β-galactosidase α peptides as non-antibiotic selection markers and their uses

Abstract

Provided herein are methods for using nucleic acid constructs as selectable markers. The nucleic acid constructs include an isolated β-galactosidase expression cassette, which includes a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter. Also provided are isolated vectors including the β-galactosidase expression cassette, methods for producing the isolated vectors, and kits including the isolated vectors.

Description

[Technical field]

[0001] The present invention relates to an isolated β-galactosidase expression cassette comprising a non-antibiotic selection marker. Specifically, the isolated β-galactosidase expression cassette comprises an amino-terminal fragment of β-galactosidase operably linked to a promoter. Also provided are isolated vectors comprising the β-galactosidase expression cassette, methods of producing the isolated vectors, and kits comprising the isolated vectors.

[0002] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing, which has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing having a size of 48 kb with filename "JBI6031USPSP1Seqlist1" and a creation date of January 17, 2019. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated by reference in its entirety herein. [Background technology]

[0003] Plasmid vectors usually contain genes that are expressed in E. coli, and when the plasmid is introduced into cells by transformation or electroporation, it provides a way to identify or select cells that contain the plasmid from those that do not. The most commonly used selectable markers are genes that confer resistance to antibiotics. However, there are some situations in which antibiotic resistance genes are undesirable. When using plasmids to generate manufacturing cell lines for biologics such as antibodies, antibiotic resistance genes are usually removed or destroyed. In gene therapy, antibiotic resistance genes are also undesirable. Kanamycin / neomycin resistance genes are often accepted by the FDA, but EU regulatory agencies are much stricter. The European Pharmacopeia states that "Antibiotic resistance genes used as selectable genetic markers, especially for clinically useful antibiotics, are not included in the vector construct unless otherwise justified and approved. Other selection techniques for recombinant plasmids are preferred" ("Gene transfer medical products for human use." European Pharmacopei 7.0 (2011)). Disruption of antibiotic selection markers may be possible if small amounts of plasmid are required for cell line development, but these techniques are not practical for gene therapy applications where larger amounts of plasmid need to be produced.

[0004] A plasmid vector with a size of less than 1 kb, including both the replication origin and the selection marker, is necessary for the development of plasmid-based gene therapy to avoid gene silencing in vivo. The plasmid backbone is longer and expressed at a higher level in mice when it is 1 kb or less, compared with conventional plasmids with a plasmid backbone of 3 kb or 4 kb or more (Lu et al., Mol. Ther. 20(11):2111-9 (2012)). It was proposed that the large block of DNA that was not expressed in vivo induced silencing. Therefore, a plasmid with a smaller plasmid backbone may be much more effective.

[0005] Smaller plasmids are also needed for applications where transient transfection is used to manufacture therapeutics. One example is the production of adeno-associated virus vectors, where large-scale transfection of plasmids is used to generate clinical material. Smaller plasmids reduce the amount of DNA that must be transfected, reducing costs. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need to generate smaller plasmids containing selectable markers that can be used for gene therapy applications. [Means for solving the problem]

[0007] In one general aspect, a method of using a nucleic acid construct as a selectable marker is provided, the method comprising: (a) contacting a host cell comprising a deletion in the lac operon with a nucleic acid construct, the nucleic acid construct comprising an isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter, and (b) growing the host cell under conditions such that the nucleic acid construct is maintained within the host cell.

[0008] In another general aspect, there is provided an isolated β-galactosidase expression cassette, the isolated cassette comprising a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter.

[0009] In certain embodiments, the amino terminal fragment of β-galactosidase comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 1. In certain embodiments, the amino terminal fragment of β-galactosidase comprises the amino acid sequence of SEQ ID NO:1.

[0010] In certain embodiments, the nucleic acid sequence further comprises an origin of replication. The origin of replication can be, for example, a high copy origin of replication. In certain embodiments, the high copy origin of replication is a pUC57 origin of replication. In certain embodiments, the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO: 19.

[0011] In certain embodiments, the isolated β-galactosidase expression cassette further comprises a dimer resolution element. The dimer resolution element can comprise, for example, a nucleic acid sequence comprising a site-specific recombinase recognition site. The dimer resolution element can further comprise a nucleic acid sequence encoding a site-specific recombinase. In certain embodiments, the host cell comprises a nucleic acid sequence encoding a site-specific recombinase. The dimer resolution element can be, for example, a ColE1 dimer resolution element. In certain embodiments, the ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO: 20.

[0012] Also provided is an isolated vector comprising the isolated β-galactosidase expression cassette of the present invention. In certain embodiments, the isolated vector is less than about 1.5 kilobases in size. In certain embodiments, the isolated vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9-13, 17, and 18.

[0013] Also provided is a method of producing an isolated vector of the invention, the method comprising: (a) contacting a host cell with the isolated vector, (b) growing the host cell under conditions to produce the vector, and (c) isolating the vector from the host cell.

[0014] In certain embodiments, the host cells are grown in a minimal medium. The minimal medium may include lactose as the only carbon source. In certain embodiments, the minimal medium includes about 1% to about 4% by weight per volume (w / v) lactose. In certain embodiments, the minimal medium includes about 2% w / v lactose.

[0015] Also provided is a kit comprising (a) an isolated β-galactosidase expression cassette of the invention, and (b) a host cell comprising a deletion in the lac operon. In certain embodiments, the kit further comprises a minimal medium comprising lactose as the sole carbon source. In certain embodiments, the vector comprises the isolated β-galactosidase expression cassette. In certain embodiments, the host cell comprises a LacZΔM15 deletion. In certain embodiments, the host cell is selected from the group consisting of an E. coli host cell and a yeast host cell. [Brief description of the drawings]

[0016] The above summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1] 1 shows a schematic diagram of the P215 plasmid. [Diagram 2] 1 shows a schematic diagram of the P216 plasmid. [Diagram 3] 1 shows a schematic diagram of the P217 plasmid. [Figure 4] 1 shows a schematic diagram of the P218 plasmid. [Diagram 5] 1 shows a schematic diagram of the P219 plasmid. [Figure 6] 1 shows a schematic diagram of the P469-2 plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Various publications, articles, and patents are cited or described in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles and the like which has been included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention(s) disclosed or claimed.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0019] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0020] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the term "about". Thus, numerical values ​​typically include ±10% of the described value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within the range, unless the context clearly indicates otherwise.

[0021] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0022] As used herein, it will be understood that the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to include a stated integer or group of integers, but not to exclude other integers or groups of integers, and are intended to be non-exclusive or non-limiting. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or not inherently present in such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0023] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be included in the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be included in the meaning and thus meets the requirements of the term "and / or."

[0024] As used herein, the term "consists of" or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates that any recited integer or group of integers is inclusive, but that no additional integer or group of integers is added to the specified method, structure, or composition.

[0025] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" as used throughout the specification and claims indicates the inclusion of any recited integer or group of integers, optionally including any recited integer or group of integers that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0026] It should also be understood that the terms "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or features of preferred inventive components, indicate that the described dimensions / features are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, and the like).

[0027] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences (e.g., amino terminal β-galactosidase peptides and the polynucleotides encoding them, the nucleic acids of the isolated vectors described herein), refers to two or more sequences or subsequences that are identical or have a specified percentage of the same amino acid residues or nucleotides, when compared and aligned for maximum correspondence as determined using one of the following sequence comparison algorithms or by visual inspection.

[0028] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on designated program parameters.

[0029] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., 2002). Sons, Inc., (1995 Supplement) (see Ausubel).

[0030] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match when aligned with words of the same length in database sequences or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0031] For nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X, when the accumulation of one or more alignments of negative scoring residues causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0032] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0033] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0034] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid, a peptide, a protein, or a cell) is substantially separated from, produced separately from, or purified from other biological components of the organism in which it naturally occurs (i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues). Thus, "isolated" nucleic acids, peptides, proteins, and cells include nucleic acids, peptides, proteins, and cells that have been purified by standard purification methods and those described herein. "Isolated" nucleic acids, peptides, proteins, and cells can be part of a composition and are still isolated even if the composition is not part of the original environment of the nucleic acid, peptide, protein, or cell. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0035] As used herein, the term "polynucleotide" is also referred to interchangeably as "nucleic acid molecule," "nucleotide," or "nucleic acid" and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that include RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contain one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. A variety of modifications can be made to DNA and RNA. Thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms having characteristics of viral and cellular DNA and RNA. "Polynucleotide" also includes relatively short nucleic acid strands, often referred to as oligonucleotides.

[0036] As used herein, the term "vector" refers to a replicon into which another nucleic acid segment can be operatively inserted to bring about the replication or expression of the segment.

[0037] As used herein, the term "expression" refers to the biosynthesis of a gene product. Such terms include the transcription of a gene into RNA. Such terms also include the translation of RNA into one or more polypeptides, and further include all naturally occurring post-transcriptional and post-translational modifications. The expressed CAR can be present in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or can be anchored in the cell membrane.

[0038] As used herein, the term "operably linked" refers to the linkage between nucleic acids (e.g., a promoter and a nucleic acid encoding a polypeptide) when they are placed in a structural or functional relationship. For example, one segment of a nucleic acid sequence can be operably linked to another segment of a nucleic acid sequence when they are placed relative to each other on the same contiguous nucleic acid sequence and have a structural or functional relationship, such as a promoter or enhancer that is placed relative to the coding sequence to facilitate transcription of the coding sequence, a ribosome binding site that is placed relative to the coding sequence to facilitate translation, or a presequence or secretory leader that is placed relative to the coding sequence to facilitate expression of a preprotein (e.g., a preprotein involved in the secretion of the encoded polypeptide). In other examples, operably linked nucleic acid sequences are not contiguous, but are placed such that they have a functional relationship to each other as nucleic acids or as proteins expressed by them. Enhancers, for example, need not be contiguous. Linkage can be achieved by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.

[0039] As used herein, the term "promoter" refers to a nucleic acid sequence that enables the initiation of transcription of a gene sequence into messenger RNA; such transcription is initiated by the binding of RNA polymerase on or near the promoter.

[0040] As used herein, the term "origin of replication" or "origin of replication" refers to a nucleic acid sequence required for replication of a plasmid. Examples of origins of replication include, but are not limited to, pBR322 origin of replication, ColE1 origin of replication, pUC57 origin of replication, pMB1 origin of replication, pSC101 origin of replication, and R6K gamma origin of replication. Origins of replication can be high copy or low copy. When present in a vector, a high copy origin of replication can result in a high copy number of vector per cell (e.g., 150-200). When present in a vector, a medium copy origin of replication can result in a medium copy number of vector per cell (e.g., 25-50). When present in a vector, a low copy origin of replication can result in a low copy number of vector per cell (e.g., 1-3).

[0041] As used herein, the term "dimer resolution element" refers to a nucleic acid sequence that facilitates the in vivo conversion of a multimer of a nucleic acid sequence (e.g., a vector or a plasmid) into a monomer in which the sequence is present. The dimer resolution element may include a nucleic acid sequence that includes a site-specific recombinase target site (e.g., LoxP target site, rfs target site, FRT target site, RP4 res target site, RK2 res target site, and res target site). The dimer resolution element may include a nucleic acid sequence that encodes a site-specific recombinase (e.g., Cre recombinase, ResD recombinase, Flp recombinase, ParA recombinase, Sin recombinase, β recombinase, γδ recombinase, tnpR recombinase, and pSK41 resolvase). The isolated vector / nucleic acid dimer may be degraded by an enzyme that acts on the target DNA sequence contained within the dimer resolution element. The enzyme recombines the target DNA sequence. As a non-limiting example, the enzymes XerC and XerD, expressed either by the host cell or by a vector containing a dimer resolution element, recognize target sites in the ColE1 dimer resolution element and function with several additional cofactors to ensure that monomers of the vector / nucleic acid are produced.

[0042] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0043] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right, and although isomeric forms of amino acids are known, it is the L-form of the amino acid that is shown unless expressly indicated otherwise.

[0044] Polynucleotides, Vectors, Host Cells, and Methods of Use In one general aspect, a method of using a nucleic acid construct as a selectable marker is provided, the method comprising: (a) contacting a host cell comprising a deletion in the lac operon with a nucleic acid construct, the nucleic acid construct comprising an isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter, and (b) growing the host cell under conditions such that the nucleic acid construct is maintained within the host cell.

[0045] In another general aspect, the invention relates to an isolated β-galactosidase expression cassette that includes a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter.

[0046] In certain embodiments, the amino terminal fragment of β-galactosidase comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 1. In certain embodiments, the amino terminal fragment of β-galactosidase comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. The amino terminal fragment of β-galactosidase may comprise SEQ ID NO:1.

[0047] In certain embodiments, the nucleic acid sequence further comprises an origin of replication. The origin of replication can be, for example, a high copy origin of replication. In certain embodiments, the high copy origin of replication is a pUC57 origin of replication. In certain embodiments, the pUC57 origin of replication comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19. In certain embodiments, the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO: 19.

[0048] In certain embodiments, the isolated β-galactosidase expression cassette may further comprise a dimer resolution element. The dimer resolution element may, for example, comprise a nucleic acid sequence comprising a site-specific recombinase recognition site. The site-specific recombinase recognition site may, for example, be selected from the group consisting of LoxP sites, rfs sites, FRT sites, RP4 res sites, RK2 res sites, and res sites. The dimer resolution element may further comprise a nucleic acid sequence encoding a site-specific recombinase. In certain embodiments, the host cell comprises a nucleic acid sequence encoding a site-specific recombinase. The site-specific recombinase may, for example, be selected from the group consisting of Cre recombinase, ResD recombinase, Flp recombinase, ParA recombinase, Sin recombinase, β recombinase, γδ recombinase, tnpR recombinase, and pSK41 resolvase.

[0049] The dimer resolution element can be, for example, a ColE1 dimer resolution element. The ColE1 dimer resolution element can comprise a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20. In certain embodiments, the ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO: 20.

[0050] In certain embodiments, the isolated vector comprises an isolated β-galactosidase expression cassette of the present invention. In view of the present disclosure, any vector known to those skilled in the art can be used, such as a plasmid, a cosmid, an artificial chromosome (e.g., bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and / or P1-derived artificial chromosome (PAC)), a transposon, a phage vector, or a viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or reconfigurable promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce amino-terminal fragments of β-galactosidase peptides. Conventional cloning techniques, or artificial gene synthesis, can be used to generate recombinant expression vectors according to embodiments of the present invention.

[0051] In certain aspects, the isolated vector is less than about 1.5 kilobases in size. The isolated vector can be, for example, about 700 base pairs, about 800 base pairs, about 900 base pairs, about 1000 base pairs (about 1 kilobase), about 1100 base pairs (about 1.1 kilobase), about 1200 base pairs (about 1.2 kilobase), about 1300 base pairs (about 1.3 kilobase), about 1400 base pairs (about 1.4 kilobase), or about 1500 base pairs (about 1.5 kilobase) in length. In certain embodiments, the isolated vector is less than about 1 kilobase in size. In certain embodiments, the isolated vector is less than about 900 base pairs in size. In certain embodiments, the isolated vector is less than about 800 base pairs in size.

[0052] In certain embodiments, the isolated vector comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleic acid selected from the group consisting of SEQ ID NOs: 9-13, 17, and 18. In certain embodiments, the isolated vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9-13, 17, and 18.

[0053] Also provided is a method of producing an isolated vector of the invention, the method comprising: (a) contacting a host cell with the isolated vector, (b) growing the host cell under conditions to produce the vector, and (c) isolating the vector from the host cell.

[0054] In certain embodiments, the host cells are grown in a minimal medium. The minimal medium may include lactose as the only carbon source. In certain embodiments, the minimal medium includes about 1% to about 4% by weight per volume (w / v) lactose. In certain embodiments, the minimal medium includes about 1% to about 4% w / v, about 1% to about 3% w / v, about 1% to about 2% w / v, about 1.5% to about 4% w / v, about 1.5% to about 3% w / v, about 1.5% to about 2% w / v, about 2% to about 4% w / v, about 2% to about 3% w / v, about 2.5% to about 4% w / v, about 2.5% to about 35% w / v, or about 3% to about 4% w / v lactose. In certain embodiments, the minimal medium includes about 2% w / v lactose.

[0055] In certain embodiments, the present invention relates to a host cell comprising the isolated vector of the present invention. In view of the present disclosure, any host cell known to those skilled in the art may be used to contain the isolated vector of the present invention. Suitable host cells include cells that have a LacZΔM15 deletion, but with the remaining lactose biosynthetic pathway intact. Strains that contain this mutation in the context of the bacteriophage Φ80 integration (i.e., the Φ80lacZΔM15 marker) contain this mutation in the context of the complete lac operon, and are therefore suitable hosts. Other hosts with different deletions in the amino-terminal (N-terminal) region of the LacZ gene that produce significant levels of β-galactosidase when transformed with a LacZ-α complementation plasmid may also be suitable hosts. Suitable host cells of the present invention may include E. coli host cells or yeast host cells.

[0056] Also provided are kits comprising (a) an isolated β-galactosidase expression cassette of the invention, and (b) a host cell comprising a deletion in the lac operon. In certain embodiments, a vector comprises the isolated β-galactosidase expression cassette. In certain embodiments, the host cell comprises a LacZΔM15 deletion. In certain embodiments, the host cell may be selected from an E. coli host cell or a yeast host cell.

[0057] In certain embodiments, the kit further comprises a minimal medium comprising lactose as the only carbon source. In certain embodiments, the minimal medium comprises about 1% to about 4% by weight per volume (w / v) lactose. In certain embodiments, the minimal medium comprises about 1% to about 4% w / v, about 1% to about 3% w / v, about 1% to about 2% w / v, about 1.5% to about 4% w / v, about 1.5% to about 3% w / v, about 1.5% to about 2% w / v, about 2% to about 4% w / v, about 2% to about 3% w / v, about 2.5% to about 4% w / v, about 2.5% to about 35% w / v, or about 3% to about 4% w / v lactose. In certain embodiments, the minimal medium comprises about 2% w / v lactose.

[0058] Embodiment The present invention provides the following non-limiting embodiments. Embodiment 1 is a method of using a nucleic acid construct as a selectable marker, the method comprising: a. contacting a host cell containing a deletion in the lac operon with a nucleic acid construct, the nucleic acid construct comprising an isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter; and b. Growing the host cells under conditions such that only the host cells containing the nucleic acid construct are maintained within the host cells.

[0059] Embodiment 2 is the method of embodiment 1, wherein the amino terminal fragment of β-galactosidase comprises an amino acid sequence having at least 75% identity to SEQ ID NO:1.

[0060] Embodiment 3 is the method of embodiment 1 or 2, wherein the amino terminal fragment of β-galactosidase comprises the amino acid sequence of SEQ ID NO:1.

[0061] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the nucleic acid sequence further comprises an origin of replication.

[0062] Embodiment 5 is the method of embodiment 4, wherein the origin of replication is a high copy origin of replication.

[0063] Embodiment 6 is the method of embodiment 5, wherein the high copy origin of replication is a pUC57 origin of replication.

[0064] Embodiment 7 is the method of embodiment 6, wherein the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO:19.

[0065] Embodiment 8 is the method of any one of embodiments 1 to 7, wherein the isolated β-galactosidase expression cassette further comprises a dimer resolution element.

[0066] Embodiment 9 is the method of embodiment 8, wherein the dimer resolution element comprises a nucleic acid sequence comprising a site-specific recombinase recognition site.

[0067] Embodiment 10 is the method of embodiment 8 or 9, wherein the dimer resolution element further comprises a nucleic acid sequence encoding a site-specific recombinase.

[0068] Embodiment 11 is the method of embodiment 8 or 9, wherein the host cell comprises a nucleic acid sequence encoding a site-specific recombinase.

[0069] Embodiment 12 is the method according to any one of embodiments 8 to 11, wherein the dimer resolution element is a ColE1 dimer resolution element.

[0070]

[0023] Embodiment 13 is the method of embodiment 12, wherein the ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO:20.

[0071] Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the host cell comprises a LacZΔM15 deletion.

[0072] Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the isolated vector comprises an isolated β-galactosidase expression cassette.

[0073] Embodiment 16 is the method of embodiment 15, wherein the isolated vector is less than about 1.5 kilobases in size.

[0074] Embodiment 17 is the method of embodiment 15 or 16, wherein the isolated vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9-13, 17, and 18.

[0075] Embodiment 18 is a method for producing an isolated vector according to any one of embodiments 15 to 17, the method comprising: a. contacting a host cell with an isolated vector; b. growing a host cell under conditions to produce the vector; and c. Isolating the vector from the host cell.

[0076] Embodiment 19 is the method of embodiment 18, wherein the host cells are grown in minimal medium.

[0077] Embodiment 20 is the method of embodiment 19, wherein the minimal medium comprises lactose as the sole carbon source.

[0078] Embodiment 21 is the method of embodiment 20, wherein the minimal medium comprises about 1% to about 4% weight per volume (w / v) lactose.

[0079] Embodiment 22 is the method of embodiment 21, wherein the minimal medium comprises about 2% w / v lactose.

[0080] Embodiment 23 is an isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding an amino terminal fragment of β-galactosidase operably linked to a promoter.

[0081] Embodiment 24 is an isolated β-galactosidase expression cassette according to embodiment 23, wherein the amino terminal fragment of β-galactosidase comprises an amino acid sequence having at least 75% identity to SEQ ID NO:1.

[0082] Embodiment 25 is an isolated β-galactosidase expression cassette according to embodiment 23 or 24, wherein the amino terminal fragment of β-galactosidase comprises the amino acid sequence of SEQ ID NO:1.

[0083] Embodiment 26 is an isolated β-galactosidase expression cassette according to any one of embodiments 23 to 25, wherein the nucleic acid sequence further comprises an origin of replication.

[0084] Embodiment 27 is an isolated β-galactosidase expression cassette as described in embodiment 26, wherein the origin of replication is a high copy origin of replication.

[0085] Embodiment 28 is an isolated β-galactosidase expression cassette as described in embodiment 27, wherein the high copy origin of replication is a pUC57 origin of replication.

[0086] Embodiment 29 is an isolated β-galactosidase expression cassette as described in embodiment 28, wherein the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO:19.

[0087] Embodiment 30 is an isolated β-galactosidase expression cassette according to any one of embodiments 23 to 29, wherein the isolated β-galactosidase expression cassette further comprises a dimer resolution element.

[0088] Embodiment 31 is an isolated β-galactosidase expression cassette according to embodiment 30, wherein the dimer resolution element comprises a nucleic acid sequence comprising a site-specific recombinase recognition site.

[0089] Embodiment 32 is an isolated β-galactosidase expression cassette according to embodiment 30 or 31, wherein the dimer resolution element further comprises a nucleic acid sequence encoding a site-specific recombinase.

[0090] Embodiment 33 is an isolated β-galactosidase expression cassette according to any one of embodiments 30 to 32, wherein the dimer resolution element is a ColE1 dimer resolution element.

[0091] Embodiment 34 is an isolated β-galactosidase expression cassette according to embodiment 33, wherein the ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO:20.

[0092] Embodiment 35 is an isolated vector comprising the isolated β-galactosidase expression cassette according to any one of embodiments 23 to 34.

[0093] Embodiment 36 is the isolated vector of embodiment 35, wherein the isolated vector is less than about 1.5 kilobases in size.

[0094] Embodiment 37 is the isolated vector of embodiment 35 or 36, wherein the isolated vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9-13, 17, and 18.

[0095] Embodiment 38 is a. an isolated β-galactosidase expression cassette according to any one of embodiments 23 to 37; and b. A kit comprising a host cell containing a deletion in the Lac operon.

[0096] Embodiment 39 is the kit of embodiment 38, further comprising a minimal medium comprising lactose as the sole carbon source.

[0097] Embodiment 40 is a kit according to embodiment 38 or 39, wherein the vector comprises an isolated β-galactosidase expression cassette.

[0098] Embodiment 41 is a kit according to any one of embodiments 38 to 40, wherein the host cell comprises a LacZΔM15 deletion.

[0099] Embodiment 42 is the kit of embodiment 41, wherein the host cell is selected from the group consisting of an E. coli host cell and a yeast host cell. EXAMPLES

[0100] Example 1: Plasmid selection via α-complementation of β-galactosidase instead of antibiotic selection in TOP10 cells material Cells: One Shot Top10 competent cells (Thermo-Fisher; Waltham, MA, Catalog No. C404003). NEB 5-α (New England Biolabs, Ipswich, MA, Catalog No. (C2987). GT115 (InvivoGen, San Diego, CA, Catalog No. GT115-21). NEB Stable (New England Biolabs, Catalog No. C3040H). Stellar (Takara Bio USA, Mountain View, CA, Catalog No. 636766). DH10B (Thermo-Fisher, Catalog No. 18297010). Stbl3 (Thermo-Fisher, Catalog No. C737303). Xli-blue (Agilent, Santa Clara, CA, Catalog No. 200236). Plasmids: pUC19 (Thermo-Fisher Scientific, Catalog No. SD0061); pBluescript II.KS(-) (Agilent; Santa Clara, CA, Catalog No. 212208). Clone P215 (SEQ ID NO: 9) and P216 (SEQ ID NO: 10). GWIZ-Luciferase (Genlantis Corporation; San Diego, CA; P030200); P219 (SEQ ID NO: 13, Figure 5). P469-2 (SEQ ID NO: 17, Figure 6). Media: M9 + lactose medium (Teknova, Hollister CA, Catalog No. M1348-04 (plates)): 0.3% KH2PO4, 0.6% Na2HPO 4、 0.5% (85 mM) NaCl, 0.1% NH4Cl, 2 mM MgSO4, 50 mg / L L-leucine, 50 mg / L isoleucine; 1 mM thiamine, 2% lactose, and 1.5% agar. M9 + glucose medium (Teknova Hollister CA, Catalog No. M1346-04 (plates)): 0.3% KH2PO4, 0.6% Na2HPO 4、0.5% (85 mM) NaCl, 0.1% NH4Cl, 2 mM MgSO4, 50 mg / L L-leucine, 50 mg / L isoleucine, 1 mM thiamine, 1% glucose, and 1.5% agar. LB-carbenicillin (100) plates (Teknova, Hollister CA, Cat. No. L1010). LB plates (Teknova Hollister CA L1100). LB + 60 μg / mL X-Gal, 0.1 mM IPTG (Teknova Hollister CA L1920). SOC media (Thermo-Fisher 15544034). LB broth (Thermo-Fisher 10855021); D-PBS, pH 7.1, Mg 2+ None, Ca 2+ None (ThermoFisher 14200-075)

[0101] result Plasmids without antibiotic selection markers are desirable for gene therapy applications and for developing cell lines for therapeutic products. It has also been reported that plasmid backbones of 1 kb or less were useful for avoiding gene silencing when delivered to animals in vivo. The purpose of these experiments was to explore new strategies for developing small metabolic selection markers for selecting plasmid-containing cells in E. coli.

[0102] It was hypothesized that a plasmid expressing the α-peptide of β-galactosidase would complement the LacZΔ15 allele in TOP10 cells, completing the lactose operon and enabling the cells to grow in minimal medium with lactose as the sole carbon source.

[0103] Plasmids pUC19 and pBluescript II, both of which express a β-galactosidase α-peptide fusion protein, were tested for their ability to complement the lac mutation in the Top10 host strain, allowing growth on minimal medium.

[0104] To test whether pUC19 and / or pBluescript II were able to complement the LacZΔ15 mutation in TOP10 cells, these plasmids were transformed into cells using the following procedure.

[0105] Two transformation mixtures were prepared in sterile microcentrifuge tubes as follows: 1) 1 μL (100 pg) pBluescript II plasmid + 50 μL One Shot TOP10 cells. 2) 1 μL (10 pg) pUC19 plasmid + 50 μL One Shot TOP10 cells. The transformation mixtures were incubated on ice for 30 minutes and then heat shocked at 42° C. for 30 seconds. After heat shock, the transformation mixtures were incubated on ice for 1 minute. To the transformation mixtures, 450 μL SOC medium was added and the cells were incubated at 37° C. for 1 hour with shaking. The transformation mixtures containing the cells were centrifuged and the cells were resuspended in 500 μL sterile D-PBS buffer. The cells were centrifuged and resuspended two more times. For each sample, two 1:10 serial dilutions of the cells were made in D-PBS. 200 μL of each dilution was spread onto M9+lactose plates. 200 μL of the first two dilutions were also spread onto LB-carbenicillin (100) plates. The plates were incubated at 37° C. overnight.

[0106] After overnight incubation, there were many colonies from both transformations plated onto LB-carbenicillin (100) plates, and these plates were stored at 4° C. There were no visible colonies from either transformation plated onto M9+lactose plates, and these plates were incubated for an additional 24 hours at 37° C. No colonies were visible on the M9-lactose plates. The cells were grown for an additional 48 hours at 30° C. No colonies were visible on these plates, even after the extended incubation period.

[0107] None of the cloning vectors expressing the LacZ-α fusion peptide complemented the Lac mutation in the TOP10 host strain and allowed growth on minimal medium containing lactose as the sole carbon source.

[0108] It is possible that expression of LacZ-α peptide fusion proteins by the pUC19 and pBluescript II cloning vectors was not high enough to adequately complement the lac mutation in the host strains tested: both vectors produce fusion proteins that are transcribed through the multiple cloning region, and such fusion proteins may be suboptimal for complementing the LacZΔ15 mutation.

[0109] Example 2: LacZ expression plasmid used as a metabolic selection marker in E. coli. Two LacZ-α expression cassettes with medium and strong promoters (LacZYA and OmpF, respectively) were designed. The OmpF promoter sequence was based on the OmpF promoter used by Stavropoulos et al. (Stavropoulos and Strathdee, Genomics 72(1):99-104 (2001)). The LacZYA promoter was derived from the sequence of pBluescript with the lac operator sequence bound by the lac repressor.

[0110] Regarding the open reading frame (ORF) of the LacZα peptide, Reddy (Reddy, Biotechniques 37(6):948-52 (2004) reported that the plasmid pUC19 yielded approximately 10-fold more β-galactosidase activity than pBluescript. These plasmids have the same promoter element driving the lacZα peptide. However, pBluescript has a much longer polylinker than pUC19, and pUC19 encodes non-lacZ C-terminal residues. It is unclear what among these differences results in the high pUC19 β-galactosidase activity. Nishiyama et al. found that a 60 amino acid N-terminal α peptide had the greatest β-galactosidase activity in their assay (Nishiyama et al., Protein Sci. 24(5):599-603(2015)). The following wild-type LacZα region from strain MG1655, truncated at residue 60, was used: MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTD RPSQQLRSLNGEWR (sequence number 1).

[0111] The terminator sequence was derived from the rrnBT2 terminator described by Orosz et al. (Orosz et al., Eur. J. Biochem. 201(3):653-9 (1991)).

[0112] The P215 (SEQ ID NO:9) (Figure 1) and P216 (SEQ ID NO:10) (Figure 2) plasmids were constructed by gene synthesis in GeneWiz (South Plainfield, NJ). The plasmids contain an ampicillin resistance cassette and a 4.9 kb transgene.

[0113] result Plasmids without antibiotic selection markers are desirable for gene therapy applications and for developing cell lines for therapeutic products. It has also been reported that plasmid backbones of 1 kb or less were useful for avoiding gene silencing when delivered to animals in vivo. The purpose of these experiments was to explore new strategies for developing small metabolic selection markers for selecting plasmid-containing cells in E. coli.

[0114] It was hypothesized that a plasmid expressing the α-peptide of β-galactosidase would complement the LacZΔ15 allele in Top10 cells, completing the lactose operon and enabling the cells to grow in minimal medium with lactose as the sole carbon source.

[0115] In Example 1, we tested whether pUC19 and pBluescript vectors expressing a lacZα fusion peptide were able to complement TOP10 cells and enable them to grow in minimal medium with lactose. These experiments were unsuccessful.

[0116] Based on the hypothesis that the lacZα fusion proteins encoded by these vectors were suboptimal in complementing the LacZΔ15 mutation and were not expressed at high enough levels to allow growth on lactose-containing minimal medium, vectors were synthesized with a new lacZα expression cassette. The ability of these vectors to complement the LacZΔ15 mutation was tested. Ten nanograms (ng) of plasmids P215 and P216, as well as pBluescript II, were transformed into 50 μL of OneShot Top10 cells. The cells were incubated with DNA on ice for 20 minutes, heat shocked at 42°C for 30 seconds, and returned to incubation on ice for 1 minute. 450 μL of SOC was added to the cells and the cells were incubated at 37°C for 1 hour with shaking. 250 μL of cells were removed and the remaining cells were returned to the incubator. The extracted cells were washed twice with 500 μL of D-PBS and resuspended in 200 μL of D-PBS after the last wash. 50 μL of cells were plated onto LB-carbenicillin (100), M9+glucose, and M9+lactose plates and the plates were incubated at 37° C. After 4.5 hours of heat shock, the remaining cells from the incubator were washed as above and plated onto M9+glucose and M9+lactose plates. The plates were incubated overnight at 37° C.

[0117] Transformants plated on M9+glucose produced a lawn of cells, indicating that Top10 host cells were able to grow on these plates. Transformants plated on LB-carbenicillin (100) produced many colonies as well. LB-carbenicillin plates were stored at 4°C. M9+lactose plates were kept at 37°C and incubated for an additional 24 hours.

[0118] Transformants were allowed to recover for either 1 hour or 4 hours, and both produced numerous colonies when plated onto M9+lactose plates. There were no colonies on the pBluescript II transformants, confirming the results of Example 1 and indicating that pBluescript II was unable to produce sufficient β-galactosidase through complementation of the LacZΔ15 mutation to allow growth on lactose minimal medium. Plates were stored at 4° C.

[0119] While native plasmids such as ColE1 are efficiently maintained in E. coli hosts in the absence of antibiotic selection, vectors of the pUC series can be lost from cells at high rates in the absence of selection (Summers, Molecular Microbiology 29:1137-1145 (1998)). However, given the very slow growth rates of P215 and P216 transformed cells in minimal versus rich LB medium, it would be much faster and less expensive for plasmid DNA purification to grow cell cultures in LB in the absence of selection, if the frequency of plasmid loss is not too high. β-galactosidase α-complementing plasmid-containing cells are easily distinguished from plasmid-free cells grown on LB-IPTG-XGAL plates because β-galactosidase hydrolyzes the XGAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) indicator, which turns the cells blue. This assay was used to investigate the frequency of plasmid loss when these cells were grown in LB medium without antibiotics.

[0120] A pure population of cells was obtained by streaking the cells onto LB-IPTG-XGAL plates, and colonies containing the plasmid turned blue. Most of the colonies streaked onto the plates were blue, as expected.

[0121] After obtaining a pure population of cells, a continuous cell culture was grown. A single blue colony was picked and grown in 2 mL of LB medium in a 15 mL tube. The culture was incubated overnight at 37°C with shaking.

[0122] Cells from the culture were streaked onto LB-IPTG-XGAL plates and the plates were incubated overnight at 37° C. Colonies on the restreaked plates were blue. A single colony was inoculated into 50 mL of LB in a 250 mL flask and incubated overnight at 37° C. with shaking.

[0123] 10 of overnight culture -4 50 μL of the dilution was plated on LB-IPTG-XGAL plates. The plates were incubated overnight at 37° C. 1 μL of the 50 mL culture was diluted into a fresh culture of 50 mL LB (50,000-fold dilution). The culture was grown overnight at 37° C.

[0124] After overnight incubation, all colonies on the plate were observed to be blue. -4 50 μL of the dilution was plated on LB-IPTG-XGAL plates. 1 μL of the 50 mL overnight culture was diluted into a fresh culture of 50 mL LB (50,000-fold dilution). Cultures were grown overnight at 37° C.

[0125] After overnight incubation, 50 μL of 10 -4 Approximately 1000 colonies were observed on the plates containing the dilutions. All colonies of the P215 transformant were blue, and only three white colonies were observed on the P216 transformant plate.

[0126] The results showed that plasmids P215 and P216 were stable in the absence of selection. These plasmids are 7.2 and 7.3 kb for P215 and P216, respectively. When diluted into 50 mL from a single colony, then diluted 1:50,000, and grown to confluence twice, cells grew at 1.25 x 10 ng / mL without selection while still retaining the plasmid in most of the cells. 8 This suggests that the cells can be grown to volumes of up to liters. Transformation efficiency was similar when cells were allowed to recover in SOC medium for 1 hour versus 4 hours after heat shock.

[0127] The constructed α-complementing plasmids complement the LacZΔ15 mutation in Top10 cells, allowing growth on minimal medium with lactose as the sole carbon source. These plasmids were also found to be stable in LB liquid cultures in the absence of selective pressure.

[0128] Example 3: Reducing the size of the β-galactosidase-α complementation plasmid Previous experiments had shown that expression of the β-galactosidase α peptide from the P215 and P216 plasmids was useful as a selectable marker on the plasmid, replacing the antibiotic resistance gene. Next, it was sought to define which regions of the plasmid are essential for plasmid selection and replication in E. coli, with the aim of defining the smallest possible replicon.

[0129] result Using standard cloning techniques, the mCherry and puromycin resistance genes were removed from plasmid P215 to generate plasmid P217 (SEQ ID NO: 11) (Figure 3).

[0130] The ampicillin resistance gene was removed from plasmid P217 using standard cloning techniques. The ligated DNA was transformed into 50 μL of TOP10 cells, incubated on ice for 20 minutes, heat shocked for 30 seconds, and incubated on ice for an additional 3 minutes. After incubation, 450 μL of SOC was added to the cells and the cells were incubated at 37° C. for 1 hour with shaking. The cells were pelleted and washed 3 times with 1 mL of d-PBS. The cells were plated on M9-lactose plates and incubated at 37° C. for 2 days. Colonies were picked from the transformations and streaked onto LB-IPTG-XGAL plates. The resulting colonies were blue for each clone. A single clone was picked (clone P218 (SEQ ID NO: 12, FIG. 4)) and DNA sequencing confirmed that the desired deletion was made.

[0131] To further reduce the size of the β-galactosidase selection cassette, the rrnBT2 transcription terminator (SEQ ID NO: 7) was deleted. In addition to the possibility that this sequence was not necessary to maintain transcript stability, it has been reported that read-through transcription from an upstream promoter of the pUC57 / pMB1 origin can increase copy number by increasing transcription through the replication primer region of the origin (Panayotatos, Nucleic Acid Res. 12(6):2641-8 (1984); Oka et al., Mol Gen Genet. 172(2):151-9 (1979)).

[0132] Standard cloning techniques were used to obtain colonies of the deletion construct P219 (SEQ ID NO: 13, Figure 5). The deletion was confirmed by DNA sequencing.

[0133] The minimal β-galactosidase expression cassette / origin of replication cassette (SEQ ID NO: 18) elucidated in this work is 938 bp, which meets the objective of being smaller than 1 kb to avoid DNA silencing in mammalian cells associated with larger plasmid backbones (Lu et al., Mol. Ther. 20(11):2111-9 (2012)).

[0134] Example 4: Construction of a β-galactosidase-α complementation vector carrying a firefly luciferase expression cassette In the examples provided above, plasmids were constructed that use alpha complementation of the beta-galactosidase mutation as a selection marker in place of an antibiotic resistance gene. To determine whether DNA replication was still efficient when plasmid size was increased, the minimal beta-galactosidase expression cassette / origin of replication sequence defined above (SEQ ID NO: 18) was used to replace the antibiotic selection marker and origin of replication of existing plasmids using standard cloning techniques.

[0135] The CMV promoter-luciferase-polyA expression cassette was cloned from the GWIZ-luciferase plasmid (SEQ ID NO: 16) into P219 using standard cloning techniques. Transformation into One Shot TOP10 cells, plating onto M9+lactose plates, and incubation at 37° C. for 2 days produced large colonies. Colonies were restreaked onto LB-IPTG-XGAL plates and incubated overnight at 37° C.

[0136] Blue colonies from the transformation reaction were screened for inserts using primers CNFOR (SEQ ID NO: 14) and P455R2 (SEQ ID NO: 15). Two PCR-positive colonies were picked and inoculated into 6 mL LB cultures grown at 37° C. DNA was isolated from the cultures and DNA yields were determined by measuring their OD using a spectrophotometer. 260 was estimated by measuring (Table 1).

[0137] [Table 1]

[0138] 200 mL of LB in a 500 mL flask was inoculated with a single blue colony of clone P469-2 and grown for 18 hours at 37° C. in a shaker incubator. DNA was purified from this culture using the Qiagen HiSpeed ​​MaxiPrep kit, and 440 μg of DNA was recovered.

[0139] Plasmid P469-2 (SEQ ID NO: 17) was sequenced and verified with GeneWiz.

[0140] In this example, the kanamycin resistance gene and GWIZ-luciferase origin of replication were successfully replaced with the minimal β-galactosidase / origin of replication defined above, and acceptable plasmid yields were achieved when this clone was grown in LB medium without selective pressure.

[0141] Example 5: Testing β-galactosidase-α complementation vector function in various E. coli strains To identify additional E. coli strains in which the β-galactosidase α peptide could be used as a selectable marker in place of an antibiotic resistance gene, one of the plasmids constructed above was tested by DNA transfection into eight different strains.

[0142] [Table 2]

[0143] result 50 μL of E. coli strain from Table 2 was incubated with 1 ng of plasmid P469-2 in a sterile microcentrifuge tube for 30 minutes on ice. Cells were heat shocked at 42°C for 30 seconds and incubated on ice for 1 minute. 450 μL of SOC medium was added to all cells except NEB-Stable cells. 450 μL of NEB-Stable outgrowth medium (supplied by manufacturer) was added to transformed NEB-Stable cells. Cells were incubated at 37°C for 1 hour with shaking. Cells were pelleted and washed 3 times with 1 mL of D-PBS. Cells were plated on M9-lactose plates and incubated at 37°C for 3 days.

[0144] As expected, no colonies were detected on plates from Stbl3 transformed cells, which were included as negative controls. Five of the strains (Top10, GT115, NEB-Stable, Stellar, and DH10B) had normal sized colonies. Two strains (NEB-Alpha and XL1-Blue) had small colonies. This was expected because strains like NEB-alpha (DH5alpha) and XL1-Blue contain a mutation in the purB gene that retards growth on minimal medium (Jung et al. Appl Environ. Micro. 76:6307-6309 (2010)).

[0145] The XL1-blue and NEB-Alpha plates were further incubated at 37° C. for one day.

[0146] Pure colonies were obtained by streaking colonies from M9-lactose plates onto LB-IPTG-XGAL plates and incubating at 37° C. Blue colonies (plasmid-containing cells) were streaked (a second time) onto LB-IPTG-XGAL plates and incubated at 37° C., which produced mostly blue cells.

[0147] All of the test strains contained the Φ80dlacZΔM15 marker, transformed with the β-galactosidase α peptide expression plasmid P469-2 and could be selected on M9 minimal medium with lactose as the sole carbon source. q Plasmid P469-2 transfectants of strain XL1-blue containing ZΔM15 were also selectable on M9-lactose plates, thus demonstrating that the seven commercially available E. coli strains are compatible with the β-galactosidase selectable marker.

[0148] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this description. The invention described in this specification may include the following aspects. [1] 1. A method for using a nucleic acid construct as a selectable marker, the method comprising: a. contacting a host cell containing a deletion in the lac operon with the nucleic acid construct, the nucleic acid construct comprising an isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase operably linked to a promoter; and b. growing the host cell under conditions such that the nucleic acid construct is maintained within the host cell. The method comprising: [2] The method according to [1] above, wherein the amino-terminal fragment of β-galactosidase comprises an amino acid sequence having at least 75% identity to SEQ ID NO:1. [3] The method according to [1] or [2] above, wherein the amino-terminal fragment of β-galactosidase comprises the amino acid sequence of SEQ ID NO:1. [4] The method according to any one of the above [1] to [3], wherein the nucleic acid sequence further comprises an origin of replication. [5] The method according to [4] above, wherein the replication origin is a high-copy replication origin. [6] The method according to [5] above, wherein the high copy replication origin is a pUC57 replication origin. [7] The method according to [6] above, wherein the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO: 19. [8] The method according to any one of [1] to [7] above, wherein the isolated β-galactosidase expression cassette further comprises a dimer resolution element. [9] The method according to [8] above, wherein the dimer resolution element comprises a nucleic acid sequence comprising a site-specific recombinase recognition site.

[10] The method according to [8] or [9] above, wherein the dimer resolution element further comprises a nucleic acid sequence encoding a site-specific recombinase.

[11] The method according to [8] or [9] above, wherein the host cell comprises a nucleic acid sequence encoding a site-specific recombinase.

[12] The method according to any one of [8] to

[11] above, wherein the dimer resolution element is a ColE1 dimer resolution element.

[13] The method according to

[12] above, wherein the ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO: 20.

[14] The method according to any one of the above [1] to

[13] , wherein the host cell comprises a LacZΔ15 deletion.

[15] The method according to any one of the above [1] to

[14] , wherein an isolated vector comprises the isolated β-galactosidase expression cassette.

[16] The method according to

[15] above, wherein the isolated vector is less than about 1.5 kilobases in size.

[17] The method according to

[15] or

[16] above, wherein the isolated vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 to 13, 17, and 18.

[18] A method for producing the isolated vector according to any one of

[15] to

[17] above, comprising: a. contacting a host cell with the isolated vector; b. growing the host cell under conditions to produce the vector; and c. isolating the vector from the host cell. The method comprising:

[19] The method according to

[18] above, wherein the host cells are grown in a minimal medium.

[20] The method according to

[19] above, wherein the minimal medium contains lactose as the only carbon source. [twenty one] The method according to

[20] above, wherein the minimal medium contains about 1% by weight to about 4% by weight (w / v) lactose per volume. [twenty two] The method according to

[21] above, wherein the minimal medium contains about 2% w / v lactose. [twenty three] A kit comprising: a. An isolated β-galactosidase expression cassette according to any one of [1] to

[13] above, and b. Host cells containing a deletion in the lac operon The kit comprising: [twenty four] The kit according to

[23] above, further comprising a minimal medium containing lactose as the sole carbon source. [twenty five] The kit according to

[23] or

[24] above, wherein the vector comprises the isolated β-galactosidase expression cassette.

[26] The kit according to any one of

[23] to

[25] above, wherein the host cell comprises the LacZΔ15 deletion.

[27] The kit according to

[26] above, wherein the host cell is selected from the group consisting of an E. coli host cell and a yeast host cell. [Table 3] JPEG0007631201000004.jpg217143JPEG0007631201000005.jpg214143JPEG0007631201000006.j pg219141JPEG0007631201000007.jpg217141JPEG0007631201000008.jpg217142JPEG0007631201 000009.jpg217141JPEG0007631201000010.jpg217141JPEG0007631201000011.jpg217141JPEG00 07631201000012.jpg217142JPEG0007631201000013.jpg217142JPEG0007631201000014.jpg21514 1JPEG0007631201000015.jpg216142JPEG0007631201000016.jpg219141JPEG0007631201000017. jpg214144JPEG0007631201000018.jpg217142JPEG0007631201000019.jpg216142JPEG0007631201 000020.jpg217142JPEG0007631201000021.jpg216140JPEG0007631201000022.jpg217141JPEG00 07631201000023.jpg219144JPEG0007631201000024.jpg217141JPEG0007631201000025.jpg99141

Claims

1. 1. A method for using an isolated vector comprising a nucleic acid construct as a selectable marker, the method comprising: a. contacting a host cell comprising LacZΔM15 in its lac operon with a nucleic acid construct, wherein the nucleic acid construct comprises an isolated β-galactosidase expression cassette comprising a nucleic acid sequence operably linked to a LacZYA promoter or an OmpF promoter, the nucleic acid sequence encoding an amino terminal fragment of β-galactosidase having the amino acid sequence of SEQ ID NO:1, and the isolated vector is less than 1.2 kilobases; b. growing the host cell under conditions such that the nucleic acid construct is maintained within the host cell. The method comprising:

2. The method of claim 1 , wherein the nucleic acid sequence further comprises an origin of replication.

3. The method of claim 2, wherein the origin of replication is a high copy origin of replication.

4. The method of claim 3, wherein the high copy origin of replication is a pUC57 origin of replication.

5. 5. The method of claim 4, wherein the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO:

19.

6. The method of any one of claims 1 to 5, wherein the isolated β-galactosidase expression cassette further comprises a dimer resolution element.

7. The method of claim 6 , wherein the dimer resolution element comprises a nucleic acid sequence comprising a site-specific recombinase recognition site.

8. The method of claim 6 or 7, wherein the dimer resolution element further comprises a nucleic acid sequence encoding a site-specific recombinase.

9. The method of claim 6 or 7, wherein the host cell comprises a nucleic acid sequence encoding a site-specific recombinase.

10. The method according to any one of claims 6 to 9, wherein the dimer resolution element is a ColE1 dimer resolution element.

11. The method of claim 10 , wherein the ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO:

20.

12. 2. The method of claim 1, wherein the isolated vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12-13 and 18.

13. 2. A method for producing the isolated vector of claim 1, said method comprising: a. contacting a host cell with the isolated vector; b. growing the host cell under conditions to produce the vector; and c. isolating the vector from the host cell. The method comprising:

14. The method of claim 13 , wherein the host cells are grown in minimal medium.

15. 15. The method of claim 14, wherein the minimal medium contains lactose as the sole carbon source.

16. 16. The method of claim 15, wherein the minimal medium comprises 1% to 4% lactose by weight per volume (w / v).

17. 17. The method of claim 16, wherein the minimal medium comprises 2% w / v lactose.

18. A kit for using a nucleic acid construct as a selectable marker, the kit comprising: a. an isolated β-galactosidase expression cassette comprising a nucleic acid sequence operably linked to a LacZYA promoter or an OmpF promoter, the nucleic acid sequence encoding an amino-terminal fragment of β-galactosidase having the amino acid sequence of SEQ ID NO:1; and b. Host cells containing deletions in the lac operon Including, The nucleic acid construct is less than 1.2 kilobases.

19. 20. The kit of claim 18, further comprising a minimal medium containing lactose as the sole carbon source.

20. The kit of claim 18 or 19, wherein a vector comprises the isolated β-galactosidase expression cassette.

21. The kit according to any one of claims 18 to 20, wherein the host cell comprises the LacZΔM15.

22. 22. The kit of claim 21, wherein the host cell is selected from the group consisting of an E. coli host cell and a yeast host cell.

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