 hour after the addition of the one or more inducers of the inducible origin of replication.

10. The method according to any one of claims 3 to 9, wherein the bacterial cells are separated from the culture medium up to 6 hours after addition of one or more inducers of the inducible origin of replication.

11. A vector of at least 16 kb in size comprising an inducible origin of replication, the inducible origin of replication comprising: - the RNAI promoter is deleted or mutated so that its function is abolished or reduced, forming a truncated RNAII pre-primer, and - the nucleic acid sequence encoding RNAI is deleted or mutated so that the function of RNAI is abolished or reduced, forming a truncated RNAII pre-primer; and - a vector comprising a pMB1 origin of replication, wherein at least one LacO1 operator is introduced upstream of a nucleic acid sequence encoding said truncated RNAII preprimer, said at least one LacO1 operator being operably linked to a nucleic acid sequence encoding said truncated RNAII preprimer.

12. The vector of claim 11, wherein the endogenous RNAII promoter of the pMB1 origin of replication is replaced by a promoter that is not the endogenous promoter of the RNAII preprimer of the pMB1 origin of replication, and the promoter that is not the endogenous promoter of the RNAII preprimer of the pMB1 origin of replication is operably linked to at least one LacO1 operator.

13. 13. The vector of claim 12, wherein the endogenous RNAII promoter of the pMB1 origin of replication is replaced by a promoter selected from the group consisting of an RNAI promoter, a lac promoter, a trp promoter, a trc promoter and a T7 promoter.

14. The vector of claim 13, wherein the endogenous RNAII promoter of the pMB1 origin of replication is replaced by the L8 / UV5 lac promoter.

15. - the RNAI promoter is deleted or mutated so that its function is abolished or reduced, forming a truncated RNAII pre-primer, and - the nucleic acid sequence encoding RNAI is deleted or mutated so that the function of RNAI is abolished or reduced, forming a truncated RNAII pre-primer; and - at least one LacO1 operator is introduced upstream of the nucleic acid sequence encoding said truncated RNAII pre-primer, said at least one LacO1 operator being operably linked to said nucleic acid sequence encoding said truncated RNAII pre-primer; and - the nucleic acid corresponding to the nucleic acid at position 322 of the wild-type pMB1 origin of replication shown in SEQ ID NO: 10 is mutated from A to G and / or the nucleic acid corresponding to the nucleic acid at position 543 of the wild-type pMB1 origin of replication shown in SEQ ID NO: 10 is mutated from C to T Inducible origins of replication, including the pMB1 origin of replication.

16. The vector according to any one of claims 11 to 14, or the inducible origin of replication according to claim 15, wherein the inducible origin of replication comprises a nucleic acid sequence having at least 95% sequence identity with the nucleic acid sequence shown in SEQ ID NO:

9.

17. The method according to any one of claims 1 to 10, wherein the vector of at least 16 kb in size is a vector according to any one of claims 11 to 14 or claim 16.

Images