Plasmid and method for production of DNA vaccines and DNA products

Abstract

A method for producing replicating DNA molecules is described, producing DNA products, such as vaccines or biotherapeutics. A parental DNA template comprises a sequence of interest, and a modified Col E1 origin lacking RNA I inhibition, having an inducible switch for recombinase translation control. The method permits control of plasmid DNA production during host fermentation by controlling relocation of a plasmid RNA II promoter to upstream of the RNA II primer template sequence through recombination, where RNA II initiates plasmid replication. The bacterial origin of a linearized plasmid is stably inserted into the host chromosome by separating the origin's promoter from the RNA II primer template sequence, allowing antibiotic-free fermentation while the plasmid is hidden within the chromosome. The excision of the targeted plasmid DNA is induced to control recombination, forming a plasmid with a functional bacterial origin lacking negative regulation, driving run-away amplification and high plasmid DNA production.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[001] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 556,628 filed February 22, 2024 entitled PLASMID AND METHOD FOR PRODUCTION OF DNA VACCINES AND DNA PRODUCTS, the entirety of which is hereby incorporated by reference. FIELD

[002] The present disclosure relates generally to plasmids and the production thereof, as may be used in the preparation of DNA vaccines or other DNA products. BACKGROUND

[003] Antimicrobial Resistance in Plasmids and Size Considerations. First generation plasmid production methods utilize an antimicrobial resistance cassette to maintain plasmid populations in the fermentation host bacteria. The antimicrobial resistance cassette is typically contained in the product plasmid, which is perceived to pose a risk of spreading antimicrobial resistance to bacteria in the environment / microbiota through horizontal gene transfer. Encoding for antimicrobial resistance requires a significant amount of nucleotide sequence on the product plasmid. Larger plasmids with extraneous bacterial sequences are known to have reduced transfection and translation efficiency. However, bacterial sequences of smaller than 1,000 bp may be below the known threshold of inhibition. Antimicrobial agents used in fermentation can significantly increase the cost of manufacturing a product plasmid. Antimicrobial-free fermentation is an active focus of plasmid research and development. Existing techniques may include tRNA deletion and substitution, RNA interference to prevent the translation of a toxin / enzyme producing toxin, and the utilization of recombination to generate minicircular DNA. This plasmid and platform for its preparation and use bypasses the need for antimicrobial resistance in the product plasmid.

[004] Col E1 Origin. The Col E1 plasmid origin does not require an associated protein to initiate DNA replication. The Col E1 origin utilizes a forward promoter to generate an RNA primer (RNA II) from the RNA II promoter to create an advanced RNA secondary structure that is recognized and digested by RNase H. This generates a free 3’ hydroxyl group which in turn acts as the RNA primer for DNA polymerase to initiate DNA replication of the plasmid.

[005] The advanced RNA structure created by RNA II can be de-stabilized by the production of an interfering RNA molecule from the opposite strand of DNA, called RNA I. RNA I promotes an alternative RNA structure in RNA II, which prevents its recognition by RNase H, thereby preventing plasmid replication from the origin. However, without the generation of RNA I, the plasmid will amplify uncontrollably, creating a parasitic DNA molecule in the host fermentation bacteria that can quickly become cytotoxic.

[006] Cre LoxP Recombination System. The Cre Lox system is an effective recombinase pathway that has been used to generate knockouts and to design genetic circuits. Cre is the recombinase enzyme that recognizes two LoxP sites and excises or integrates surrounding DNA sequences based on that specific site sequence. Wildtype LoxP (LoxP-Wt) sequences are the template to both repeatable excision and integration through the Cre recombinase, while the Lox66 and Lox71 site sequences generate a Lox72 site that is no longer recognizable by Cre. LoxP-Wt is 34 bp long and contains a 13 base palindrome that does not have directionality.

[007] Phi-BT1 Integrase and Att Recombination System. The phage Phi-BT 1 encodes a site-specific recombinase (Integrase-1 nt), and an integrase cofactor that controls directionality of recombination (GP3) and contains an attachment site on the phage genome (AttP). Int creates site-specific recombination between the phage and a specific attachment site in the bacterial genome (AttB). Both the AttP and AttB sites contain unique sequences that allow easy identification and identification by the phage specific recombinase. Once the phage is integrated into its host chromosome, the AttP and AttB sites recombine to generate the attachment left (AttL) and attachment right (AttR) sequences. The AttR and AttL sites are also recognized by Int, which circularizes all genetic material in between the two to regenerate the AttP and AttB sites. GP3 and Int control the excision, circularization, and covalently closing of circular DNA to generate the phage.

[008] Synthetic RNA thermometers. An RNA thermometer can be used to control translation on the basis of temperature. Translational control on a sequence of interest, such as a gene of interest, has been shown by the translation of the PrfA transcription factor from Listeria monocytogenes. PrfA translation is controlled by a secondary RNA structure in the 5’ UTR of the transcript. At temperatures below 30°C, the RNA structure forms a tight hairpin structure that closes off the ribosomal binding site (RBS, also known as the Shine Dalgarno (SD) sequence). At 37°C, the base pairing in the hairpin structure partially melts, exposing the RBS site for the host ribosome to load onto the transcript and translate the virulence factor. The PrfA 5’ UTR has been isolated from the bacteria and has been used as a thermoswitch to control the translation of downstream genes. This work has continued in the creation of synthetic RNA thermometers that contain minimal sequences required to control translation.

[009] Lambda Red Recombination. Lambda red recombination involves the controlled expression of a cassette of three genes from the lambda phage to facilitate the recombination between a linear DNA template and the host chromosome. The expression of the Lambda red cassette is controlled by the arabinose inducible promoter pBAD, which activates the Lambda red recombinase cassette after the addition of L-arabinose to the media. Lambda red recombination requires nucleotide homology of at least 35bp at the very 5’ and 3’ ends of its target sequence.

[0010] Flagella genes in Fermentation Bacteria. The flagella of E coli. is important to the bacteria in natural settings but is dispensable in fermentation settings. Flagella are a significant metabolic burden on the bacteria to produce and maintain. Disrupting the production of the flagella results in an improved growth prospect for fermentation bacteria, potentially correlating to an increase in plasmid production.

[0011] Previous Systems for Plasmid Production. Systems are known for production of plasmids, with a focus on reducing antibiotic resistance use and increasing production yields. Recombinases have been used to make mini circular DNA, for example by using temperature-induction of recombinase. The Cre lox system and the Phi-BT1-lnt recombinase and AttR / AttL system have been described previously. Reference is made to U.S. Patent No. 9,862,954, Patent Publication US 2008 / 0057545 A1, PCT Publication WO2021 / 016075 A1. Mutations in stem loops of the Col E1 origin have been described, see for example JP2014012023A. Recombinase may be used to remove bacterial sequences, such as described in US Patent No. 9,233,174. Technologies aimed at bypassing the use of antibiotic resistance with an RNA selective marker are known, such as in US Patent No. 11,098,313. The origin may be modified to generate high levels of plasmid by using the R6K origin, such as described in Patent Publication US 2021 / 0010021 A1. Reference is made to Patent Publication US 2013 / 0266983 A1, pertaining to super-stable ColE1 plasmids prepared by duplication of SL1-4 sequence and point mutations.

[0012] There remains a need and desire for alternatives to the previous technologies to obviate or mediate one or more shortcomings of previous technologies for an aggressively replicating DNA molecule without the use of antimicrobial resistance markers and active plasmid selection. SUMMARY

[0013] A method is provided herein for production of a product DNA plasmid comprising a sequence of interest.

[0014] There is described herein a method for production of a product DNA plasmid comprising a sequence of interest, said method comprising:

[0015] preparing a parental DNA template comprising a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a RNA II primer template sequence; a recombinase coding sequence encoding an associated recombinase that is associated with the first and the second recombinase recognition sites; an inducible switch for control of recombinase translation; and the sequence of interest;

[0016] wherein the sequence of interest and the modified Col E1 origin comprise or are flanked by Lox or Att sites, which are recognized by the associated recombinase for excision to reconstitute the RNA II promoter upstream of the RNA II primer template sequence, thereby producing the RNA II primer, and to thereby form a self-replicating product DNA plasmid; wherein translation of the recombinase is controlled by the inducible switch under a control condition; and

[0017] wherein the modified Col E1 origin comprises Col E1 RNA II and Col E1 RNA I promoters, and the modified Col E1 is modified by at least one mutation or deletion in the Col E1 RNA I promoter to reduce or eliminate RNA I inhibition to increase replication of the origin post-recombination;

[0018] preparing a host bacterium having the parental DNA template inserted into the chromosome of said host organism;

[0019] fermenting the host organism under the control condition that induces the inducible switch to express the recombinase, to excise the product plasmid from the host organism;

[0020] producing the product DNA plasmid in the host organism; and

[0021] producing the product DNA plasmid comprising the sequence of interest and the modified origin, that is excised from the chromosome of the host organism.

[0022] Further, there is described herein a parental DNA template for integration into a host organism for production of a sequence of interest, said plasmid comprising:

[0023] the sequence of interest,

[0024] a modified Col E1 origin comprising a first recombinase recognition site, wherein said origin is modified to reduce or eliminate RNA I inhibition;

[0025] a second recombinase recognition site that is distal to the modified Col E1 origin;

[0026] a RNA II promoter upstream of the second recombinase recognition site;

[0027] a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites;

[0028] Lox or Att sites within or flanking the modified Col E1 origin and the sequence of interest for recognition by the recombinase; and

[0029] an inducible switch for control of recombinase translation under a controllable condition.

[0030] The parental DNA template prepared in accordance with the method described herein is also encompassed, as is the product DNA plasmid so produced.

[0031] The product DNA plasmid comprises a sequence of interest and a modified Col E1 origin for activation at a targeted time during fermentation within a host cell. The method comprises: preparing a parental DNA template comprising a sequence of interest; a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites; an inducible promoter thermoswitch for temperature-induced control of recombinase translation, and the sequence of interest; wherein the sequence of interest and the modified Col E1 origin comprise or are flanked by LoxP or AttL / AttR sites, which are recognized by the associated recombinase for excision to reconstitute and activate the RNA II promoter and thereby form a self-replicating product plasmid; wherein translation of the recombinase is controlled by the thermoswitch; and wherein the modified Col E1 origin optionally comprises at least one mutation or deletion in a RNA I promoter to reduce or eliminate RNA I inhibition to increase replication of the origin post recombination; preparing a host bacterium having the parental DNA template inserted into the chromosome of said host bacterium; fermenting the host bacterium at a temperature that induces the thermoswitch to excise the product plasmid from the host bacterium; producing the product DNA plasmid in the host bacterium; and producing the product DNA plasmid that is excised from the chromosome of the host bacterium; wherein the modified Col E1 origin encompasses a Col E1 modified as: pMB1, pBR322, or pUC origin, preferably a modified Col E1 origin wherein the RNA II and RNA I promoters comprise Col E1 RNA II and Col E1 RNA I promoters.

[0032] There is also provided herein a parental DNA template with sequence specific homology for permitting recombination to a host bacteria genome. The plasmid comprises: a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites; an inducible promoter for control of recombinase translation; and a sequence of interest.

[0033] Further, there is provided herein a plasmid comprising: a sequence of interest; a modified Col E1 origin; a recombinase coding sequence; a thermoswitch for temperature-induced control of translation; wherein the sequence of interest and the modified Col E1 origin contain or are flanked by LoxP or AttL / AttR sites, which are recognized by the recombinase of the recombinase encoding sequence, for excision to complete the origin; wherein translation of the recombinase is controlled by the thermoswitch; and wherein the modified Col E1 origin comprises at least one mutation or deletion in the Col E1 RNA I promoter to reduce or eliminate RNA I inhibition and increase replication of the origin.

[0034] A product DNA plasmid produced by the described method is provided, and a vaccine produced with the DNA from the product plasmid is provided.

[0035] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[0036] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached figures.

[0037] Figure 1 illustrates a map of the parental DNA template, which is a circular plasmid.

[0038] Figure 2 is an annotated diagram of a plasmid landing pad.

[0039] Figure 3 is a plasmid map highlighting the LoxP region where recombination will occur.

[0040] Figure 4 illustrates a product plasmid generated through Cre-LoxP recombination.

[0041] Figure 5 provides an illustration of RNA II and the added modifications (SEQ ID NO:8, and SEQ ID NO:17 including the 5 indicated modifications).

[0042] Figure 6A shows length analysis of the subject plasmid.

[0043] Figure 6B shows the agarose gel electrophoresis of the subject plasmid.

[0044] Figure 7A illustrates the agarose gel electrophoresis of plasmid recovered from recombinant cells.

[0045] Figure 7B illustrates the length of plasmid recovered from recombinant cells.

[0046] Figure 7C illustrates the abundance analysis of plasmid recovered from recombinant cells.

[0047] Figure 8A shows agarose gel electrophoresis of progeny plasmid from recombinant bacteria.

[0048] Figure 8B shows production of progeny plasmid from recombinant bacteria.

[0049] Figure 9A illustrates the integration of landing pad (LP), and the SacB negative selection cassette (1.1), specifically: LP, 1.1, and [LP+1.1],

[0050] Figure 9B shows the integration of [LP+1.1], PrfA, and [[LP+1.1] + PrfA],

[0051] Figure 9C shows the integration of [[LP+1.1]+PrfA], BPE, and [[[LP+11 ,]+PrfA] + BPE], the PrfA expression cassette (PrfA), and the final BPE construct that to be excised into the progeny plasmid.

[0052] Figure 10 shows a plasmid map of progeny plasmid from BPE linear.

[0053] Figure 11A illustrates a U12 (published) secondary RNA structure (SEQ ID NO:9).

[0054] Figure 11B illustrates the U12-Anti AUG secondary RNA structure of the design of the present invention, including the Shine-Dalgarno region and the Start codon (SEQ ID NO:10).

[0055] Figure 12 illustrates LoxP Wt and Lox recombination site variants to better stabilize the product plasmid (SEQ ID NO:11-SEQ ID NO:14).

[0056] Figure 13A provides a plasmid map according to Example 6.

[0057] Figure 13B illustrates a detailed view of the plasmid landing pad between insertion sites according to Example 6.

[0058] Figure 13C provides a linear map of the modified landing pad and inserted parental plasmid into the chromosome of the target bacteria in accordance with Example 6. DETAILED DESCRIPTION

[0059] A method to control the production of plasmid DNA during bacterial / host cell fermentation is described herein. The method involves controlling the relocation of a plasmid RNA II promoter to upstream of the RNA II primer template sequence through recombination, whereby the RNA II primer is responsible for initiating plasmid replication. The method accomplishes stable insertion of the bacterial origin of a linearized plasmid into the host bacterial chromosome by separating the origin’s promoter from the RNA II primer template sequence, allowing the option of antibiotic-free fermentation of the host while the plasmid is hidden within the host chromosome. At a desired time, the excision of the targeted plasmid DNA is triggered through controlled recombination, resulting in the formation of a plasmid with a fully functional bacterial origin that is lacking (or reduced in) negative regulation, thereby driving run-away amplification and significant plasmid DNA production for such uses as for biotherapeutic use.

[0060] The generation and use of antimicrobial-free fermentation of a circular, covalently linked replicating DNA molecule is described herein. The covalently linked circular DNA molecule comprises a modified Col E1 origin, and other replicative motifs. A modified prophage is artificially generated in a host cell’s bacterial chromosome that contains the sequence of interest, for example within an expression cassette, and modified Col E1 origin. The cassette and modified Col E1 origin are flanked by LoxP, AttL / AttR, or any other recombination sites, which are recognized by an associated recombinase to enable excision out of the chromosome to thereby create a replicating DNA molecule suitable for use as a production grade plasmid. The translation of the recombinase required for the excision may be controlled by a trans-acting 5’ UTR synthetic RNA thermoswitch or controlled by an inducible promoter, such as the lambda promoter under the regulated control of the heat liable cl857 repressor.

[0061] A method is described for production of a product DNA plasmid comprising a sequence of interest, said method comprising: preparing a parental DNA template comprising a sequence of interest; a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites; an inducible promoter thermoswitch for temperature-induced control of recombinase translation, and the sequence of interest. The sequence of interest (which may be within an expression cassette) and the modified Col E1 origin comprise or are flanked by LoxP or AttL / AttR sites, which are recognized by the associated recombinase for excision to reconstitute and activate the RNA II promoter and thereby form a self-replicating product plasmid; wherein translation of the recombinase is controlled by the thermoswitch. The wherein the modified Col E1 origin optionally comprises at least one mutation or deletion in a RNA I promoter to reduce or eliminate RNA I inhibition to increase replication of the origin post recombination. The method further comprises preparing a host bacterium having the parental DNA template inserted into the chromosome of said host bacterium; fermenting the host bacterium at a temperature that induces the thermoswitch to excise the product plasmid from the host bacterium; producing the product DNA plasmid in the host bacterium, and producing the product DNA plasmid that is excised from the chromosome of the host bacterium. The modified Col E1 origin comprises a modified Col E1, pMB1, pBR322, or pUC origin, preferably a modified Col E1 origin in which case the RNA II and RNA I promoters comprise Col E1 RNA II and Col E1 RNA I promoters. Such origins are considered to be Col E1-like, and in this context the term “modified Col E1 origin” is used as an umbrella term to encompass pUC19, pBR322, and other RNA dependent origins, which may also be referred to as “Col E1 like”. Col E1 is believed to be an adequate term to cover all other RNA l / RNA II based origins. Another term adequately representative of appropriately modified origins encompassed by the term “modified Col E1 origin” so as to be equivalent thereto is: “RNA dependent-protein free bacterial origin”, as supported by del Solar et al., (1998).

[0062] The parental DNA template may comprise a recombinase cassette containing sequence specific homology to enable targeted recombination into the bacterial genome or a phage attachment site. A serine / tyrosine recombinase may be used to integrate the parental DNA template into the chromosome. The sequence of interest and modified Col E1 origin may be flanked by a recombinase recognition site which can excise the product plasmid, post recombinase expression. The product plasmid resulting from the method comprises at least the modified Col E1 origin and sequence of interest.

[0063] The parental DNA template may comprise a recombinase cassette, and sequence specific homology to enable recombination to the bacterial genome; wherein the sequence of interest is flanked by a site specific to the recombinase, to excise the product plasmid, said product plasmid comprising the modified Col E1 origin and the sequence of interest. The parental DNA template may be, for example, a circular DNA template (a plasmid), or may be a linear DNA template.

[0064] The recombinase may be any suitable recombinase, such as the Cre recombinase for Lox-Lox excision. Further, the recombinase may be a Phi-BT1 Integrase (INT) for AttL / AttR excision.

[0065] The plasmid may be circular.

[0066] The product plasmid can be advantageously produced in an antimicrobial free environment.

[0067] Thermoswitch may be, for example, a trans-acting 5’ UTR synthetic RNA thermoswitch or a naturally derived thermoswitch. The thermoswitch may be a PrfA RNA thermoswitch. Further, the thermoswitch may comprise synthetic RNA hairpins in the 5’ untranslated region (5’ UTR) of the cassette, and optionally, the production of recombinase mRNA may be under a weak promoter. The temperature that induces the thermoswitch may, for example, be a temperature that is greater than 30 °C.

[0068] The modified Col E1 origin may comprise a recombinase recombination site; a forward promoter sequence to generate the RNA II primer (RNA II, such as Col E1 RNA II); and a sequence-specific stretch of DNA to generate stable hairpin structures to initiate replication of the excised plasmid, whereby an RNA-DNA hybrid structure is formed that initiates DNA synthesis after RNase H digestion. Further, the Col E1 promoter may be located distal to the RNA II and the sequence-specific stretch of DNA prior to excision from the host chromosome, such that after excise of the product plasmid, the Col E1 promoter comprises the aforementioned at least one mutation or deletion, and is relocated upstream of the sequence-specific stretch of DNA, to initiate DNA replication of the production plasmid.

[0069] The at least one mutation or deletion of the RNA I promoter may be present and could produce significant yields of product DNA plasmid at a targeted time during fermentation.

[0070] The origin may further comprise a primosomal assembly site (PAS) for assisting in replication of the product DNA plasmid, such as Col E1 PAS.

[0071] The sequence of interest is not limited and may be any desired gene, for example: one that encodes an immunogen, a monoclonal antibody, a prokaryotic gene expression cassette, a supplementary eukaryotic gene cassette, a DNA template for mRNA synthesis, or an immune modulating molecule to generate or suppress an immune response.

[0072] The method may involve harvesting from the host bacterium the plasmid DNA using alkaline lysis.

[0073] A parental DNA template is described herein, with sequence specific homology for permitting recombination to a host bacteria genome The parental DNA template comprises: a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites; an inducible promoter for control of recombinase translation; and a sequence of interest.

[0074] The recombinase recognition sites are LoxP sites, or may be another origin.

[0075] The modified Col E1 origin may, for example, include the recombinase recognition sites AttL / AttR sites, or other acceptable recognition sites, such as Lox.

[0076] The inducible promoter may be a temperature-sensitive inhibitor. Further, the inducible promoter may be a lambda promoter.

[0077] A plasmid is described herein comprising: a sequence of interest; a modified Col E1 origin; a recombinase coding sequence; and a thermoswitch for temperature-induced control of translation. In such a plasmid, the sequence of interest (which may be within an expression cassette) and the modified Col E1 origin contain or are flanked by LoxP or AttL / AttR sites, which are recognized by the recombinase of the recombinase encoding sequence, for excision to complete the origin; the translation of the recombinase is controlled by the thermoswitch; and the modified Col E1 origin comprises at least one mutation or deletion in the Col E1 RNA I promoter to reduce or eliminate RNA I inhibition and increase replication of the origin.

[0078] A product DNA plasmid can be prepared, as described herein, when produced by any variant of the method as described herein.

[0079] A vaccine may be produced with DNA from the product DNA plasmid as described herein.

[0080] A stem loop II sequence of the RNA II of the modified Col E1 origin may be replaced with a Lox recombination site in the product plasmid.

[0081] The method may involve the addition of an AttL, AttR, AttP or AttB recombination site in the RNA II of the modified Col E1 origin, post-recombination in the product plasmid. Further, the method may involve replacing plasmid DNA sequences located 5’ of the RNA II promoter of the modified Col E1 origin with a Lox, AttL, AttR, AttP, AttB recombination site, post-recombination in the product plasmid.

[0082] The method may further comprise the replacement of up to the first 150 nucleotides of the RNA II promoter of the modified Col E1 origin with a Lox, AttL, AttR, AttP or AttB recombination site sequence, post-recombination in the product plasmid.

[0083] It is understood that the origin of the product plasmid may be any suited origin such as the Col E1 -like origins of Col E1, pMB1, pBR322, or pUC origin. Such origins are considered to be Col E1 -like, and within the scope of “modified Col E1 origin” as are other RNA dependentprotein free bacterial origins.

[0084]

[0085] The strength of the RNA II promoter of the modified Col E1 origin may increase DNA replication in the product plasmid, which allows for some precision control of replication.

[0086] Optionally, the DNA replication of any excised DNA may be controlled through reorientation of the RNA II promoter to be upstream of the origin post-excision.

[0087] Optionally, the recombinase expression to excise the plasmid may be controlled by an inducible promoter, such as the cl857 temperature sensitive repressor and control of the lambda R promoter.

[0088] In an exemplary embodiment, a Col E1 promoter is located distal from the origin within the host chromosome during fermentation. Excision of the product plasmid generates a complete origin, post-recombination, by placing the Col E1 RNA II promoter upstream of the modified Col E1 origin. The modified Col E1 origin within the chromosome remains unactive, allowing it to be carried in the host genome during fermentation. Post recombination, the Col E1 RNA II promoter activates the origin by relocating upstream of the sequence through recombination, in turn replicating the production plasmid post excision. Post recombination, the Col E1 RNA II promoter generates RNA through the site of recombination and into the downstream origin, allowing the generation of the secondary RNA structure necessary to be cleaved by RNase H and enabling subsequent DNA replication.

[0089] The modified Col E1 origin may be mutated to reduce the expression of the inhibitory RNA I molecule which will increase plasmid replication post-excision, making aggressive replication possible. The aggressive replication of the origin results in significant yields of plasmid DNA from the fermentation process, for example within host bacteria, which allows a further significant reduction in cost of goods.

[0090] Chromosomal integration by-passes the need for antimicrobial resistance and active plasmid selection, which advantageously drives down the cost of goods for production. Bypassing antimicrobial resistance selection advantageously aids in the acceptance of products by regulatory bodies. The chromosomal insertion of the origin also allows the modification of the negative regulator RNA I promoter, since the origin is not active in the chromosome due to the distal promoter.

[0091] Product plasmids are covalently linked circular DNA molecules containing a modified Col E1 origin. To generate the production plasmid, a modified pro-phage-like cassette is generated in a bacterial host’s chromosome containing the sequence of interest (optionally within an expression cassette of interest) with a modified Col E1 origin, flanked by sites (ie- LoxP or AttL / AttR sites) that are recognized by recombinases to enable excision of a plasmid product from the bacterial host’s chromosome. Advantageously, in one embodiment the plasmid product so produced is a circular, covalently linked DNA molecule containing only the sequence of interest and the modified Col E1 origin.

[0092] A trans-acting 5’ UTR RNA thermoswitch (downstream of a weak promoter) is used to control the production method via a temperature change from about 25-30 °C to 37 °C. The RNA thermoswitch enables translation of the recombinase at higher temperatures, allowing the excision of the production plasmid. The RNA thermoswitch conversely masks the translation of the recombinase during the low temperature growth phase, allowing the production plasmid to remain stably inserted in the chromosome, allowing antimicrobial free fermentation. Alternatively, temperature-sensitive inhibitors may be used with a lambda promoter to control recombinase expression. A thermal permissive terminator was found, which is a sequence of DNA located through natural selection, that allows fine gene expression control of the recombinase. The process of natural selection yielded a sequence that contained 3 promoters upstream of a coding sequence, followed by two terminators. These two terminators are directly upstream of the recombinase. These terminators allow for the insulation of a promoter that can control the expression of the recombinase. Natural selection showed that these two terminators were slightly permissive to expression from the upstream promoters at higher temperatures, but specifically when there was no promoter between the terminators and the recombinase. It was found that the two terminators between upstream promoters and the coding sequence for the recombinase shows no expression at 25°C and just enough expression at 37°C for the recombinase to perform the excision of the plasmid. This technique works across two different loci within the genome.

[0093] Additional mutations / genetic deletions in the Col E1 RNA I promoter can be introduced, which can reduce expression of the inhibitory RNA I molecule, thus aggressively increasing plasmid replication post-excision, resulting in a high yield of plasmid DNA. Advantageously, chromosomal integration bypasses the need for active plasmid selection (ie- no antimicrobial resistance needed) and RNA I inhibition, to prevent metabolic burden on fermentation bacteria (no RNA I needed), increases replication of the plasmid post excision, leading to high yields of plasmid DNA from fermentation.

[0094] While recombinases have been known for use in making mini circular DNA, such as by using temperature-induction of recombinase, the use of an RNA thermometer / thermoswitch allows control of the translation of the recombinase to excise and complete the bacterial origin.

[0095] Ongoing development of product platforms is important for the advancement of DNA vaccines and other DNA products. The plasmid and platform for its preparation and use, as described herein, utilizes both inter- and intra-chromosomal recombination to generate a production plasmid that aggressively replicates in the host bacterial strain in an antimicrobial- free environment. The sequence of interest, optionally within a eukaryotic expression cassette, inserted into the host chromosome uses both positive and negative selection to generate a recombinant bacterium that harbours the pre-product plasmid in its genome. Inserting the pre-production plasmid by utilizing the positive and negative selection cassettes enables the final product plasmid to contain no antimicrobial resistance genes, allowing for a streamlined and cheaper production of the biological product.

[0096] The pre-production plasmid is excised from the recombinant bacterial genome using a site-specific recombinase that generates a circular, covalently linked DNA molecule (product plasmid) that contains only the sequence of interest, or the eukaryotic expression cassette pertaining to the sequence of interest, a modified Col E1 origin and other DNA elements to assist in production and transfection. Synthetic-temperature-sensitive-RNA hairpins are utilized in the 5’ untranslated region (5’ UTR) of the recombinase cassette to act as a trans acting RNA thermoswitch to control the translation of the recombinase. The production of recombinase mRNA is under the control of a weak promoter.

[0097] The platform for preparation and use of the plasmid described herein was designed and synthesized de novo, resulting in a novel plasmid that contains the recombinase cassette, sequence specific homology to enable recombination to the bacterial genome, and the sequence of interest flanked by the recombinase specific site. The design referenced herein as “BPE V2.2”, which is a self-amplifying plasmid that contains the prophage cassette within the plasmid, along with the sequences required to generate the production plasmid. The design referenced herein as “BPE linear” utilizes linear synthetic sequences to build the operon within the bacterial genome. In these specific designs, the Cre-Lox system is used for BPE V2.2, and the Phi-BT1 Integrase (with the co-factor GP3) AttL / AttR system for BPE Linear. All sequences are generated synthetically, allowing for the streamlining of the production of genetic material, and to optimize the genetic machinery used in this invention.

[0098] The Col E1 origin requires two sequences to initiate replication of the excised plasmid. The first is the forward promoter required to generate the RNA primer (RNA II) generating a required higher level-secondary RNA structure, and the second is the sequence specific stretch of nucleotides required to generate the RNA secondary structure. The secondary RNA structure contains sequence specific homologous sites that allow for the RNA to fold onto itself, creating stable hairpin structures. The RNA structure then develops into the RNA-DNA hybrid that initiates DNA synthesis post-RNase H digestion. The RNA II promoter is always active and before the recombination event, the promoter is producing the antibiotic resistance gene. After the excision / recombination event, the active promoter is relocated from upstream of the antibiotic resistance gene to upstream of the RNA II primer sequence, which then generates the RNA II primer from the DNA template. This RNA II primer is then digested with RNAse H to initiate DNA replication, an event that only occurs when the RNA II promoter is upstream of the RNA II primer template sequence. The RNA II promoter is relocated from a distal downstream gene to upstream of the RNA II primer template sequence. The RNA sequence from this template is what creates the DNA replication fork, and triggers replication of the product plasmid. Excision of the sequence of interest and the modified Col E1 origin via the flanking sites permits reconstitution of the RNA II promoter upstream of the RNA II primer template sequence, thereby producing the RNA II primer. Thus, a self-replicating product plasmic can be formed, by activation of the inducible switch, which exerts control over the translation of the recombinase.

[0099] As described herein, the RNA II promoter is physically separated from the sequence specific stretch of nucleotides, allowing stable integration into the genome of the host fermentation bacteria. Native Col E1 origins generate the secondary RNA structure in the bacterial genome that can result in instability of the chromosome and lead to acute toxicity for recombinant bacteria. After the recombinase is translated from the weak promoter and the 5’ UTR is “melted” at high temperatures (37°C - 42°C), the recombinase enzyme circularizes and excises all DNA between the Lox sites and AttL / AttR sites. This recombination event moves the promoter from its distal location to a site just upstream (proximal) of the sequence specific stretch of nucleotides that regenerates the replicon. The recombination event will leave a 34-base pair (bp) Lox site, or a 73-bp AttP site between the promoter and replication specific sequence. The present disclosure illustrates that this sequence has no consequential effect on the replication of the plasmid. This is the case in both the progeny plasmid and parental DNA template (parental plasmid).

[00100] Placing the Col E1 promoter distal to the sequence specific stretch of DNA enables stable insertion of the pro-production (parental) plasmid (parental DNA template) into the host chromosome. There is a second promoter in the Col E1 origin that generates RNA I, a negative regulator of the Col E1 origin. Without RNA I expression the plasmid would replicate aggressively in the host bacteria until it becomes cytotoxic from “run away amplification”. The promoter for RNA I is roughly 20-100 times stronger than that of RNA II, which enables more “inhibitory” RNA I to be produced than RNA II. Controlling the migration of the promoter from distal to proximal also allows the removal of the promoter for the inhibitory RNA I. Destroying the RNA I promoter (removing the RNA I inhibition) enables the aggressive run-away replication of the production plasmid post-excision.

[00101] Aggressive replication enables the production of significant yields of the product DNA plasmid at a specifically targeted time in the fermentation process. The origin of replication may also contain a Col E1 primosomal assembly site (PAS) that will assist in replication of the production plasmid. Other DNA motifs may be utilized, to increase transfection efficiency through binding of eukaryotic factors to assist in the migration to the nucleus post-transfection (Nf-kB sites, etc.). Further, non-eukaryotic sequences in the product plasmid may advantageously be designed to be less than 1,000 bp, which will allow optimal transfection and translation of the product.

[00102] Applications. The plasmid and the platform for its preparation and use, as described herein, is fundamentally built on the genetic circuit of temperature-controlled recombinase translation. Temperature regulated recombination can lead to the building or destruction of an expression cassette / origin through the moving of distal / proximal sequences. This permits generation of plasmid DNA, for use and application in numerous fields. For example, the product plasmid may contain a sequence of interest encoding an immunogen, a monoclonal antibody, a prokaryotic gene expression cassette, a supplementary eukaryotic gene cassette, a DNA template for mRNA synthesis, or an immune modulating molecule to generate or suppress an immune response, among other options described herein.

[00103] Vaccinology. Significant levels of product / progeny plasmid can be generated that will encode either a viral or bacterial sequence of interest that can be used to stimulate an immune response against the targeted antigen. It is advantageous to generate large amounts of covalently closed self-replicating DNA for this purpose using a bacterial origin that is controlled through temperature-sensitive recombination. An expression cassette that may be utilized as described herein, contains a sequence of interest which may be a gene of interest (GOI) from a human or animal pathogen against which an immune response is created. The GOI can be against any immune epitope. Plasmid DNA can be generated that contains an optimized eukaryotic sequence of interest, or an expression cassette, with minimal bacterial components (less than 1,000 bp). This platform also allows use of other molecular methodologies in the field, such as encoding the VEEV (Venezuelan Equine Encephalitis Virus) replicon that generates a strong immune response against the GOI.

[00104] Apart from pathogens, the platform may be used to create components used for vaccines inducing immunity against tumour associated antigens.

[00105] Further, several proteins can be expressed simultaneously allowing the creation of advanced protein structures, such as virus like particles (VLPs). VLPs are highly immunogenic and known to generate immunity to antigenic landscapes that are not represented by individual proteins. Utilizing this platform, immune stimulatory or immune modulatory molecules can be encoded to co-express with the GOI. This will permit control of specific immune responses against the GOI.

[00106] Gene Therapy. The economical production of large amount of plasmid DNA, can be utilized to produce animal or human therapeutics that can replace or supplement proper gene function, or that can express biological therapeutics in target cells. For example, the product / progeny plasmid can be modified to contain the Epstein Barr Nuclear antigen-1 (EBNA-1) and Epstein Barr virus origin (OriP) to create a self maintaining extra chromosomal plasmid in target cells. These episomal vectors have been used previously to encode for immunoglobulins. Combining such a technology with the present platform can make bulk production of episomal vector therapies more practical. The progeny plasmid can be captured in a lipid bilayer and targeted to a specific cell type using an antibody or viral attachment protein. Utilizing current biological techniques, a monoclonal antibody can be encoded against a specified antigen, with consideration given to the targeted antigen, constraints of regulatory bodies, and the known coding sequences of the monoclonal antibody of interest. Many monoclonal antibodies are approved as biologies and lay the groundwork for DNA therapy to express a monoclonal antibody against a pathogenic antigen (HIV, TNF-alpha). Furthermore, tissue specific promoters can be included in progeny plasmids in the expression cassette of the present platform that can add an additional layer of control for the expression of the sequence of interest. The mRNA generated by the expression cassette can also contain a native exon junction complex (EJC) to assist in gene expression.

[00107] Template for mRNA Synthesis. mRNA production requires a template plasmid that contains a strong promoter upstream of the sequence of interest to be transcribed into mRNA. A plasmid could be produced in bulk to serve as the template for in vitro transcription (IVT) of mRNA. Such sequences would not impair the formation of the cassette in the chromosome, nor effect the formation of the progeny plasmid. The BPE platform described herein could be used to generate significant amounts of template DNA for mRNA synthesis at manufacturing scale.

[00108] In Overview. A method for producing a replicating DNA molecule is described, used for producing DNA products such as vaccines. The method generates a parental DNA plasmid (parental DNA template) comprised of a sequence of interest located in an eukaryotic expression cassette, a modified Col E1 origin containing or flanked by LoxP or AttL or AttR or AttP or AttB sites which are recognized by an associated recombinase, and a thermoswitch for temperature-induced control of downstream recombinase translation; preparing a pro-phage like cassette in the host bacteria with the parental plasmid (parental DNA template); fermenting the host bacteria at a temperature that induces the thermoswitch to excise a smaller product DNA plasmid from the host bacteria; and the production of the product plasmid in the recombinant bacteria.

[00109] The expression cassette and the modified Col E1 origin of the parental DNA template contain or are flanked by a LoxP or AttL or AttR or AttP or AttB recombinase recognition site, which prevents the RNA II promoter of the Col E1 origin from initiating replication. This modified Col E1 origin, currently lacking the required RNA II promoter, allows stable, anti-microbial free growth of the bacteria when the parental DNA template is inserted into the bacterial chromosome.

[00110] A thermoswitch sequence in the bacterial chromosome-integrated parental DNA template allows downstream translation of a recombinase when incubated at a certain temperature. The LoxP or AttL or AttR or AttP or AttB site is then recognized by this recombinase to excise a smaller product plasmid containing a reconstituted, modified Col E1 origin in which the RNA II promoter has been relocated to its correct upstream position, allowing initiation of its replication. The modified Col E1 origin also includes additional alterations to reduce RNA I inhibition which increases product plasmid replication rates significantly post-excision.

[00111] Additional aspects (options and features) are described in the section entitled “Aspects” below.

[00112] Table 1 lists sequences referenced herein. Table 1 Sequences SEQ ID NO Sequence SEQ ID NO:1 TTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTGGCAAACAAAAAAACCACCGCTACCAA CGGTGGTTTGTTTGC TATAGCATACAT TATAC GAAGT TATAAGGTAAC T GGC T T CAGCAGAG C GCAGATAC CAAATAC T GT T C T T C TAGT GTAGC C GTAGT TAGGC CAC CAC T T CAAGAAC T C T GTAGCAC C GC C TACATAC CTCGCTCTGC TAAT C C T GT TAC CAGT GGC T GC T GC CAGT GGC GA TAAGT CGTGTCTTACCGGGTT GGAC T CAAGAC GATAGT TAC C GGATAAGGC GCAGC GGT C GG GC T GAAC GGGGGGT T C GT GCACACAGC C CAGC T T GGAGC GAAC GAC C TACAC C GAAC T GAGA TACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA T C C GGTAAGC GGCAGGGT C GGAACAGGAGAGC GCAC GAGGGAGC T T C CAGGGGGAAAC GC C T GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC T C GT CAGGGGGGC GGAGC C TAT GGAAAAAC GC CAGCAAC GCGGCCTTTTTACGGTTCCTGGC CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACC GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG T CAGT GAGC GAGGAAGC GGAAGAGC GC C CAATAC GCAAAC CGCCTCTCCCCGCGCGTTGGCC GAT T CAT TAAT GCAGC C GGCAT GGC GTAC T T T T CATAGC GGGCAGT SEQ ID NO:2 TTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTG SEQ ID NO:3 TATAGCATACAT TATAC GAAGT TAT SEQ ID NO:4 AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTT CTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATA CCGCTCGCCGCAGCC GAAC GAC C GAGC GCAGC GAGT CAGT GAGC GAGGAAGC GGAAGAGC GC CCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAG SEQ ID NO:5 C GC GGAAC C C C TAT T T GT T TAT T T T T C TAAATACAT T CAAATAT GTAT C C GC T CAT GAGACA ATAAC C C T GATAAAT GC T T CAATAATAT T GAAAAAGGAAGAGT GCAAACAAAAAAAC CAC C G C TAC CAAC GGTGGTTTGTTT GCATAAC T T C GTATAGCATACAT TATAC GAAGT TATAAGGTA AC T GGC T T CAGCAGAGC GCAGATAC CAAATAC TGTCCGGCTTGTGTTGCC GTAGT C GGGC CA C TACAT CAAGC C C T C T GTAGCAC C GT T T GT GC CAT CATCGCTCTGC TAAT C C GGT TAC CAGT GGC T GC T GC CAGT GGC GT TAAGGC GT GC C T TAC C GGGT T GGAC T CAAGAC GATAGT TAC C GG ATAAGGC GCAGC GGT C GGGC T GAAC GGGGGGT T C GT GCACACAGC C CAGC T T GGAGC GAAC G AC C TACAC C GAAC T GAGATAC CAACAGC GT GAGC TAT GAGAAAGC GCCACGCTTCCC GAAGG GAGAAAGGC GGACAGGTAT C C GGTAAGC GGCAGGGT C GGAACAGGAGAGC GCAC GAGGGAGC T T C CAGGGGGAAAC GCCTGGTAGCTT TATAGT CCTGTCGGGTTTCGCCACCTCT GAC T T GAG CGTCTATTTTTGT GAT GCTCGTCAGGGGGGC GGAGC C TAT GGAAAAAC GC C T GC TAC GT GGC CTTCTTCCTGTTCCTGGTCTTTTGCTCACATGTTCTTTCCGGCCTTATCCCCTGATTCTGTG GATAACTGTGTTACCGTTTTTG SEQ ID N0:6 AACGCCTGCTACGTGGCCTTCTTCCTGTTCCTGGTCTTTTGCTCACATGTTCTTTCCGGCCT TATCCCCTGATTCTGTGGATAACTGTGTTACCGTTTTTG SEQ ID N0:7 TTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTGAAGACGTTTCGGGTGCTGGGTTGTTG T C T C T GGACAGT GAT C CAT GGGAAAC TACT CAGCAC CAC CAAT GT T C C GCAAACAAAAAAAC CAC C GC TAC CAGC GGT GGT T T GT T T GC C GGAT CAAGAGC TAC CAAC TCTTTTTCC GAAGGTA AC T GGC T T CAGCAGAGC GCAGATAC CAAATAC TGTTCTTCTTGTGTTGCC GTAGT TAGGC CA CAACAACAAGAAC T C T GTAGCAC CGCCTACATACCTCGCTCTGC TAAT C C T GT TAC CAGT GG C T GC T GC CAGT GGC GATAAGT C GT GT C T TAC C GGGT T GGAC T CAAGAC GATAGT TAC C GGAT AAGGC GCAGC GGT C GGGC T GAAC GGGGGGT T C GT GCACACAGC C CAGC T T GGAGC GAAC GAC C TACAC C GAAC T GAGATAC C TACAGC GT GAGC TAT GAGAAAGC GCCACGCTTCCC GAAGGGA GAAAGGC GGACAGGTAT C C GGTAAGC GGCAGGGT C GGAACAGGAGAGC GCAC GAGGGAGC T T C CAGGGGGAAAC GCCTGGTATCTT TATAGT CCTGTCGGGTTTCGCCACCTCT GAC T T GAGC G T C GAT T T T T GT GAT GCTCGTCAGGGGGGC GGAGC C TAT GGAAAAAC GC CAGCAAC GC GGC C T TTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCT GATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAAC GAC C GAGC GCAGC GAGT CAGT GAGC GAGGAAGC GGAAGAGC GC C CAATAC GCAAAC C GC C T C TCCCCGCGCGTTGGCCGATTCATTAATGCAG SEQ ID N0:8 (Fig 5) As shown with modifications indicated in Figure 5 GCAAACAAAAAAACCACCGCUACCAACGGUGGUUUGUUUGCCGGAUCAAGAGCUAC CAACUCUUUUUCCGAAGGUAACUGGCUUCAGCAGAGCGCAGAUACCAAAUACUGUC CUUCUAGUGUAGCCGUAGUCGGGCCACUACUUCAAGAACUCUGUAGCACCGUUUGU GCCAUCAUCGCUCUGCUAAUCCGGUUACCAGUGGCUGCUGCCAGUGGCGUUAAGGC GUGCCUUACCGGGUUGGACUCAAGACGAUAGUUACCGGAUAAGGCGCAGCGGUCGG GCUGAACGGGGGGUUCGUGCACACAGCCCAGCUUGGAGCGAACGACCUACACCGAA CUGAGAUACCAACAGCGUGAGCUAUGAGAAAGCGCCACGCUUCCCGAAGGGAGAAA GGCGGACAGGUAUCCGGUAAGCGGCAGGGUCGGAACAGGAGAGCGCACGAGGGAGC UUCCAGGGGGAAACGCCUGGUAUCUUUAUAGUCCUGUCGGGUUUCGCCACCUCUGA CUUGAGCGUCUAUUUUUGUGAUGCUCGUCAGGGGGGCGGAGCCUAUGGAAA SEQ ID N0:9 (Fig 11 A) GGAUCCUCUCCUUCACUAGUCUGCAGAAGGAGAUAUACCC SEQ ID NO:10 (Fig 11B) AT T GGC CAT GGAT CCTCTCCTTCAC TAGT C T CAGAAGGAGATATAC C CAT GGC CAAT T T GC T GAC C GT AC AC SEQ ID N0:11 Lox66 Correct Orientation as shown in Figure 12 TACCGTTCGTATAATGTATGCTATACGAAGTTAT SEQ ID N0:12 Lox71 Correct Orientation as shown in Figure 12 ATAACTTCGRATAATGTATGCTATACGAACGGTA SEQ ID N0:13 Lox72 Correct Orientation as shown in Figure 12 TACCCGTTCGTATAATGTATGCTATACGAACGGTA SEQ ID N0:14 LoxP Wt Correct Orientation as shown in Figure 12 ATAAC T T C GTATAAT GTAT GC TATAC GAAGT TAT SEQ ID NO:15 SEQ ID N0:16 BPE RNA Switch V2.31 as in Example 4 BPE RNA Switch V2.41 as in Example 5 ID NO: 17 GCAAACAAAAAAACCACCGCUACCAACGGUGGUUUGUUUGCCGGAUCAAGAGCUAC CAACUCUUUUUCCGAAGGUAACUGGCUUCAGCAGAGCGCAGAUACCAAAUACUGUC CUUCUUGUGUUGCCGUAGUCGGGCCACAACAACAAGAACUCUGUAGCACCGUUUGU GCCAUCAUCGCUCUGCUAAUCCGGUUACCAGUGGCUGCUGCCAGUGGCGUUAAGGC GUGCCUUACCGGGUUGGACUCAAGACGAUAGUUACCGGAUAAGGCGCAGCGGUCGG GCUGAACGGGGGGUUCGUGCACACAGCCCAGCUUGGAGCGAACGACCUACACCGAA CUGAGAUACCAACAGCGUGAGCUAUGAGAAAGCGCCACGCUUCCCGAAGGGAGAAA GGCGGACAGGUAUCCGGUAAGCGGCAGGGUCGGAACAGGAGAGCGCACGAGGGAGC UUCCAGGGGGAAACGCCUGGUAUCUUUAUAGUCCUGUCGGGUUUCGCCACCUCUGA CUUGAGCGUCUAUUUUUGUGAUGCUCGUCAGGGGGGCGGAGCCUAUGGAAA ID NO:18 Example 6 - "Bla_term_ci857_term_only" 2051 bp DNA linear acggacgaat gtcactttcg ccgctaacac tggctgtcgg ttcgtctttg acttcgctgt caccattcaa cagcgcatct tctagagttt acagctagct cagtcctagg tataatgcta gcttttaaat cctttttttc tgcgcgtatg atgctgctgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtaggagg acgatacatg agcacaaaaa agaaaccatt aacacaagag cagcttgagg acgcacgtcg ccttaaagca atttatgaaa aaaagaaaaa tgaacttggc ttatcccagg aatctgtcgc agacaagatg gggatggggc agtcaggcgt tggtgcttta tttaatggca tcaatgcatt aaatgcttat aacgccgcat tgcttacaaa aattctcaaa gttagcgttg aagaatttag cccttcaatc gccagagaaa tctacgagat gtatgaagcg gttagtatgc agccgtcact tagaagtgag tatgagtacc ctgttttttc tcatgttcag gcagggatgt tctcacctaa gcttagaacc tttaccaaag gtgatgcgga gagatgggta agcacaacca aaaaagccag tgattctgca ttctggcttg aggttgaagg taattccatg accgcaccaa caggctccaa gccaagcttt cctgacggaa tgttaattct cgttgaccct gagcaggctg ttgagccagg tgatttctgc atagccagac ttgggggtga tgagtttacc ttcaagaaac tgatcaggga tagcggtcag gtgtttttac aaccactaaa cccacagtac ccaatgatcc catgcaatga gagttgttcc gttgtgggga aagttatcgc tagtcagtgg cctgaagaga cgtttggctg ataaccggag gaggatcgaa ccatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgataa ccacacttag accagatgtc cgtgacgtct agcttgagct gtcagcccgc ctaatgagcg ggcttttttt tgaaaagggc tcgcgcttaa cagtgtcgtg agaaggccat cctgacggat ggccttttcc cggatggaat tcatggccaa tttgctgacc gtacaccaga acttgcctgc attaccagtc gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga t SEQ ID NO:19 Example 6 - “fliM CDS” Product: Flagellar motor switch protein (gene fliM) Protein id="WP_001350520.1 MGDSILSQAEIDALLNGDSEVKDEPTASVSGESDIRPYDPNTQRRWRERLQALEI INERFARHFRMGLFNLLRRSPDITVGAIRIQPYHEFARNLPVPTNLNLIHLKPLRG TGLWFSPSLVFIAVDNLFGGDGRFPTKVEGREFTHTEQRVINRMLKLALEGYSDA WKAINPLEVEYVRSEMQVKFTNITTSPNDIWNTPFHVEIGNLTGEFNICLPFSMI EPLRELLVNPPLENSRNEDQNWRDNLVRQVQHSQLELVANFADISLRLSQILKLNP GDVLPIEKPDRIIAHVDGVPVLTSQYGTLNGQYALRIEHLINPILNSLNEEQPK SEQ ID NQ:20 Example 6 -="KanR / NeoR CDS" Product: KanR / NeoR; Protein id = “AIZ65988.1" MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPVLFVKTDLSGA LNELQDEAARLSWLATTGVPCAAVLDWTEAGRDWLLLGEVPGQDLLSSHLAPAEK VSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQGLAPA ELFARLKASMPDGEDLWTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIALA TRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF SEQ ID N0:21 Example 6 -= Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences " Product: site-specific recombinase; Label = “CreNoCodonOPTI" MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWC KLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNA VSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAY NTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSG QRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMV RLLEDGD SEQ ID NO:22 RNA thermometer for Cre expression, split ori (tGFP F2 modified) BPE-RNA switch) of Example 1 1 gatatcatgg cagataatcc agacccttca tcgctcctgc cggacgtgtt ttcaccaccg 61 gtccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg 121 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt 181 ctgcgtttat aaaaccgctg cgtcaggtac aggccgatct gaagtaatca aggttatctc 241 ccgcaatggt ttatcgttgc gggagtgtaa aaaacatcat ttagcgtgac tttctttcaa 301 cagctaacaa ttgttgttac tgcctaatgt ttttagggta ttttaaaaaa gggcgataaa 361 aaacgattgg gggatgagac atgaacgctc aagcagaaga attcatggcc aatttactga 421 ccgtacacca aaatttgcct gcattaccag tcgatgcaac gagtgatgag gttcgcaaga 481 acctgatgga catgttcagg gatcgccagg cgttttctga gcatacctgg aaaatgcttc 541 tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa tggtttcccg 601 cagaacctga agatgttcgc gattatcttc tatatcttca ggcgcgcggt ctggcagtaa 661 aaactatcca gcaacatttg ggccagctaa acatgcttca tcgtcggtcc gggctgccac 721 gaccaagtga cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa gaaaacgttg 781 atgccggtga acgtgcaaaa caggctctag cgttcgaacg cactgatttc gaccaggttc 841 gttcactcat ggaaaatagc gatcgctgcc aggatatacg taatctggca tttctgggga 901 ttgcttataa caccctgtta cgtatagccg aaattgccag gatcagggtt aaagatattt 961 cacgtactga cggtgggaga atgttaatcc atattggcag aacgaaaacg ctggttagca 1021 ccgcaggtgt agagaaggca cttagcctgg gggtaactaa actggtcgag cgatggattt 1081 ccgtatctgg tgtagctgat gatccgaata actacctgtt ttgccgggtc agaaaaaatg 1141 gtgttgccgc gccatctgcc accagccagc tatcaactcg cgccctggaa gggatttttg 1201 aagcaactca tcgattgatt tacggcgcta aggatgactc tggtcagaga tacctggcct 1261 ggtctggaca cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa 1321 taccggagat catgcaagct ggtggctgga ccaatgtaaa tattgtcatg aactatatcc 1381 gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc gattaaacgt 1441 aaataaatat cctttgtatt catttgttta tagtcagaag gccatcctga cggatggcct 1501 tttgactcgc atcctcacga taatatccgg gtaggcgcaa tcactttcgt ctactccgtt 1561 acaaagcgag gctgggtatt tcccggcctt tctgttatcc gaaatccact gaaagcacag 1621 cggctggctg aggagataaa taataaacga ggggctgtat gcacaaagca tcttctgttg 1681 agttaagaac gagtatcgag atggcacata gccttgctca aattggaatc aggtttgtgc 1741 caataccagt agctcgagtg ataacttcgt atagcataca ttatacgaag ttataaggta 1801 actggcttca gcagagcgca gataccaaat actgtccggc ttgtgttgcc gtagtcgggc 1861 cactacatca agccctctgt agcaccgttt gtgccatcat cgctctgcta atccggttac 1921 cagtggctgc tgccagtggc gttaaggcgt gccttaccgg gttggactca agacgatagt 1981 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 2041 agcgaacgac ctacaccgaa ctgagatacc aacagcgtga gctatgagaa agcgccacgc 2101 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 2161 gcacgaggga gcttccaggg ggaaacgcct ggtagcttta tagtcctgtc gggtttcgcc 2221 acctctgact tgagcgtcta tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 2281 acgcctgcta cgtggccttc ttcctgttcc tggtcttttg ctcacatgtt ctttccggcc 2341 ttatcccctg attctgtgga taactgtgtt accgtttttg cccgggggca ttgattattg 2401 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 2461 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 2521 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 2581 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 2641 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 2701 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 2761 accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 2821 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 2881 cgggactttc caaaatgtcg taacaactgc gatcgcccgc cccgttgacg caaatgggcg 2941 gtaggcgtgt acggtgggag gtctatataa gcagagctga cgtggctagc ggatcattat 3001 cggaaggggc ggcagcaaaa gcagggtaga taatcactca atgaggtaag tttagtcttt 3061 ttgtctttta tttcaggtcc cggatccggt ggtggtgcaa atcaaagaac tgctcctcag 3121 tggatgttgc ctttacttct agtgacatcg aaaccatgga gagcgacgag agcggcctgc 3181 ccgccatgga gatcgagtgc cgcatcaccg gcaccctgaa cggcgtggag ttcgagctgg 3241 tgggcggcgg agagggcacc cccgagcagg gccgcatgac caacaagatg aagagcacca 3301 aaggcgccct gaccttcagc ccctacctgc tgagccacgt gatgggctac ggcttctacc 3361 acttcggcac ctaccccagc ggctacgaga accccttcct gcacgccatc aacaacggcg 3421 gctacaccaa cacccgcatc gagaagtacg aggacggcgg cgtgctgcac gtgagcttca 3481 gctaccgcta cgaggccggc cgcgtgatcg gcgacttcaa ggtgatgggc accggcttcc 3541 ccgaggacag cgtgatcttc accgacaaga tcatccgcag caacgccacc gtggagcacc 3601 tgcaccccat gggcgataac gatctggatg gcagcttcac ccgcaccttc agtctgcgcg 3661 acggcggcta ctacagctcc gtggtggaca gccacatgca cttcaagagc gccatccacc 3721 ccagcatcct gcagaacggc ggccccatgt tcgccttccg ccgcgtggag gaggatcaca 3781 gcaacaccga gctgggcatc gtggagtacc agcacgcctt caagaccccg gatgcagatg 3841 ccggtgaaga ataaggaaaa atacccttgt ttctactgag ctcctgtgcc ttctagttgc 3901 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 3961 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 4021 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 4081 catgctgggg atgcggtggg ctctatgggg gactttccgg tcgggacttt ccaattgcgg 4141 gactttcctc ttcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 4201 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtgca 4261 aacaaaaaaa ccaccgctac caacggtggt ttgtttgcat aacttcgtat agcatacatt 4321 atacgaagtt ataggaggtc tgaatgagta ttcaacattt ccgtgtcgcc cttattccct 4381 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 4441 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 4501 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 4561 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 4621 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 4681 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 4741 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 4801 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 4861 acgacgagcg tgacaccacg atgcctgtag cgatggcaac aacgttgcgc aaactattaa 4921 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 4981 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 5041 ccggagccgg tgagcgtggt tctcgcggta tcatcgcagc gctggggcca gatggtaagc 5101 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 5161 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaataactg tcagcccgcc 5221 taatgagcgg gctttttttt ggatcctgcg tgaggcggga ttttcaagtt cgccacaagg 5281 gctggattgg ggataggata tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5881 gcctatggaa a

[00113] Example 1

[00114] Plasmid BPE V2.2

[00115] BPE Version 2.2 (also referenced herein as V2.2) is a self-maintaining plasmid that contains the entire operon required to generate the progeny plasmid. The plasmid contains two sites of origin, the first is fully intact and operational and the second has the RNA II promoter and the specific stretch of DNAthat generates the secondary structure physically separated. The second origin moves in location from distal to proximal to the sequence specific stretch of nucleotides that regenerates the replicon after the Cre-Lox recombination.

[00116] BPE V2.2 contains 50 nucleotide stretches that are homologous to the Lhr gene in the host chromosome and in the BPE landing pad (described below). The 50 bp homology boxes are flanked by two blunt end cutting EcoRV sites. Digesting the plasmid with EcoRV liberates the homologous stretches of Lhr. Digesting with EcoRV also removes the plasmid’s intact site of origin, allowing the insertion of the linearized plasmid into the host’s chromosome without an active site of origin.

[00117] The plasmid also contains the Cre recombinase required for the Lox-Lox excision of the progeny plasmid. Cre expression is prevented in the parental DNA template by insulating terminators that are upstream of the Cre ORF. Once integrated into the host chromosome, a weak promoter is inserted, and a strong synthetic RNA thermometer is positioned upstream of Cre to control the expression of the recombinase. The transcription of the Cre recombinase can also be controlled by an inducible promoter, such as the lambda promoter and the temperature sensitive inhibitor cl857. The translation of Cre is controlled by this RNA thermoswitch that demonstrates minimal expression at 25 / 30°C and maximal expression at 37 / 42°C. Downstream of Cre is the LoxP site followed by the distal origin. This sensitive sequence is also insulated with terminators to prevent RNA production of the secondary structure responsible for plasmid replication.

[00118] The parental DNA template thus contains the sequence specific stretch of nucleotides that generates the secondary RNA structure necessary for DNA replication that is lacking the RNA II promoter. The RNA II promoter is located upstream of the distal LoxP or AttL recombination site, that will be placed upstream of the origin post excision. The BPE origin is designed to activate post-recombination. In this exemplary plasmid, stem loop II in RNA II is specifically replaced with the LoxP site, due to stem loop II showing remarkable similarity to the secondary structure of LoxP. Further, the RNA I promoter is destroyed, through key mutations in the -10 and -35 nucleotide positions of the promoter box, which will thus allow for run away amplification of the progeny plasmid in the fermentation bacteria, post-excision. The start of RNA II is just downstream of the Amp promoter, which prior to recombination is remote to the rest of RNA II. After recombination, the Amp promoter is now re-located to just upstream of RNA II to produce the first nucleotides of RNA II, the LoxP site, and the remainder of the essential RNA II sequence. Recombination produces a smaller (~2.6kb) progeny plasmid, which amplifies to a greater level than the parental DNA template. The progeny plasmid origin also contains sequences known to promote plasmid replication, such as the primosomal assembly site (PAS), which assists in lagging strand synthesis.

[00119] Another component of the BPE parental DNA template may be an antibiotic resistance cassette. This contains the Amp promoter, which generates an mRNAthat contains the LoxP site in its 5’ UTR, along with the beta-lactamase (BLA) enzyme required for beta-lactam antibiotic resistance. Before recombination, the Amp promoter ensures that the plasmid is ampicillin resistant, but post-recombination, the antimicrobial resistance cassette is broken. The progeny plasmid will thus contain just the eukaryotic expression cassette along with the Amp promoter that also generates RNA to read through the LoxP site and into the modified Col E1 origin. This promoter can also be replaced by the native Col E1 promoter to allow further control of progeny plasmid replication. The ampicillin resistance cassette will remain on the chromosome of the fermentation plasmid, allowing the generation of pDNA that does not contain any antimicrobial resistance.

[00120] Building the Parental DNA Template (Plasmid). The plasmid was synthesized De Novo synthetic DNA technologies from commercial companies. By necessity, due to its length and secondary structure, the parental DNA template (plasmid) sequence was produced in 4 different gBIock fragments that were then digested and ligated together. The fragments were designed to be cloned using the directional restriction enzymes Nhel, Sacl, Agel, and Xhol (Figure 1). Due to the temperature sensitive nature of the RNA thermoswitch, the creation and modification of the plasmid was completed at 25°C. The plasmid encodes for Beta-lactam resistance, and therefore can be selected for using BLA expression. Creating the plasmid using 4 gBIocks also allows modification of any part of the plasmid easily by switching out a specific gBIock sequence.

[00121] Eukaryotic Expression Cassette. The purpose of developing the progeny plasmid is to generate aggressive amounts of the sequence of interest, or gene of interest (GOI) product in vaccinated host cells to stimulate a desired biological response. The expression cassette pertaining to the sequence of interest may encode an immunogen, a monoclonal antibody, a prokaryotic gene expression cassette, a supplementary eukaryotic gene cassette, a DNA template for mRNA synthesis, or an immune modulating molecule to generate or suppress an immune response. The eukaryotic promoter of choice in this example is the CMV enhancer and promoter that generates an mRNA with a 5’ and 3’ UTR generated from Influenza A virus (IAV) nucleoprotein (NP). The IAV NP mRNA shows robust translation independent of infection that assists in the translation of the target protein. The RNA transcript also contains an intron from the SV40 virus that promotes splicing. Splicing results in the formation of the exon junction complex, which promotes mRNA export, stability, and translation. The development GOI is tGFP currently to allow quantification of transfection easily by measurement of fluorescence of this protein. The GOI insertion site is flanked by the Nhel and Sacl restriction sites, allowing quick replacement of the GOI with directional cloning. Finally, the standard SV40 terminator signals the end of the transcriptional unit and is utilized to synthesize the Poly A tail required for proper mRNA function.

[00122] Figure 1 shows a plasmid map of the parental DNA template (plasmid) for use as described in this example. The AmpR promoter generates mRNA to synthesize the beta-lactamase (BLA) with the LoxP site in the 5’ UTR. The BPE ori from nucleotides ~1,800-2,400 is insulated from any RNA read through from the PrfA promoter by the presence of the rrnB T2 terminator, tIMM terminator, and the Lambda tL3 terminator. There is a cryptic promoter in the LoxP site that is uninsulated. The functional origin on this plasmid is found from nucleotides ~5,300-5,900 and is flanked by two EcoRV sites used to remove the origin. Upstream and downstream of the EcoRV sites are homologous sequences to Lhr, allowing recombination into the genome using lambda red recombinase from the helper plasmid pRR008 Amp KO. The map shown in Figure 1 is generated from full plasmid sequencing of the parental DNA template (plasmid) from an Oxford Nanopore™ sequencer. This image was generated in Geneious™ R 11.0.15 (www.geneious.com).

[00123] Workflow and Production Methodology. The aim in this example is to generate significant levels of product / progeny plasmid from fermentation bacteria. To complete this without antibiotics, the operon must first be integrated into the chromosome of the host bacteria. The DH5 alpha bacterial host strain was selected, due to its extensively studied genome and publicly available genome sequence. The modified pRR008-Amp KO plasmid is introduced to the DH5 alpha bacteria, which provides the lambda red recombination machinery required for homologous recombination.

[00124] Figure 2 is an annotated diagram of a plasmid landing pad. The farthest left sequence is homologous to the FliK ORF in the bacterial chromosome. Downstream of this is the BLA and R105 terminators to insulate the BPE insert. The annotated Start Lhr and Stop Lhr is homologous to BPE V2.2, along with the EcoRV scar to allow homologous recombination with EcoRV digested BPE V2.2. The AttL and GP3 and PrfA promoter are all for the building of BPE Linear and not BPE V2.2. The ProD promoter synthesizes the resistance gene to spectinomycin / streptomycin and a GFP molecule. After recombination with BPE V2.2, the bacteria are no longer resistant to spectinomycin / streptomycin and no longer fluorescent green. This image was generated in Geneious™ R 11.0.15.

[00125] Figure 3 provides a plasmid map highlighting the LoxP where recombination will occur. Ore activation will excise the product / progeny plasmid from both the parental DNA template (plasmid) and the chromosome, generating the smaller plasmid. This image was generated in Geneious™ R 11.0.15.

[00126] The bacterial genome is manipulated to contain a “genetic landing pad”, which contains homologous sequences to BPE V2.2, as well as BPE linear (Figure 3). The host chromosome is modified initially in this way to allow easy insertion of BPE V2.2. The first modification was completed with the BPE landing pad (Figure 3). The landing pad (LP) integrates into the Fli gene cassette (Flagella) using 65 bp of homology to the chromosome at this site (Figure 2). The landing pad also contains 50 nucleotides of homology to BPE V2.2 (Lhr) with the accompanying EcoRV cleavage scar on the 5’ and 3’ ends, allowing integration into the landing pad. The landing pad sequence contains a resistance cassette to kanamycin (KanR) to allow for selection of integrons on microbiology plates using kanamycin. This cassette also utilizes the polycistronic nature of prokaryotic translation by encoding a tGFP downstream of KanR.

[00127] This cassette is then replaced by BPE V2.2. BPE V2.2 expresses resistance to beta-lactams, allowing for selection of integrants. Another method to confirm integration is the loss of tGFP when the KanR-tGFP cassette is replaced by BPE V2.2. The LP also contains two terminators upstream of the BPE V2.2 integration site that insulates Ore postintegration. A final integration is performed to insert both a weak promoter and a strong RNA thermometer upstream of Ore to allow for temperature regulated translation control. After this replacement has been completed, pRR008 Amp-KO, the helper plasmid that encodes the recombinant machinery for lambda red, can be cured from the bacteria using 5% sucrose. pRR008 Amp-KO encodes for SacB (levansucrase), an enzyme that converts sucrose to long chain levans that are toxic to E. Coli. Once the bacteria are cured of the helper plasmid, a cell bank of modified bacteria is generated for use in generating seed cultures for fermentation.

[00128] For expressing GOI (or sequence of interest) an RNA thermometer for Ore expression with split origin may be as found in SEQ ID NO:22

[00129] Figure 4 illustrates a product plasmid generated through Cre-LoxP recombination. The AmpR promoter produces the RNA required to generate the secondary structure for plasmid replication. The second stem loop of RNA II was replaced with LoxP to minimize the effects of sequence changes on RNA folding. This plasmid is also generated by the parental DNA template (plasmid) that is incubated at 37°C, as seen in Figure 1. The map of Figure 4 is generated from full plasmid sequencing from an Oxford Nanopore™ sequencer, and the image was generated in Geneious™ R 11.0.15.

[00130] Figure 5 provides an illustration of RNA II and the added modifications. The five mutations are annotated in the -10 and -35 box for RNA I to reduce the expression of RNA I. In BPE V2.2, Stem loop II is replaced with LoxP, which also forms a strong stem loop structure. In BPE Linear, the transcription start site is the first nucleotide of AttP, which is added to the 5’ sequence of the entirety of RNA II. The image is modified from the publication of Masukata H., Tomizawa J. (1986).

[00131] Figure 5 shows the LoxP insertion into the RNA II sequence in stem loop II. The LoxP sequence mimics the secondary structure of stem loop. Unique mutations made in the RNA I promoter serve to maintain the secondary structure of the RNA primer.

[00132] The AttP sequence is attached on the 5’ sequence of RNA II, which showed 100% tolerability to the insertion.

[00133] The following sequences are represented in Figure 5, with the promoter and primosomal sequence. The full sequence of Figure 5, including the indicated modifications is deemed SEQ ID NO:8.

[00134] The following sequence is the pUC origin containing the LoxP sequence instead of stem loop II. This origin also utilizes the Col E1 promoter to generate the RNA II primer. RNA I is intact. The primosomal sequence is also included in the 3' end of the sequence.

[00135] The BPE: Origin, pUC modified Stem Loop II LoxP replacement, Col E1 promoter is as follows: TTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTGGCAAACAAAAAAACCACCGCTACCAAC GGT GGT T T GTT T GCTATAGCATACATTATACGAAGTTATAAGGTAACT GGCT TCAGCAGAGCG CAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTA AGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGG GGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCC TTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAG GAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC AGCCGGCATGGCGTACTTTTCATAGCGGGCAGT (SEQ ID NO :1) .

[00136] SEQ ID NO:2 is the first underlined sequence within SEQ ID NO:1, representing the addition of AttP: TTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTG (SEQ ID NO :2) .

[00137] SEQ ID NO:3 is the second underlined sequence within SEQ ID NO:1, representing LoxP: TATAGCATACATTATACGAAGTTAT (SEQ ID NO:3).

[00138] SEQ ID NO:4 is the third underlined sequence, Col E1 promoter, within SEQ ID NO:1: AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTC TTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCA ATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAG (SEQ ID NO :4) .

[00139] The following sequence (SEQ ID NO:5) is the Col E1 origin containing the LoxP sequence instead of stem loop II. This origin utilizes the Amp P promoter to generate the RNA II primer. The RNA I promoter is mutated to no longer function. The primosomal sequence is also included in the 3' end of the sequence.

[00140] BPE Origin, Col E1 modified stem loop II LoxP replacement, Amp Promoter: CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAA TAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTGCAAACAAAAAAACCACCGCT ACCAACGGTGGTTTGTTTGCATAACTTCGTATAGCATACATTATACGAAGTTATAAGGTAACT GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCGGCTTGTGTTGCCGTAGTCGGGCCACTAC ATCAAGCCCTCTGTAGCACCGTTTGTGCCATCATCGCTCTGCTAATCCGGTTACCAGTGGCTG CTGCCAGTGGCGTTAAGGCGTGCCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CCGAACTGAGATACCAACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTAGCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCTATTTT T GT GAT GCT CGT CAGGGGGGCGGAGCCTAT GGAAAAACGCCTGCTACGTGGCCTTCTTCCTGT TCCTGGTCTTTTGCTCACATGTTCTTTCCGGCCTTATCCCCTGATTCTGTGGATAACTGTGTT ACCGTTTTTG (SEQ ID NO:5).

[00141] SEQ ID NO:3, LoxP, is included within SEQ ID NO:5, as the first underlined sequence.

[00142] SEQ ID NO:6, the Amp promoter, is included within SEQ ID NO:5, as the second underlined sequence: AACGCCTGCTACGTGGCCTTCTTCCTGTTCCTGGTCTTTTGCTCACATGTTCTTTCCGGCCTT ATCCCCTGATTCTGTGGATAACTGTGTTACCGTTTTTG (SEQ ID NO:6).

[00143] The following sequence (SEQ ID NO:7) is the pUC origin containing a 5’ AttP sequence. The origin utilizes the native Col E1 promoter to generate the RNA II primer. The RNA I promoter is mutated to no longer function. The primosomal sequence is also included in the 3' end of the sequence.

[00144] BPE Origin, pUC modified 5’ AttP sequence, Col E1 promoter, is represented by SEQ ID NO:7. The shaded and underlined portion represents a region of 5 mutation locations A>U; A>U; U>A; U>A; and U>A. The RNA I promoter is mutated, at key mutation locations in the -10 and -35 nucleotide positions of the promoter box to permit run away amplification of the progeny plasmid in the fermentation bacteria, post-excision. TTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTGAAGACGTTTCGGGTGCTGGGTTGTTGT CTCTGGACAGTGATCCATGGGAAACTACTCAGCACCACCAATGTTCCGCAAACAAAAAAACCA CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT GGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTTGTGTTGCCGTAGTTAGGCCACAAC AACAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCG CAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCG GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGA AACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTG TGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGC GAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG CCGATTCATTAATGCAG (SEQ ID NO :7) .

[00145] SEQ ID NO:7 includes SEQ ID NO:2 (AttP) and SEQ ID NO:4 (Col E1 promoter), as shown as first and second underlined regions of SEQ ID NO:7.

[00146] Bacteria are fermented at <30°C until their optimal cell density has been achieved, at that point, the incubating temperature is increased to >37°C to melt the secondary RNA structure in the 5’ UTR of the Cre recombinase. The RNA thermoswitch may also be replaced with an inducible promoter, such as the lambda promoter and the temperature sensitive inhibitor cl857. Once the temperature sensitive RNA thermoswitch secondary structure has been melted or the temperature sensitive inhibitor cl857 dimers have been destabilized, the Cre recombinase then excises the progeny (or “product”) plasmid from the host chromosome through the two LoxP sites. The product / progeny plasmid then has the Amp / Col E1 promoter located upstream of the modified Col E1 origin, which generates the secondary RNA structure, RNA II, needed for plasmid replication (Figure 4). The product / progeny plasmid will then amplify aggressively due to the lack of the inhibitory RNA I promoter (Figure 5). After letting the bacteria ferment at 37°C for an additional number of hours, the bacteria is then harvested and the plasmid DNA is extracted through slow alkaline lysis.

[00147] Cre-Lox Recombination and Progeny Plasmid Replication. The production and replication of the progeny plasmid is demonstrated in both the final cassette and from the parental DNA template (plasmid). Firstly, to validate the functionality of the thermoswitch and recombinase activity of Cre, an older version of the parental DNA template (plasmid) that contains Cre under control of the PrfA promoter and RNA thermometer in fermentation bacteria was incubated at 37°C and analysis of extracted plasmid materials was performed.

[00148] Figure 6A and Figure 6B show length and abundance analysis, respectively, of BPE V2.2. In Figure 6A, a Maxi-prep extraction of the BPE V2.2 plasmid was sequenced using Oxford Nanopore™ technology. The number of reads show a non-biased measurement of abundance of each plasmid. The product / progeny plasmid is ~2.5 kb, the parental DNA template (plasmid) is ~5.9 kb, and the 2x parental artifact plasmid is ~11,8kb. The stock of plasmid was grown at 37°C and in the presence of ampicillin. In Figure 6B, the plasmid was analyzed on a DNA agarose gel with a supercoiled DNA plasmid ladder as reference.

[00149] Both the progeny and parental plasmid were identified in the sample, along with a duplication of the parental plasmid into what was termed 2x parental plasmid. Oxford Nanopore™ sequencing was performed on the plasmid to confirm the sequence of the products. Oxford Nanopore™ sequencing confirmed the sequences, but it was noted that there were more reads aligned to the progeny plasmid than the parental. The sample was also analyzed by gel electrophoresis, which demonstrates the recombination of the parental DNA template (plasmid) into the progeny plasmid. The recombination also produces a ~3.35 kb product that is non-replicative. Taken together, these data show that the parental DNA template (plasmid) successfully recombines into the progeny plasmid and this progeny plasmid is replicating to a high degree (the progeny plasmid being significantly more abundant than the product left-over from recombination). This confirms integration of BPE V2.2 into the bacterial chromosome, to allow for antimicrobial-free fermentation and the destruction of any contaminating plasmids, can proceed.

[00150] BPE V2.2 was inserted into the bacterial chromosome using the described methods above with the weak promoter and strong RNA thermoswitch. The recombinant bacteria was incubated at 25 °C (no Cre translation) and 42 °C (weak Cre translation) for 12 hours. The bacteria excised the progeny plasmid and it showed rapid replication.

[00151] Figure 7A, Figure 7B and Figure 7C illustrate the agarose gel electrophoresis, length, and abundance analysis of BPE V2.2, respectively, recovered from recombinant cells. Recombinant DH5 alpha cells harbouring the pre-plasmid were heat shocked at 42°C to promote the expression of Cre, which in turn excised the plasmid from the chromosome. The plasmid self replicated and was analyzed through gel electrophoresis (Figure 7A), Sequencing read length and depth (Figure 7B), and nucleotide sequence confirmation of progeny plasmid (Figure 7C).

[00152] Figure 8A and Figure 8B show production of progeny plasmid from recombinant bacteria. Gel electrophoresis analysis (Figure 8A) of plasmid generated from recombinant bacteria without helper plasmid is shown. Recombinant bacteria was cured of the helper plasmid and incubated at 37°C to promote the Cre mediated excision of the progeny plasmid from the host chromosome. In Figure 8B, a visualization of the Cre expression cassette and the progeny plasmid in the host chromosome before excision.

[00153] Example 2

[00154] BPE Linear

[00155] BPE Linear utilizes linear gBIocks from IDT to expedite recombination. BPE linear has a larger cassette in the chromosome that allows more advanced recombinant machinery to be used. The U12 5’ Thermoswitch-Cre-LoxP system initially tested in earlier versions of BPE V2.2 had a weakness in that recombination was shown to occur at inappropriate times, creating an unstable production / progeny plasmid. As seen in Figure 6A and Figure 6B, there is a peak of plasmid found in the sample analysis that is the result of two parental DNA template (plasmids) recombining and forming a “2xsize plasmid”. Another weakness of the Cre-LoxP system is the presence of a cryptic promoter, that can not be insulated against, that is located in the LoxP sequence in the chromosome. To overcome these weaknesses, identified in earlier tests, BPE Linear utilizes a different recombinase, the integrase (Int) from the Phi-BT1 phage. The Phi-BT1 phage has unique attachment sites (Att) that allow for directional recombination and the AttP site that does not contain a cryptic promoter. BPE Linear still utilizes the PrfA RNA thermoswitch to control the expression of the recombinase, which in turn circularizes the genetic material between the AttR and AttL sites. This regenerates the Col E1 origin with the promoter with the AttP site residing in RNA II.

[00156] Utilizing GP3, the cofactor to the recombinase Int, the directionality of recombination can be controlled. Thus, a stable progeny plasmid can be created that does not undergo further recombination events and is unlikely to re-integrate into the host chromosome.

[00157] An artificial pro-phage is introduced into the host chromosome, containing the sequence of interest and modified Col E1 origin that are flanked by AttL and AttR sites, for controlling its excision through Int translation controlled by an RNA thermoswitch. Once the plasmid has been excised, the native RNA II promoter is then located proximal to the RNA II encoding sequence so it is able to generate the secondary RNA structure required for DNA replication.

[00158] Unlike BPE V2.2 progeny, the BPE linear progeny plasmid has the promoter upstream of the RNA secondary structure. BPE V2.2 utilizes the AmpR promoter, while BPE Linear uses the native Col E1 promoter and any of its modifications or deviations.

[00159] A further difference is the scar left from recombination: BPE V2.2 contains a 34-bp LoxP site in stem loop II in RNA II, while BPE linear has a 73-bp AttP site at the very 5’ end of RNA II (Figure 8). Both the LoxP site and AttP sequence are compatible with the Col E1 origin.

[00160] Finally, BPE Linear utilizes the BPE Landing pad described above, which destroys the expression of FliK, an essential component of the flagella (Fli) machinery. Destroying the flagella cassette provides a new bacterial phenotype consisting of a modified bacteria that is non-motile which can be assayed in soft agar plugs. Furthermore, disabling the flagella of the bacteria frees up precious metabolism to further allow plasmid production. The final integron of BPE linear removes significantly more Fli coding capacity from the genome. BPE V2.2 can delete 1,058 bp from the Fli cassette through the landing pad, while BPE linear removes 6,487 bp.

[00161] Building the BPE Linear Cassette. The BPE linear cassette requires the synthesis of two critical enzymes to generate product / progeny plasmid from the altered bacterial chromosome. These two enzymes are Phi-BT1 Int and GP3, which mediate the recombination between AttL and AttR to produce AttB and AttP. AttP will be located on the resulting product plasmid in the RNA II sequence. The INT and GP3 mediated recombination event will reconstitute the Col E1 origin, bringing the distal Col E1 promoter up stream of the RNA II sequence, resulting in product plasmid replication. Several gBIocks are used as linear templates for recombination to allow introduction of the full BPE cassette, which encodes the integrase (INT) recombinase, GP3, and the sequence of interest and modified Col E1 origin flanked by the AttL and AttR sites, into the chromosome, bypassing the need for the parental DNA template (plasmid).

[00162] The first gBIock is BPE Landing Pad, which contains partial sequences for GP3 and PrfA-Int, along with full sequences for AttL and insulating terminators. After confirming the insertion of the BPE landing pad into the bacterial chromosome through PCR validation, these bacteria are used to insert the next gBIock, termed BPE 1.1, or herein “1.1”.

[00163] The second gBIock, BPE 1.1, contains the same promoters for the expression cassette, but instead produces SacB and KanR, along with complete the ORF for GP3 and half the AttR site. SacB expression allows use of negative selection to exchange the GOI eukaryotic expression cassette with SacB in the future and permits selection on sucrose plates. The second insertion is screened to confirm integration.

[00164] The third gBIock, the integration cassette, expresses the Phi-BT 1 integrase followed by the beta-lactamase gene to allow for the positive selection of successful integrons. Int remains under the 5’ UTR of PrfA to maintain its thermoregulation. The third insertion is screened to confirm integration.

[00165] The final gBIock integration required for BPE linear is the GOI expression cassette that can be PCR amplified from an intermediate plasmid. The intermediate plasmid allows the building of larger and more complex GOI expression cassettes, bypassing the 3 kb limit imposed onto the synthesis of gBIocks. The fourth and final insertion is screened to confirm integration.

[00166] Eukaryotic Expression Cassette. The eukaryotic expression cassette used in BPE Linear is the same as that of BPE V2.2.

[00167] Workflow and Production Methodology. The workflow and production methodology are similar to that of BPE V2.2 regarding the requirement for Lambda red mediated recombination. The landing pad is compatible with the generation of both BPE V2.2 and BPE linear. BPE linear requires 3 integration events, noted below as Integration Event 1,2, and 3, to build the entire chromosomal cassette with controlled translation of the Phi-BT1 Int.

[00168] Integration Event 7 - LP + 1.1. Landing pad (LP) contains stretches of homology with integron 1.1 at the promoter ProD and for the cofactor GP3. The integration of 1.1 completes the coding capacity of the cofactor GP3. Furthermore, the integration of 1.1 replaces the SmR and tGFP cassette with the negative selection cassette for SacB and KanR. 1.1 also contains a homologous site for BPE integration. Bacteria that contain only LP are fluorescent from the expression of tGFP, which allows screening for both LP integration, and the replacement of LP with 1.1.

[00169] Integration Event 2 - LP + 1.1 + PrfA. After LP has 1.1 integrated into it (LP + 1.1), the next integrant contains the complete coding sequence for the Phi-BT1 Integrase open reading frame under the expression and translational control of the PrfA promoter and 5’ UTR. The PrfA cassette (PrfA) contains homologous sites to LP and an upstream sequence in the bacterial chromosome in the Fli cassette. This cassette also utilizes BLA as a positive selection marker, with these bacteria now resistant to kanamycin, ampicillin, and sensitive to sucrose (through negative selection with SacB). These recombinant bacteria are used to generate a significant cell bank that can be used for future recombinant events with specific BPE GOI expression cassettes. A new recombinant bacterium can be prepared for each BPE product produced.

[00170] Integration Event 3 - LP + 1.1 + PrfA + BPE core. After the bacteria have been modified with LP, 1.1, and PrfA, the BPE core sequence is introduced that will replace the SacB and KanR cassette. Replacing the SacB cassette enables screening for successful recombinant bacteria on sucrose plates.

[00171] The pRR008 Amp KO helper plasmid may be removed in this step, both curing the bacteria and selecting for successful integration. The core BPE cassette contains homologous sequences to the AttL sequence in the LP, and the AttR sequence that was inserted in 1.1. The AttR sequence in 1.1 specifically does not contain the left flank to prevent the excision of the cassette between the AttL and partial AttR sequences at inappropriate times. The integration of the BPE cassette completes the AttR site, allowing for successful excision of the final recombinant bacteria when incubated at 37°C. The final BPE construct can be built in a plasmid, allowing the creation of a larger construct than the current limitations of synthetic biology methodologies will allow. The LP is designed to contain the EcoRV scar on the flanks of each homologous stretch to enable digestion out of the BPE insert from the plasmid using the blunt ended cutter EcoRV.

[00172] Figures 9A, 9B and 9C are illustrations of the integration of landing pad (LP), The SacB negative selection cassette (1.1), the PrfA expression cassette (PrfA), and the final BPE construct from which the progeny plasmid is excised. Sites of homology are annotated with black lines and subheadings. Figure 9A shows LP, 1.1, and [LP+1.1 ]. Figure 9B shows [LP+1.1], PrfA, and [[LP+1.1] + PrfA], Figure 9C shows [[LP+1.1]+PrfA], BPE, and [[[LP+11.]+PrfA] + BPE],

[00173] Figure 10 shows a plasmid map of progeny plasmid from BPE linear. The native Col E1 promoter starts transcription at the first nucleotide of AttP and reads through the entire origin. The mutations created to destroy the RNA I promoter are listed in blue at nucleotides —175. A Kpnl site is also introduced to the Ori sequence downstream of the RNA-DNA hybrid to allow for screening of directionality when building the origin-promoter cassette. The map is generated In Silica and remains as the hypothetical plasmid generated by BPE linear. This image was generated in Geneious™ R 11.0.15.

[00174] Examples

[00175] Modifications of the Core BPE

[00176] Various modifications may be utilized to further increase the productivity, directionality, and stability of the parental, chromosomal, and production / progeny DNA templates (plasmids). Several different pathways are utilized to complete a bacterial operon that is regulated by the temperature of incubation. The translation of the recombinase results in the excision of the progeny plasmid with a completed origin that can result in run away amplification. There are molecular modifications that can be used to optimize this process. Some of these deviations are listed below.

[00177] Modifications to RNA Thermoswitch Translation and Transcription. Improved translational control of the recombinase downstream of the 5’ UTR RNA thermoswitch can be achieved. The 5’ UTR contains a stem loop structure as discussed previously that partially melts in higher incubation temperatures. There are nucleotides that can be mutated to improve the stability of the secondary RNA structure to reduce the expression of the downstream recombinase. These mutations create Watson-Crick base pairing between the top and bottom strand of RNA which prevent the Shine-Dalgarno sequence from being recognized by the host ribosome. Further modifications include secondary structures that anneal to the AUG start codon, along with the upstream Shine-Dalgarno sequence. This hybrid structure prevents the 16S rRNA and anti AUG tRNAfrom binding the mRNA encoding the recombinase.

[00178] Another option to reduce the expression of a target protein is to decrease the strength of the transcribing promoter. Future molecular deviations may utilize a weaker upstream promoter to reduce the amount of RNA transcribed. A weak DAM (DNA Adenosine methylase) promoter may be used, that has been identified to have roughly 1% of the promoter activity of the Lac promoter.

[00179] A further modification for the recombinase itself is its codon optimization. Both codon optimized and non-codon optimized recombinase enzymes can be expressed. Expression of a native, non-codon optimized recombinase will have lower translation efficiencies than an expressed, codon optimized version.

[00180] Figure 11A and Figure 11B illustrate a U12 secondary RNA structure. Figure 11A (SEQ ID NO:9) represents the U12 secondary RNA structure from the publication of Neupert et al. (2008), wherein the U12 RNA thermometer folds in on itself to prevent the recognition of the Shine-Dalgarno sequence. In Figure 11B (SEQ ID NO: 10), the RNA thermometer, (referenced as “U12-Anti AUG”) extends the RNA thermoswitch across the start codon. The melting temperature for the ATG hybridization sequence of U12-Anti AUG RNA thermometer is 37°C, allowing RNA melting and exposure of the start codon at even higher temperatures. The secondary RNA structure of the U12-Anti AUG RNA thermometer was computed using the RNA energy model from Andronescu et a / .(2007) at the temperature of 25°C. This image was generated in Geneious™ R 11.0.15.

[00181] SEQ ID NO:10 is shown in Figure 11B, having a Shine-Dalgarno sequence upstream of the start codon ATG, both of which are underlined below: ATTGGCCATGGATCCTCTCCTTCACTAGTCTCAGAAGGAGATATACCCATGGCCAATTTGCTGACCGT ACAC (SEQ ID NO:10) .

[00182] Modifications of the Recombinase Sites. BPE V2.2 utilizes the Cre-LoxP system to generate the progeny plasmid. Here, the recombination between two LoxP sites yields two identical LoxP sites that are indistinguishable from each other, allowing for further recombination between the two freshly created sites. This allows the product plasmid to recombine with a second plasmid to generate a 2x plasmid that contains two copies of the original plasmid. The wild-type LoxP sites can be modified to contain the Lox66 and Lox71 sites in the artificial inter chromosomal pro-phage that recombine to generate the LoxP-Wt (on the chromosome) and Lox72 sequences (on the progeny plasmid) (Figure 10). Lox72 is unable to recombine with Lox-Wt or Lox72 sequences, which could potentially increase the stability of the progeny plasmid in the presence of Ore. Modifying LoxP may also change the potential cryptic promoter in the Lox-Wt site that may generate RNA II of the modified Col E1 origin in the product plasmid, initiating plasmid replication. These modifications may further be used to control plasmid replication post excision.

[00183] Figure 12 illustrates LoxP Wt and deviations of the recombination sites. The left of Lox66 (SEQ ID NO:11) and the right flank of Lox71 (SEQ ID NO: 12) recombine with each other to generate LoxP Wt (SEQ ID NO: 14) and the Lox72 (SEQ ID NO: 13) sequences. The Lox72 sequence is unable to be recognized by the Ore recombinase, allowing for increased plasmid stability of the progeny plasmid post-excision. This image was generated in Geneious™ R 11.0.15.

[00184] Modification to the Origin of Replication in the Progeny Plasmid. The Origin of replication generated on the progeny plasmid is responsible for the aggressive replication and ultimate yields of product DNA. The aim is to maximize DNA replication in the host fermentation bacteria to lower the cost of goods per dose. There are three significant variables to be assayed to determine the best combination of variables. The three variables are as follows: RNA II promoter strength, location of AttP / LoxP, and deletion / modification of RNA I promoter (Figure 5).

[00185] RNA II Promoter Strength. The strength of the RNA II promoter determines how much RNA is generated from template DNA that produces the secondary RNA structure which induces DNA synthesis. A promoter that is too weak will not generate enough RNA to create the RNA structure, while a promoter that is too strong will rapidly generate RNA that will not be processed by RNase H, inhibiting the maturation of the RNA primer. Plasmid copy number can be finely controlled through minimal mutation of the RNA II promoter. Screening for a promoter strength that works best with the other listed variables post-excision can be undertaken.

[00186] Location of AttP / LoxP Site. The location of AttP / LoxP may affect the secondary structure of RNA necessary for RNase H digestion. Without thorough investigation, these effects will remain unknown. The effect of the location of these RNA structures can be functionally read out by determining plasmid copy number per cell in a multi-variate analysis of a large screen of a series of different locations of AttP and LoxP. For example, AttP / LoxP can either be placed at the very 5’ sequence of RNA II, can replace a stem loop, or can replace all sequences up to the Beta sequence in RNA II (Figure 5).

[00187] RNA I Promoter. The RNA I promoter generates the inhibitory RNA I moleculethat prevents run-away replication. Advantageously, the generation of a maximum amount of progeny plasmid DNA is possible from the fermentation bacteria, as described herein. To do so, a deletion / reduction in the expression of RNA I can be done. Specific residues in the RNA I promoter can be mutated to abolish the -10 and -35 boxes of the RNA I promoter, preventing any RNA from being generated from this promoter region. Different RNA I promotors can be utilized to optimize the formation of the RNA II primer. One option is to replace the first 197 nucleotides of RNA II with the AttP / LoxP site, which would completely remove the RNA I promoter.

[00188] Multiple Integrations of Pro-phage Cassette. Integrating several alliterations of the BPE pro-phage cassette into the genome would give more than one copy to excise from the genome. The excision of the pro-phage cassette may have a high rate of success, however it is unlikely to be 100%. Providing more than one LoxP / AttL-AttR cassette to excise can enable near 100% progeny plasmid generation post-excision. There are several dispensable sequences within the genome offermentation bacteria, allowing incorporation of several copies of the pro-phage cassette by modifying the site of recombination of this parental DNA template (plasmid).

[00189] Host-specific Plasmid Replication. Other technologies that generate significant amounts of DNA vector therapeutics may have designed a bacterial origin to require a protein that is specifically synthesized from the host fermentation bacteria. This prevents the product plasmid from replicating in any host that uptakes the plasmid in the environment. The RNA II promoter is specifically engineered to be active in the fermentation bacteria. Further modifications that can result in this include the controlled expression of a viral polymerase, such as T3, that replaces the AmpR promoter or native Col E1 promoter. This can restrict the plasmid to only replicate in cells that encode this specific polymerase, further increasing the safety profile of the product line.

[00190] Example 4

[00191] Exemplary Plasmid Sequence: PBE RNA Switch V2.31

[00192] An exemplary plasmid sequence “BPE RNA Switch V2.31” is provided below as SEQ ID NO: 15. This exemplary sequence, for expressing a sequence of interest, or gene of interest (GOI), comprises an RNA thermometer for Cre recombinase expression, which acts upon the LoxP recombinase recognition site. The modified Col E1 origin comprises of the LoxP site and pUC19 sequences, while the RNA II promoter is found 5' of the distal LoxP site found in the BLA cassette. SEQ ID NO: 15 is as follows:

[00193] TTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGG CGGTTTTTC GAAGGT T C T C T GAGC TAC CAAC T C T T T GAAC C GAGGTAAC T GGC T T GGAGGAGC GCAGT CAC CAAA ACTTGTCCTTTCAGTT TAGC C T TAAC C GGC GCAT GAC T T CAAGAC TAAC T C C T C TAAAT CAAT TAC CAGT GGC T G C T GC CAGT GGTGCTTTT GCAT GT C T T T C C GGGT T GGAC T CAAGAC GATAGT TAC C GGATAAGGC GCAGC GGT C GG AC T GAAC GGGGGGT T C GT GCATACAGT C CAGC T T GGAGC GAAC T GC C TAC C C GGAAC T GAGT GT CAGGC GT GGAA T GAGACAAAC GC GGC CATAACAGC GGAAT GACAC C GGTAAAC C GAAAGGCAGGAACAGGAGAGC GCAC GAGGGAG C C GC CAGGGGAAAC GCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC CAC T GAT T T GAGC GT CAGAT T T C GT GAT GC T T GT CAGGGGGGC GGAGC C TAT GGAAAAAC GGCTTTGCCGCGGCCCTCT CAC TTCCCTGT TAAGTAT C T T CCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAG C GAGT CAGT GAGC GAGGAAGC GGAATATAT C C T GTAT CACATAT T C T GC T GAC GCAC C GGT GCAGC CTTTTTTCT C C T GC C GGCAT GGC GTAC T T T T CATAGATAT CAT GGCAGATAAT C CAGAC C C T T CAT C GC T C C T GC C GGAC GT GT T T T CAC CAC C GGT GAGAC GACAT C TAT GGAT GTAC T CAGC GAGCAGAT GC T C T C C T GT CAGC C C GC C TAAT GAGC GGGCTTTTTTTTCCAC CAAAAC GAC C C T GAAGGC C CAT GGAAAAGGGC T C GC GC T TAACAGT GT C GT GAGAAGGC CAT C C T GAC GGAT GGCCTTTTCCC GGAT GGAAT T CAT GGC CAAT T TAC T GAC C GTACAC CAAAAT T T GC C T GCAT TAC CAGT C GAT GCAAC GAGT GAT GAGGT T C GCAAGAAC C T GAT GGACAT GT T CAGGGAT C GC CAGGC GTTTTCTG AGCATAC C T GGAAAAT GCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGT GCAAGT T GAATAAC C GGAAAT GGT T T C C C GCAGAAC C T GAAGAT GT T C GC GAT TAT CTTCTATATCTT CAGGC GCGCGGTCT GGCAGTAAAAAC TAT C C AGCAACAT T T GGGC CAGC TAAACAT GC T T CAT C GT C GGT C C GGGC T GC CAC GAC CAAGT GACAGCAAT GC T GT T T CAC T GGT TAT GC GGC GGAT C C GAAAAGAAAAC GT T GAT GC C GGT GAAC GT GCAAAACAGGC TCTAGCGTTC GAAC GCAC T GAT T T C GAC CAGGT T C GT T CAC T CAT GGAAAATAGC GAT C GC T GC CAGGATATAC GTAAT C T GGCAT T T C T GGGGAT T GC T TATAACAC C C T GT TAC GTATAGC C GAAAT T GC CAGGAT CAGGGT TAAAGATAT T T CAC GTAC T G AC GGT GGGAGAAT GT TAAT C CATAT T GGCAGAAC GAAAAC GC T GGT TAGCAC C GCAGGT GTAGAGAAGGCAC T TA GC C T GGGGGTAAC TAAAC T GGT C GAGC GAT GGAT T T C C GTAT C T GGT GTAGC T GAT GAT C C GAATAAC TAC C T GT T T T GC C GGGT CAGAAAAAAT GGT GT T GC C GC GC CAT C T GC CAC CAGC CAGC TAT CAAC T C GC GC C C T GGAAGGGA T T T T T GAAGCAAC T CAT C GAT T GAT T TAC GGC GC TAAGGAT GAC T C T GGT CAGAGATAC CTGGCCTGGTCT GGAC ACAGTGCCCGTGTC GGAGC C GC GC GAGATAT GGCCCGCGCT GGAGT T T CAATAC C GGAGAT CAT GCAAGC T GGT G GC T GGAC CAAT GTAAATAT T GT CAT GAAC TATAT C C GTAAC C T GGATAGT GAAACAGGGGCAAT GGT GC GC C T GC T GGAAGAT GGC GAT TAAAC GTAAATAAATAT C C T T T GTAT T CAT T T GT T TATAGT CAGAAGGC CAT C C T GAC GGA TGGCCTTTT GAC T C GCAT C C T CAC GATAATAT C C GGGTAGGC GCAAT CACTTTCGTCTACTCCGT TACAAAGC GA GGCTGGGTATTTCCCGGCCTTTCTGTTATCC GAAAT C CAC T GAAAGCACAGC GGC T GGC T GAGGAGATAAATAAT AAAC GAGGGGC T GTAT GCACAAAGCAT CTTCTGTT GAGT TAAGAAC GAGTAT C GAGAT GGCACATAGC C T T GC T C AAAT T GGAAT CAGGT T T GT GC C AATAC CAGTAGC T C GAGT GATAAC T T C GTATAGCATACAT TATAC GAAGT TAT AAGGTAAC T GGC T T CAGCAGAGC GCAGATAC CAAATAC T GT T C T T C TAGT GTAGC C GTAGT TAGGC CAC CAC T T C AAGAAC T C T GTAGCAC CGCCTACATACCTCGCTCTGC TAAT C C T GT TAC CAGT GGC T GC T GC CAGT GGC GATAAG T C GT GT C T TAC C GGGT T GGAC T CAAGAC GATAGT TAC C GGATAAGGC GCAGC GGT C GGGC T GAAC GGGGGGT T C G TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC AC GC T T C C C GAAGGGAGAAAGGC GGACAGGTAT C C GGTAAGC GGCAGGGT C GGAACAGGAGAGC GCAC GAGGGAG CTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTG T GAT GC T C GT CAGGGGGGC GGAGC C TAT GGAAAAAC GC CAGCAAC GCGGCCTTTTTACGGTTCCTGGCCTTTTGC TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA GCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATA C GCAAAC CGCCTCTCCCCGCGCGTTGGCC GAT T CAT TAAT GCAGC C GGCAT GGC GTAC T T T T CATAGC GGGCAGT AGC C C GGGGGCAT T GAT TAT T GAC TAGT TAT TAATAGTAAT CAAT TAC GGGGT CAT TAGT T CATAGC C CATATAT GGAGT T C C GC GT TACATAAC T TAC GGTAAAT GGCCCGCCTGGCT GAC C GC C CAAC GAC CCCCGCCCATT GAC GT C AATAAT GAC GTAT GT T C C CATAGTAAC GC CAATAGGGAC T T T C CAT T GAC GT CAAT GGGT GGAGTAT T TAC GGTA AAC T GC C CAC T T GGCAGTACAT CAAGT GTAT CATAT GC CAAGT CCGCCCCCTATT GAC GT CAAT GAC GGTAAAT G GC C C GC C T GGCAT TAT GC C CAGTACAT GAC C T TAC GGGAC TTTCCTACTT GGCAGTACAT C TAC GTAT TAGT CAT C GC TAT TAC CAT GGT GAT GC GGT T T T GGCAGTACAC CAAT GGGC GT GGATAGC GGT T T GAC T CAC GGGGAT T T C C AAGT C T C CAC C C CAT T GAC GT CAAT GGGAGT T T GT T T T GGCAC CAAAAT CAAC GGGAC T T T C CAAAAT GT C GTAA CAAC TGC GAT CGCCCGCCCCGTT GAC GCAAAT GGGC GGTAGGC GT GTAC GGT GGGAGGT C TATATAAGCAGAGC T GAC GT GGC TAGC GGAT CAT TAT C GGAAGGGGC GGCAGCAAAAGCAGGGTAGATAAT CAC T CAAT GAGGTAAGT T T AGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTT GCCTTTACTTCTAGT GACAT C GAAAC CAT GGAGAGC GAC GAGAGC GGC C T GC C C GC CAT GGAGAT C GAGT GC C GC AT CAC C GGCAC C C T GAAC GGC GT GGAGT T C GAGC TGGTGGGCGGC GGAGAGGGCAC C C C C GAGCAGGGC C GCAT G AC CAACAAGAT GAAGAGCAC CAAAGGC GC C C T GAC CTTCAGCCCCTACCTGCT GAGC CAC GT GAT GGGC TAC GGC T T C TAC CAC T T C GGCAC C TAC C C CAGC GGC TAC GAGAAC CCCTTCCT GCAC GC CAT CAACAAC GGC GGC TACAC C AACAC C C GCAT C GAGAAGTAC GAGGAC GGC GGC GT GC T GCAC GT GAGC T T CAGC TAC C GC TAC GAGGC C GGC C GC GT GAT C GGC GAC T T CAAGGT GAT GGGCAC C GGC T T C C C C GAGGACAGC GT GAT C T T CAC C GACAAGAT CAT C C GC AGCAAC GC CAC C GT GGAGCAC C T GCAC C C CAT GGGC GATAAC GAT C T GGAT GGCAGC T T CAC C C GCAC C T T CAGT C T GC GC GAC GGC GGC TAC TACAGC T C C GT GGT GGACAGC CACAT GCAC T T CAAGAGC GCCATCCACCC CAGCAT C C T GCAGAAC GGCGGCCCCATGTTCGCCTTCCGCCGCGT GGAGGAGGAT CACAGCAACAC C GAGC TGGGCATCGTG GAGTAC CAGCAC GC C T T CAAGAC C C C GGAT GCAGAT GC C GGT GAAGAATAAGGAAAAATAC CCTTGTTTCTACTG AGCTCCTGT GCAAC T T GT T TAT T GCAGC T TATAAT GGT TACAAATAAAGCAATAGCAT CACAAAT T T CACAAATA AAGCAT T T T T T T CAC T GCAT T C TAGT T GT GGT T T GT C CAAAC T CAT CAAT GTAT C T TAGGGAC TTTCCGGTCGGG ACTTTCCAATTGCGCGGCCGCTCTTTTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTGGCAAACAAAAAAAC CAC C GC TAC CAAC GGT GGT T T GT T T GCATAAC T T C GTATAGCATACAT TATAC GAAGT TATAGGAGGTAC GC GTA TGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA GC GGTAAGAT C C T T GAGAGT TTTCGCCCC GAAGAAC GT T T T C CAAT GAT GAGCAC T T T TAAAGT T C T GC TAT GT G GC GC GGTAT TAT C C C GTAT T GAC GC C GGGCAAGAGCAAC T C GGT C GC C GCATACAC TAT T C T CAGAAT GAC T T GG T T GAGTAC T CAC CAGT CACAGAAAAGCAT C T TAC GGAT GGCAT GACAGTAAGAGAAT TAT GCAGT GC T GC CATAA C CAT GAGT GATAACAC T GC GGC CAAC T TAC T T C T GACAAC GAT C GGAGGAC C GAAGGAGC TAAC CGCTTTTTTGC ACAACAT GGGGGAT CAT GTAAC T C GC C T T GAT C GT T GGGAAC C GGAGC T GAAT GAAGC CATAC CAAAC GAC GAGC GT GACAC CAC GAT GC C T GTAGC GAT GGCAACAAC GT T GC GCAAAC TAT TAAC T GGC GAAC TAC T TAC T C TAGC T T C C C GGCAACAAT TAATAGAC T GGAT GGAGGC GGATAAAGT T GCAGGAC CAC TTCTGCGCTCGGCCCTTCCGGCTG GC T GGT T TAT T GC T GATAAAT C C GGAGC C GGT GAGC GTGGTTCTCGCGGTAT CAT C GCAGC GC T GGGGC CAGAT G GTAAGC CCTCCCGTATC GTAGT TAT C TACAC GAC GGGGAGT CAGGCAAC TAT GGAT GAAC GAAATAGACAGAT C G C T GAGATAGGT GC C T CAC T GAT TAAGCAT T GGTAATAAAGAAAC T C C GCAGT C C GAAC C C TAAGTAGTAT C TAT T CGGGTTCTCGGAATGCATTGGCGCTGTCAGCCCGCCTAATGAGCGGGCTTTTTTTTGGATCCTGCGTGAGGCGGG AT T T T CAAGT T C GC CACAAGGGC T GGAT T GGGGATAGGATAT C.

[00194] Examples

[00195] Exemplary Plasmid Sequence: BPE RNA Switch V2.41

[00196] An exemplary Plasmid sequence “BPE RNA Switch V2.41” is provided below as SEQ ID NO:16. The exemplary V2.41 sequence uses the INT recombinase, which acts upon the AttL / R, the modified Col E1 origin comprises the AttL sequence, while AttR is found in the BLA cassette with the 5' RNA II promoter. SEQ ID NO:16 is as follows:

[00197] TTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAG C GGTAT CAGC T CAC T CAAAGGC GGTAATAC GGT TAT C CACAGAAT CAGGGGATAAC GCAGGAAAGGGAT C TAACA AC T TATAT C GTAT GGGGC T GAC T T CAGGT GC TACAT T T GAAGAGATAAAT T GCAC T GAAAT C TAGTAATAT T T TA TCTGATTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAATCTCTTGCTCTGAAAACGAAAAAACCG CCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGC AGT CAC CAAAAC TTGTCCTTTCAGTTTAGCCT TAAC C GGC GCAT GAC T T CAAGAC TAAC T C C T C TAAAT CAAT TA C CAGT GGC T GC T GC CAGT GGT GC T T T T GCAT GT C T T T C C GGGT T GGAC T CAAGAC GATAGT TAC C GGATAAGGC G CAGC GGT C GGAC T GAAC GGGGGGTTCGT GCATACAGT C CAGC T T GGAGC GAAC T GC C TAC C C GGAAC T GAGT GT C AGGC GT GGAAT GAGACAAAC GC GGC CATAACAGC GGAAT GACAC C GGTAAAC C GAAAGGCAGGAACAGGAGAGC G CAC GAGGGAGC C GC CAGGGGGAAAC GC C T GGTAT C T T TATAGT CCTGTCGGGTTTCGC CAC CAC T GAT T T GAGC G TCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTG TTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACG AC C GAGC GTAGC GAGT CAGGAGGAAGC GGAATATAT CCCCTAGGTC TAC T T T T T T C T C C T GC C GGCAT GGC GTAC T T T T CATAGATAT CAT GGCAGATAAT C CAGAC CCTTCATCGCTCCTGCC GGAC GT GT T T T CAC CAC C GGT GAGAC GACAT C TAT GGAT GTAC T CAGC GAGCAGAT GCTCTCCTC GGTAC CAAAT T C CAGAAAAGAGGC C GC GAAAGC GGC CTTTTTTCGTTTTGGTCCCCACCAAAACGACCCTGAAGGCCCATGGAAAAGGGCTCGCGCTTAACAGTGTCGTGA GAAGGCCATCCTGACGGATGGCCTTTTCCCGGATGGAATTCATGAGCCCGTTCATTGCACCAGATGTCCCAGAAC ACCTTCTGGACACCGTCCGTGTCTTCCTCTACGCACGTCAGTCCAAGGGTCGTTCTGATGGCTCTGATGTATCCA C C GAAGCACAAC TCGCGGCTGGTCGTGCTCTGGTTGCGTCCC GTAAC GC T CAGGGC GGTGCTCGTT GGGT AGT C G CAGGC GAGT T T GT GGAT GT T GGT C GCAGC GGC T GGGAC C C GAAC GT CAC T C GT GC T GAT T T T GAGC GTAT GAT GG GC GAAGT GCGTGCTGGC GAAGGT GAT GTAGT GGTAGT GAAC GAAC T CAGC CGCCTCACTC GCAAAGGT GC T CAC G AT GC T C T GGAGAT T GATAAC GAGT T GAAGAAACAT GGCGTCCGCTT TAT GAGC GT T C T GGAGC C GT T T C T GGATA C T T C CAC T C C TAT C GGT GT GGCAAT CTTTGCTCTCATCGCCGCACTTGC CAAACAGGATAGC GAC C T GAAAGC GG AAC GC C T GAAAGGT GCAAAAGAC GAAAT T GC T GCAC TGGGTGGCGTTCACTCCTCTTCT GCAC C C T T C GGTAT GC GT GC T GT GC GTAAGAAAGT GGACAAT C T GGT TAT CTCCGTGCT GGAGC CAGAT GAGGACAAC C CAGAC CAC GT GG AAC T GGT T GAAC GTAT GGC GAAAAT GT C C T T T GAAGGAGT C T C GGACAAC GCAAT C GCAAC CAC T T T T GAAAAAG AGAAAAT TCCTTCTCCGGGTATGGCT GAAC GC C GT GC CAC C GAAAAGC GT C T GGCAAGC GTAAAAGC C C GT C GC C T GAAT GGT GC T GAAAAAC C TAT CAT GT GGC GT GC C CAGAC C GT T C GC T GGATAC T GAAC CAC C CAGC CAT T GGT G GCTTTGCCTTT GAGC GC GT CAAACAC GGTAAAGCACACAT CAAT GTAAT C C GC C GT GAC C CAGGGGGTAAAC CAC T GAC C C C GCACAC T GGTAT C C T GAGC GGTAGCAAGT GGC T T GAAC T T CAGGAAAAGC GTAGC GGTAAAAAT C T C T CTGACCGTAAACCTGGTGCTGAGGTTGAGCCTACCCTGCTCTCTGGCTGGCGGTTTCTGGGCTGCCGTATCTGTG GC GGTAGCAT GGGACAGT C T CAGGGC GGT C GTAAAC GCAAC GGT GAT T TAGCAGAAGGCAAC TACAT GT GT GC CA AT C C GAAAGGT CAC GGC GGT C T CAGC GT CAAAC GT T C GGAAC T GGAC GAGT TCGTGGCTTC CAAAGT GT GGGC T C GC C T C C GCAC C GCAGACAT GGAAGAC GAACAT GAC CAAGCAT GGAT T GC T GC GGC T GC C GAAC GT T T C GCAC T T C AGCAT GAC C T T GCAGGC GT GGC T GAC GAAC GC C GT GAGCAACAGGC T CAT C T GGACAAC GT T C GGC GT T C TAT CA AAGAC C T T CAGGC T GAC C GTAAAC CAGGT C T GTAT GT T GGAC GC GAGGAAC T GGAAAC GT GGC GTAGCAC C GTAT T GCAATAC C GT T C T TAC GAAGCAGAAT GCAC GAC CCGTCTGGCT GAAC T T GAT GAAAAAAT GAAC GGAAGCAC T C GT GT T C C T T C GGAAT GGT T CAGT GGT GAAGAC C C GAC C GC T GAAGGT GGCAT C T GGGCAT C GT GGGAC GT GTAT G AAC GC C GT GAAT TTCTGTCTTTCTTTCT GGATAGC GT TAT GGT T GATAGGGGT C GC CAC C CAGAGAC CAAGAAAT ACAT C C C GC T GAAAGAC CGCGTTACTCT GAAAT GGGCAGAAC T T C T CAAAGAGGAAGAT GAGGCAT C GGAAGCAA CCGAACGTGAACTTGCCGCTCTGTAATAAACGTCTGTCAGCCCGCCTAATGAGCGGGCTTTTTTTTGAAAAGGGC T C GC GC T TAACAGT GTCGTGCTC GGTAC CAAAT T C CAGAAAAGAGGC C GC GAAAGC GGCCTTTTTTCGTTTTGGT C C GAC T C T C GAGT GGCCCGCTGCCGTCCTT GAC CAGGT T T T T GAC GAAAGT GAT C CAT GGGAAAC TACT CAGCAC CAC CAAT GT T C C GCAAACAAAAAAAC CAC C GC TAC CAGC GGTGGTTTGTTTGCC GGAT CAAGAGC TAC CAAC T C T T T T T C C GAAGGTAAC T GGC T T CAGCAGAGC GCAGATAC CAAATAC TGTTCTTCTTGTGTTGCC GTAGT TAGGC CA CAACAACAAGAAC T C T GTAGCAC C GC C TACATAC CTCGCTCTGC TAAT C C T GT TAC CAGT GGC T GC T GC CAGT GG C GATAAGT CGTGTCTTACCGGGTT GGAC T CAAGAC GATAGT TAC C GGATAAGGC GCAGC GGT C GGGC T GAAC GGG GGGT T C GT GCACACAGC C CAGC T T GGAGC GAAC GAC C TACAC C GAAC T GAGATAC C TACAGC GT GAGC TAT GAGA AAGC GC CAC GC T T C C C GAAGGGAGAAAGGC GGACAGGTAT C C GGTAAGC GGCAGGGT C GGAACAGGAGAGC GCAC GAGGGAGC T T C CAGGGGGAAAC GC C TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT GAC T T GAGC GT C G ATT T T T GTGAT GCTCGTCAGGGGGGC GGAGC C TAT GGAAAAAC GC CAGCAAC GC GGC C T T T T TAC GGT T C C T GGC CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTT T GAGT GAGC T GATAC C GC T C GC C GCAGC C GAAC GAC C GAGC GCAGC GAGT CAGT GAGC GAGGAAGC GGAAGAGC G C C CAATAC GCAAAC CGCCTCTCCCCGCGCGTTGGCC GAT T CAT TAAT GCAGC C GGCAT GGC GTAC T T T T CATAGC GGGCAGTAGC C C GGGGGCAT T GAT TAT T GAC TAGT TAT TAATAGTAAT CAAT TAC GGGGT CAT TAGT T CATAGC C CATATAT GGAGT T C C GC GT TACATAAC T TAC GGTAAAT GGCCCGCCTGGCT GAC C GC C CAAC GAC C C C C GC C CAT T GAC GT CAATAAT GAC GTAT GT T C C CATAGTAAC GC CAATAGGGAC T T T C CAT T GAC GT CAAT GGGT GGAGTAT T TAC GGTAAAC T GC C CAC T T GGCAGTACAT CAAGT GTAT CATAT GC CAAGT C C GC C C C C TAT T GAC GT CAAT GAC G GTAAAT GGC C C GC C T GGCAT TAT GC C CAGTACAT GAC C T TAC GGGAC T T T C C TAC T T GGCAGTACAT C TAC GTAT TAGT CAT C GC TAT TAC CAT GGT GAT GC GGT T T T GGCAGTACAC CAAT GGGC GT GGATAGC GGT T T GAC T CAC GGG GATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAAT GT C GTAACAAC T GC GAT CGCCCGCCCCGTT GAC GCAAAT GGGC GGTAGGC GT GTAC GGT GGGAGGT C TATATAAG CAGAGC T GAC GT GGC TAGC GGAT CAT TAT C GGAAGGGGC GGCAGCAAAAGCAGGGTAGATAAT CAC T CAAT GAGG TAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGT GGAT GT T GC C T T TAC T T C TAGT GACAT C GAAAC CAT GGAGAGC GAC GAGAGC GGCCTGCCCGCCAT GGAGAT C GA GT GC C GCAT CAC C GGCAC C C T GAAC GGC GT GGAGT T C GAGC TGGTGGGCGGC GGAGAGGGCAC C C C C GAGCAGGG C C GCAT GAC CAACAAGAT GAAGAGCAC CAAAGGC GC C C T GAC CTTCAGCCCC TAC C T GC T GAGC CAC GT GAT GGG C TAC GGC T T C TAC CAC T T C GGCAC C TAC C C CAGC GGC TAC GAGAAC C C C T T C C T GCAC GC CAT CAACAAC GGC GG C TACAC CAACAC C C GCAT C GAGAAGTAC GAGGAC GGCGGCGTGCT GCAC GT GAGC TTCAGCTACCGCTAC GAGGC CGGCCGCGTGATCGGCGACTTCAAGGTGATGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGAT CAT C C GCAGCAAC GC CAC C GT GGAGCAC C T GCAC C C CAT GGGC GATAAC GAT C T GGAT GGCAGC T T CAC C C GCAC CTTCAGTCTGCGC GAC GGC GGC TAC TACAGC T C C GT GGT GGACAGC CACAT GCAC T T CAAGAGC GC CAT C CAC C C CAGCAT C C T GCAGAAC GGCGGCCCCATGTTCGCCTTCCGCCGCGT GGAGGAGGAT CACAGCAACAC C GAGC T GGG CAT C GT GGAGTAC CAGCAC GC C T T CAAGAC C C C GGAT GCAGAT GC C GGT GAAGAATAAGGAAAAATAC C C T T GT T T C TAC T GAGC T C C T GT GCAAC T T GT T TAT T GCAGC T TATAAT GGT TACAAATAAAGCAATAGCAT CACAAAT T T C ACAAATAAAGCAT TTTTTTCACTGCATTC TAGT TGTGGTTTGTC CAAAC T CAT CAAT GTAT C T TAGGGAC T T T C C GGTCGGGACTTTCCAATTGCGCGGCCGCTCTTTTTAAATCCTTTTTTTCTGCGCGTATGATGCTGCTGAAGACGT TTCGGGTGCTGGGTTGTTGTCTCT GGACAGT GAT C CAGAT GAT C CAGC T C CACAC C C C GAAC GC GAGAGGAGGTA CGCGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATC T CAACAGC GGTAAGAT C C T T GAGAGT TTTCGCCCC GAAGAAC GT T T T C CAAT GAT GAGCAC T T T TAAAGT T C T GC TAT GT GGC GC GGTAT TAT C C C GTAT T GAC GC C GGGCAAGAGCAAC T C GGT C GC C GCATACAC TAT T C T CAGAAT G AC T T GGT T GAGTAC T CAC CAGT CACAGAAAAGCAT C T TAC GGAT GGCAT GACAGTAAGAGAAT TAT GCAGT GC T G C CATAAC CAT GAGT GATAACAC T GC GGC CAAC T TAC T T C T GACAAC GAT C GGAGGAC C GAAGGAGC TAAC C GC T T T T T T GCACAACAT GGGGGAT CAT GTAAC T C GC C T T GAT C GT T GGGAAC C GGAGC T GAAT GAAGC CATAC CAAAC G AC GAGC GT GACAC CAC GAT GC C T GTAGC GAT GGCAACAAC GT T GC GCAAAC TAT TAAC T GGC GAAC TAC T TAC T C TAGC T T C C C GGCAACAAT TAATAGAC T GGAT GGAGGC GGATAAAGT T GCAGGAC CAC TTCTGCGCTCGGCCCTTC CGGCTGGCTGGTTTATTGCT GATAAAT C C GGAGC C GGT GAGC GTGGTTCTCGCGGTAT CAT C GCAGC GC T GGGGC CAGAT GGTAAGC C C T C C C GTAT C GTAGT TAT C TACAC GAC GGGGAGT CAGGCAAC TAT GGAT GAAC GAAATAGAC AGAT C GC T GAGATAGGT GC C T CAC T GAT TAAGCAT T GGTAATAAAGAAAC T C C GCAGT C C GAAC C C TAAGTAGTA TCTATTCGGGTTCTCGGAATGCATTGGCGCTGTCAGCCCGCCTAATGAGCGGGCTTTTTTTTGTCGACTGCGTGA GGC GGGAT T T T CAAGT T C GC CACAAGGGC T GGAT T GGGGATAGGATAT C.

[00198]      Example6

[00199]      Exemplary Plasmid Sequence: PBE 2.6 with Terminator Switch

[00200] An exemplary plasmid is described, and a method to insert the parental DNA template into the chromosome.

[00201] Thermal permissive terminators may be used in the method for production of the product DNA plasmid. A terminator / promoter (promoter / terminator) control cassette may be used for recombinase expression. A thermally permissive terminator may be used to control recombinase expression (for example when located just upstream of the recombinase) that is triggered by promoters, which promoters can optionally be thermoregulated themselves, located farther upstream of the recombinase. For example, the location farther upstream may be in front of other expressed genes that are upstream from the recombinase. Optionally, a thermally controlled promoter located upstream of the recombinase or a thermally permissive terminator may be used to control expression.

[00202] Reference is made to Figure 13A, Figure 13B and Figure 13C. A plasmid map is provided of a progeny plasmid from BPE version 2.6.

[00203] Figure 13A illustrates that BPE 2.6 contains AttP sites from the C31 phage to allow integration into the AttB sites found in the genetically engineered landing pad depicted in Figure 13B. All sequences between the AttP and AttB sites are to be exchanged with each other, allowing the gene of interest to replace the integrase coding sequence within the genome. These images were generated in Geneious™ R 11.0.15 (genious.com).

[00204] In Figure 13A, a plasmid map of the parental plasmid from BPE version 2.6 is shown. BPE V2.6 utilizes two AttP sites from the C31 phage to enable directional insertion of the parental plasmid into the modified host fermentation bacteria. All genetic information between the two AttP sites will be exchanged with the genetic material between the two AttP sites in the 2.6 landing pad. The SacB coding sequence and the SacB promoter will not be integrated into the chromosome, allowing for 5% sucrose counter selection against the maintenance of the parental plasmid post chromosomal integration. Advantageously, BPE V2.6 utilizes directional recombinase (integrase) from the C31 phage into the chromosome, which streamlines the generation of recombinant bacteria. BPE V2.6 still utilizes the Ore Lox system to produce the product plasmid from the bacterial chromosome post C31-integrase mediated insertion. There are directional terminators flanking the AttP sites disallowing the expression of the kanamycin resistance open reading frame. Once the parental plasmid is inserted into the landing pad within the chromosome, the kanamycin cassette is expressed through the removal of the directional terminator and the newly placed lac promoter / AmpR promoter. This enables positive selection of the integrant through kanamycin selection.

[00205] In Figure 13B, a map is provided of the modified landing pad containing two AttB sites from the C31 phage, the coding sequence of the recombinase / integrase from the C31 phage, and a positive selection marker Chloramphenicol R (Chlor-R). The landing pad integration into the chromosome is selected through the expression of Chlor-R, which also leads to the poly-cystronic expression of the Int-recombinase from the C31 phage. The expression of the integrase enables the insertion of the parental plasmid into the landing pad sequence through the recombination between the AttP and AttB sequences found on the parental plasmid and the landing pad. This integration results in the destruction of the Integrase expression cassette, enabling the stable insertion of the parental plasmid into the host chromosome.

[00206] Figure 13C depicts a linear map of the modified landing pad and inserted parental plasmid in the chromosome of the target bacteria. The insertion of parental plasmid replaces the ChlorR and INT sequences with the parental plasmid. The landing pad contains the appropriate promoters to express the KanR cassette, allowing positive selection of the integration event. This image was generated in Geneious™ R 11.0.15

[00207] Figure 13C also represents the utilization of the thermal permissive terminator control of Ore expression from SEQ ID NO: 18 termed “Bla_term_ci857”. SEQ ID NO: 18 contains upstream 3 constitutively active promoters to a coding sequence, followed by the BLA and rrnB T2 RHO independent terminators. The expression dosage from the constitutively active promoters and the terminators prevent Ore expression at 25 °C, but enables the recombinases expression at 37 °C. This combination of upstream promoters and terminators allows fine temperature control of the expression of the Ore recombinase, which acts to directly excise the product plasmid from the host chromosome.

[00208] The sequence “Bla_term_ci857” having 2051 bp (SEQ ID NO: 18) is provided herein, with promoters at: 80..123 (j23101); 124..159 (ColE1 mutant 832); 160..264 (AmpR promoter) and 224..249; ribosomal binding sites at: 265..271 and 999..1004; terminators at: 1849..1881 (beta-lactamase “bla” terminator) and 1911..1938 (transcription terminator from T2 of E. coli rrnB, or “rrnB T2 terminator”); as well as other regulatory components such as a vertebrate consensus sequence for strong initiation located at 1953..1956. As to coding sequence features (CDS) within SEQ ID NO: 18, these include flagellar motor switch protein fliM (SEQ ID NO: 19) of Reference Sequence No: WP_005127760.1 and Protein id No: WP_001350520.1 (complement 79); KanR / NeoR (SEQ ID NO:20) and a Ore site-specific recombinase (SEQ ID NO:21) from Bacteriophage P1 wherein recombination occurs at loxP sequences.

[00209] The method described advantageously permits control of the production of plasmid DNA during bacterial fermentation by controlling the relocation of the plasmid RNA II promoter to upstream of the RNA II primer template sequence through recombination. The RNA II primer is responsible for initiating plasmid replication. The bacterial origin of the linearized plasmid was stably inserted into the host bacterial chromosome by separating the origin’s promoter from the RNA II primer template sequence, thus allowing antibiotic-free fermentation of the host while the plasmid is hidden within the host chromosome. At the desired time, the excision of the targeted plasmid DNA was triggered / induced by temperature, thus permitting controlled recombination. This resulted in the formation of a plasmid with a fully functional bacterial origin that is lacking negative regulation. This permitted run-away amplification and high / significant levels of plasmid DNA production.

[00210] Aspects

[00211] The methods and products described herein are to be understood in the context of the following aspects of the invention, as described and encompassed herein.

[00212] Aspect 1. A method for production of a product DNA plasmid comprising a sequence of interest, said method comprising:

[00213] preparing a parental DNA template comprising a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a RNA II primer template sequence; a recombinase coding sequence encoding an associated recombinase that is associated with the first and the second recombinase recognition sites; an inducible switch for control of recombinase translation; and the sequence of interest;

[00214] wherein the sequence of interest and the modified Col E1 origin comprise or are flanked by Lox or Att sites, which are recognized by the associated recombinase for excision to reconstitute the RNA II promoter upstream of the RNA II primer template sequence, thereby producing the RNA II primer, and to thereby form a self-replicating product DNA plasmid; wherein translation of the recombinase is controlled by the inducible switch under a control condition; and

[00215] wherein the modified Col E1 origin comprises Col E1 RNA II and Col E1 RNA I promoters, and the modified Col E1 is modified by at least one mutation or deletion in the Col E1 RNA I promoter to reduce or eliminate RNA I inhibition to increase replication of the origin post-recombination;

[00216] preparing a host bacterium having the parental DNA template inserted into the chromosome of said host organism;

[00217] fermenting the host organism under the control condition that induces the inducible switch to express the recombinase, to excise the product plasmid from the host organism;

[00218] producing the product DNA plasmid in the host organism; and

[00219] producing the product DNA plasmid comprising the sequence of interest and the modified origin, that is excised from the chromosome of the host organism.

[00220] Aspect 2. The method of Aspect 1, wherein the parental DNA template comprises a recombinase cassette and sequence specific homology to the host organism, such as a bacterium, to enable targeted recombination to the host genome, or a phage attachment site to utilize a serine / tyrosine recombinase to integrate the parental DNA template into a chromosome.

[00221] Aspect 3. The method of Aspect 1 or 2, wherein the recombinase is a sequence specific recombinase matched with the Lox or Att site, such as a Cre recombinase for Lox-Lox excision, for example LoxP, Lox66 or Lox 71; or is Phi-BT 1 integrase (INT) for AttL / AttR excision.

[00222] Aspect 4.    The method of any one of Aspects 1 to 3, wherein the parental DNA template is a plasmid or is a linear DNA template.

[00223] Aspect 5.    The method of any one of Aspects 1 to 4, wherein the inducible switch for control of recombinase translation is an inducible promoter, a thermal-permissive terminator, a temperature sensitive inhibitor, a small molecule inducible promoter such as the IPTG inducible Lac promoter; a pH inducible promoter, ora light inducible promoter.

[00224] Aspect 6. The method of Aspect 5, wherein the inducible promoter is temperature-inducible such as a Lambda virus pR / pL promoter-cl857, a trans-acting 5’ untranslated region (5’ UTR) synthetic RNA thermoswitch, or a PrfA RNA thermoswitch; or comprises synthetic 5’ UTR RNA hairpins wherein the production of recombinase mRNA is under a weak promoter.

[00225] Aspect 7. The method of any one of Aspects 1 to 6, wherein the modified Col E1 origin comprises a recombinase recombination site; a forward promoter sequence to generate the RNA II primer (RNA II); and a sequence-specific stretch of DNA to generate a stable hairpin structure to initiate replication of the excised product plasmid;

[00226] whereby a RNA-DNA hybrid structure is formed that initiates DNA synthesis after RNase H digestion.

[00227] Aspect 8. The method of Aspect 7, wherein the Col E1 promoter is located distal to the RNA II primer (RNA II) and the sequence-specific stretch of DNA prior to excision from the host chromosome, and

[00228] wherein excision of the product DNA plasmid relocates the RNA II promoter upstream of the RNA II primer template sequence within the product plasmid, producing the RNA II primer (RNA II), and initiating DNA replication of the product plasmid.

[00229] Aspect 9. The method of any one of Aspects 1 to 8, wherein at least one mutation or deletion in the Col E1 RNA I promoter comprises a mutation or deletion to decrease RNA I RNA production to thereby increase yield of the product DNA plasmid at a targeted time during fermentation.

[00230] Aspect 10. The method of any one of Aspects 1 to 9, wherein the origin further comprises a Col E1 primosomal assembly site (PAS) for assisting in replication of the product DNA plasmid.

[00231] Aspect 11. The method of any one of Aspects 1 to 10, wherein the sequence of interest encodes an immunogen, an antibody, a single chain or multiple chain antibody, a eukaryotic or prokaryotic gene expression cassette, a supplementary eukaryotic gene cassette, a DNA template for mRNA synthesis, a peptide, multiple open reading frames, an antibody conjugate, a vaccine, a biological, a provirus, or an immune modulating molecule for generating or suppressing an immune response.

[00232] Aspect 12. The method of any one of Aspects 1 to 11, wherein a stem loop II secondary structure of the RNA II primer of the modified Col E1 origin is replaced with a Lox recombination site in the product plasmid for control of RNA II primer production.

[00233] Aspect 12A. The method of Aspect 12, wherein the stem loop II secondary structure replaced comprises from residues -514 to -486 of SEQ ID NO:8.

[00234] Aspect 13. The method of any one of Aspects 1 to 12, further comprising the addition of an AttL, AttR, AttP or AttB recombination site in the RNA II primer sequence of the modified Col E1 origin, post-recombination in the product plasmid, for control of RNA II primer production.

[00235] Aspect 14. The method of any one of Aspects 1 to 13, wherein the parental DNA template is inserted into the host chromosome with site specific homology to the host chromosome to control the site of insertion.

[00236] Aspect 15. The method of any one of Aspects 1 to 5, wherein the inducible switch comprises an RNA thermoswitch derived from Listeria monocytogenes.

[00237] Aspect 16. The method of any one of Aspects 1 to 15, wherein the method comprises a plurality of sequences of interest and / or a plurality of parental DNA templates.

[00238] Aspect 16A. The method of any one of Aspects 1 to 5, wherein the control condition that induces the inducible switch is temperature, and the switch is a thermoswitch induced at a temperature greater than 30 °C.

[00239] Aspect 16B. The method of any one of Aspects 1 to 16, wherein the modified Col E1 origin comprises a modified pMB1, pBR322, or pUC origin.

[00240] Aspect 16C. The method of any one of Aspects 1 to 16, additionally comprising the step of harvesting from the host bacterium the plasmid DNA using alkaline lysis.

[00241] Aspect 16D. The method of any one of Aspects 1 to 5, wherein the recombinase expression to excise the product plasmid is controlled by an upstream temperature permissive terminator.

[00242] Aspect 16E. The method of any one of Aspects 1 to 16, further comprising modifying the 5’ RNA II primer sequence upstream of nucleotide -350 in the origin sequence of the modified Col E1 origin with a recombinase recognition site, for control for RNA II primer production.

[00243] Aspect 16F. The method of any one of Aspects 1 to 16, further comprising the replacement of up to the first 150 nucleotides of the RNA II promoter of the modified Col E1 origin with a Lox, AttL, AttR, AttP or AttB recombination site sequence, postrecombination in the product plasmid for control of RNA II primer production.

[00244] Aspect 16G. The method of any one of Aspects 1 to 16, wherein the modified Col E1 origin is Col E1 derived, such as a pMB1, pBR322, or pUC origin.

[00245] Aspect 16H. The method of any one of Aspects 1 to 16, wherein the strength of the RNA II promoter and RNA I promoter located in the modified Col E1 origin are altered to increase or decrease replication in the product plasmid.

[00246] Aspect 161. The method of any one of Aspects 1 to 16, wherein DNA replication of any excised DNA is controlled through reorientation of the RNA II promoter sequence to be upstream of the origin RNA II primer template DNA sequence post-excision, optionally wherein the RNA II promoter is a forward promoter that generates RNA II primer from the template DNA.

[00247] Aspect 16J. The method of any one of Aspects 1 to 16, where the inducible switch controls or limits expression of the recombinase, such as with a mutation to reduce polymerase recruitment to the promoter.

[00248] Aspect 17. A parental DNA template for integration into a host organism for production of a sequence of interest, said plasmid comprising:

[00249] the sequence of interest,

[00250] a modified Col E1 origin comprising a first recombinase recognition site, wherein said origin is modified to reduce or eliminate RNA I inhibition;

[00251] a second recombinase recognition site that is distal to the modified Col E1 origin;

[00252] a RNA II promoter upstream of the second recombinase recognition site;

[00253] a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites;

[00254] Lox or Att sites within or flanking the modified Col E1 origin and the sequence of interest for recognition by the recombinase; and

[00255] an inducible switch for control of recombinase translation under a controllable condition.

[00256] Aspect 18. The parental DNA template of Aspect 17, wherein the Lox or Att sites are LoxP sites, or AttL / AttR sites.

[00257] Aspect 18A. The parental DNA template of Aspect 17 or 18, wherein the modified Col E1 origin comprises Col E1 RNA II and Col E1 RNA I promoters; wherein the modified Col E1 origin is modified in the Col E1 RNA I promoter to reduce or eliminate RNA I inhibition and increase replication of the origin.

[00258] Aspect 18B. The parental DNA template of Aspect 17 or 18, wherein integration into a host organism comprises recombination into a bacterial genome, wherein the parental DNA template comprises sequence specific homology for permitting recombination to the bacterial genome, or a phage attachment site to allow serine / tyrosine integrase mediated integration into the bacterial genome.

[00259] Aspect 18C. The parental DNA template of Aspect 17 or 18, wherein the sequence of interest encodes an immunogen, an antibody, a single chain or multiple chain antibody, a eukaryotic or prokaryotic gene expression cassette, a supplementary eukaryotic gene cassette, a DNA template for mRNA synthesis, a peptide, multiple open reading frames, an antibody conjugate, a vaccine, a biological, a provirus, or an immune modulating molecule for generating or suppressing an immune response.

[00260] Aspect 18D. The parental DNA template of Aspect 17, comprising SEQ ID NO:18.

[00261] Aspect 19. The parental DNA template prepared in accordance with the method of any one of Aspects 1 to 16.

[00262] Aspect 19A. The parental DNA template of Aspect 17, 18 or 19, wherein the inducible switch is a temperature-sensitive inhibitor induced under the controllable condition of temperature change.

[00263] Aspect 20. The product DNA plasmid produced by the method of any one of Aspects 1 to 16.

[00264] Aspect 20A. A vaccine produced with DNA from the product DNA plasmid of Aspect 20.

[00265] Examples Only

[00266] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required.

[00267] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be made to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00268] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[00269] References

[00270] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00271] U.S. Patent No. 9,862,954 B2 (reissue as RE49,294 and RE48,908) entitled: DNA vector production system.

[00272] U.S. Patent No. 9,290,778 B2 entitled: DNA vector production system.

[00273] U.S. Publication US2008 / 0057545 A1 entitled: High copy number plasmids and their derivatives.

[00274] WO 2021 / 016075 A1 entitled: Recombinase compositions and methods of use.

[00275] JP 2014 / 012023 A entitled: Expression vectors having modified ColE1 origin of replication for control of plasmid copy number.

[00276] U.S. Patent No. 9,233,174 B2 entitled: Minicircle DNA vector preparations and methods of making and using the same.

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[00278] U.S. Publication US2021 / 0010021 A1 entitled: Viral and non-viral nanoplasmid vectors with improved production.

[00279] U.S. Publication US2013 / 0266983 A1 entitled: Creation of super-stable ColE1 plasmids by duplication of SL1-4 sequence and point mutations.

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[00285] Juhas, M., Ajioka, J.W. Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome. Microb Cell Fact 15, 172 (2016). https: / / doi.Org / 10.1186 / S12934-016-0571 -y.

[00286] Loh E, Memarpour F, et al., An unstructured 5'-coding region of the prfA mRNA is required for efficient translation. Nucleic Acids Res. 2012 Feb;40(4): 1818-27. doi: 10.1093 / nar / gkr850.

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Claims

1. A method for production of a product DNA plasmid comprising a sequence of interest, said method comprising:preparing a parental DNA template comprising a modified Col E1 origin comprising a first recombinase recognition site; a second recombinase recognition site that is distal to the modified Col E1 origin; a RNA II promoter upstream of the second recombinase recognition site; a RNA II primer template sequence; a recombinase coding sequence encoding an associated recombinase that is associated with the first and the second recombinase recognition sites; an inducible switch for control of recombinase translation; and the sequence of interest;wherein the sequence of interest and the modified Col E1 origin comprise or are flanked by Lox or Att sites, which are recognized by the associated recombinase for excision to reconstitute the RNA II promoter upstream of the RNA II primer template sequence, thereby producing the RNA II primer, and to thereby form a self-replicating product DNA plasmid; wherein translation of the recombinase is controlled by the inducible switch under a control condition; andwherein the modified Col E1 origin comprises Col E1 RNA II and Col E1 RNA I promoters, and the modified Col E1 is modified by at least one mutation or deletion in the Col E1 RNA I promoter to reduce or eliminate RNA I inhibition to increase replication of the origin post-recombination;preparing a host bacterium having the parental DNA template inserted into the chromosome of said host organism;fermenting the host organism under the control condition that induces the inducible switch to express the recombinase, to excise the product plasmid from the host organism;producing the product DNA plasmid in the host organism; andproducing the product DNA plasmid comprising the sequence of interest and the modified origin, that is excised from the chromosome of the host organism.

2. The method of claim 1, wherein the parental DNA template comprises a recombinase cassette and sequence specific homology to the host organism, such as a bacterium, to enable targeted recombination to the host genome, or a phage attachment site to utilize a serine / tyrosine recombinase to integrate the parental DNA template into a chromosome.

3. The method of claim 1, wherein the recombinase is a sequence specific recombinase matched with the Lox or Att site, such as a Cre recombinase for Lox-Lox excision, for example LoxP, Lox66 or Lox 71; or is Phi-BT 1 integrase (INT) for AttL / AttR excision.

4. The method of claim 1, wherein the parental DNA template is a plasmid or is a linear DNA template.

5. The method of claim 1, wherein the inducible switch for control of recombinase translation is an inducible promoter, a thermal-permissive terminator, a temperature sensitive inhibitor, a small molecule inducible promoter such as the IPTG inducible Lac promoter; a pH inducible promoter, ora light inducible promoter.

6. The method of claim 5, wherein the inducible promoter is temperature-inducible such as a Lambda virus pR / pL promoter-cl857, a trans-acting 5’ untranslated region (5’ UTR) synthetic RNA thermoswitch, or a PrfA RNA thermoswitch; or comprises synthetic 5’ UTR RNA hairpins wherein the production of recombinase mRNA is under a weak promoter.

7. The method of claim 1, wherein the modified Col E1 origin comprises a recombinase recombination site; a forward promoter sequence to generate the RNA II primer (RNA II); and a sequence-specific stretch of DNA to generate a stable hairpin structure to initiate replication of the excised product plasmid;whereby a RNA-DNA hybrid structure is formed that initiates DNA synthesis after RNase H digestion.

8. The method of claim 7, wherein the Col E1 promoter is located distal to the RNA IIprimer (RNA II) and the sequence-specific stretch of DNA prior to excision from the host chromosome, andwherein excision of the product DNA plasmid relocates the RNA II promoter upstream of the RNA II primer template sequence within the product plasmid, producing the RNA II primer (RNA II), and initiating DNA replication of the product plasmid.

9. The method of claim 1, wherein at least one mutation or deletion in the Col E1 RNA I promoter comprises a mutation or deletion to decrease RNA I RNA production to thereby increase yield of the product DNA plasmid at a targeted time during fermentation.

10. The method of claim 1, wherein the origin further comprises a Col E1 primosomal assembly site (PAS) for assisting in replication of the product DNA plasmid.

11. The method of claim 1, wherein the sequence of interest encodes an immunogen, an antibody, a single chain or multiple chain antibody, a eukaryotic or prokaryotic gene expression cassette, a supplementary eukaryotic gene cassette, a DNA template for mRNA synthesis, a peptide, multiple open reading frames, an antibody conjugate, a vaccine, a biological, a provirus, or an immune modulating molecule for generating or suppressing an immune response.

12. The method of claim 1, wherein a stem loop II secondary structure of the RNA II primer of the modified Col E1 origin is replaced with a Lox recombination site in the product plasmid for control of RNA II primer production.

13. The method of claim 1, further comprising the addition of an AttL, AttR, AttP or AttB recombination site in the RNA II primer sequence of the modified Col E1 origin, postrecombination in the product plasmid, for control of RNA II primer production.

14. The method of claim 1, wherein the parental DNA template is inserted into the host chromosome with site specific homology to the host chromosome to control the site of insertion.

15. The method of claim 1, wherein the inducible switch comprises an RNA thermoswitch derived from Listeria monocytogenes.

16. The method of claim 1, wherein the method comprises a plurality of sequences of interest and / or a plurality of parental DNA templates.

17. A parental DNA template for integration into a host organism for production of a sequence of interest, said plasmid comprising:the sequence of interest,a modified Col E1 origin comprising a first recombinase recognition site, wherein said origin is modified to reduce or eliminate RNA I inhibition;a second recombinase recognition site that is distal to the modified Col E1 origin;a RNA II promoter upstream of the second recombinase recognition site;a recombinase coding sequence encoding a recombinase associated with the first and the second recombinase recognition sites;Lox or Att sites within or flanking the modified Col E1 origin and the sequence of interest for recognition by the recombinase; andan inducible switch for control of recombinase translation under a controllable condition.

18. The parental DNA template of claim 17, wherein the Lox or Att sites are LoxP sites, or AttL / AttR sites.

19. The parental DNA template prepared in accordance with the method of claim 1.

20. The product DNA plasmid produced by the method of claim 1.

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