Β-galactosidase α peptide as non-antibiotic selection marker and uses thereof

Abstract

To provide methods of using a nucleic acid construct as a selectable marker.SOLUTION: A nucleic acid construct comprises an isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding the amino-terminal fragment of β-galactosidase operably linked to a promoter. Also provided are isolated vectors comprising the β-galactosidase expression cassette, methods of generating the isolated vector, and kits comprising the isolated vector.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides an isolated β-galactosidase expression cassette containing a non-antibiotic selection marker. Specifically, the isolated β-galactosidase expression cassette is and An isolated vector comprising a β-galactosidase expression cassette, Methods for producing the vectors and kits containing the isolated vectors are also provided.

[0002] (Reference to electronically submitted sequence listing) This application is filed under the file name “JBI6031USPSP1Seqlist1” and The EF file is an ASCII sequence table with a size of 48kb and was created on January 17, 2013. Contains a sequence listing submitted electronically via EFS-Web. The sequence listing provided is a part of the present specification and is incorporated herein by reference in its entirety. do. [Background technology]

[0003] Plasmid vectors usually contain genes that are expressed in E. coli. When a plasmid is introduced into a cell by transformation or electroporation, it becomes The present invention provides a method for identifying or selecting 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 plasmids are used to generate cell lines for the production of biologics such as antibodies, antibiotic resistance genes 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 E. The European regulatory authorities are much stricter. The European Pharmacopoeia states: "Unless otherwise justified and authorised, , particularly for use as selectable genetic markers for clinically useful antibiotics. The gene for resistance to the plasminogen activator is not included in the vector construct. "The technology is favorable," he said. (Gene transfer medical pr oducts for human use.”European Pharmacop ei 7.0(2011)). If small amounts of plasmid are required for cell line development, Although disruption of the material selection marker may be possible, these techniques tend to result in more plasmids. It is not practical for gene therapy applications where production is required.

[0004] A plasmid vector in which the replication origin and selection marker are less than 1 kb in total size is used. , Plasmid-based gene therapy to circumvent in vivo gene silencing The plasmid backbone is required for development. The plasmid backbone is 3 kb or 4 kb or more. Compared to conventional plasmids, the plasmids are longer and more efficient in mice when they are less than 1 kb. (Lu et al., Mol. Ther. 20(11):21 11-9 (2012)). Large blocks of DNA that were not expressed in vivo were silenced. It was proposed that this induced cisplatin synthesis. Sumido may be much more effective.

[0005] Smaller plasmids also allow for transient transfection to produce therapeutics. It also depends on the application in which it is used. One example is large-scale transfection of plasmids. The production of adeno-associated virus vectors that are used to generate clinical specimens. Smaller plasmids reduce the amount of DNA that must be transfected and Reduce costs. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, smaller vectors containing selectable markers that can be used in gene therapy applications are There remains a need to generate new plasmids. [Means for solving the problem]

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

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

[0009] In certain embodiments, the amino terminal fragment of β-galactosidase is In certain embodiments, the amino acid sequence has at least 75% identity to SEQ ID NO:1. 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. In certain embodiments, the high copy origin of replication is pUC57 In certain embodiments, the pUC57 origin of replication is the nucleic acid sequence of SEQ ID NO: 19. Contains arrays.

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

[0012] Also provided is an isolated vector comprising an isolated β-galactosidase expression cassette of the present invention. In certain embodiments, the isolated vector is about 1.5 kilobases long. In certain embodiments, the isolated vector is SEQ ID NO: 9-1. 3, 17, and 18.

[0013] Also provided is a method for producing the isolated vector of the invention. The method includes: (a) administering a host cell to the host cell; (b) contacting a host cell with the isolated vector; and (b) producing the host cell under conditions that result in the production of the vector. (c) growing the cell; and (c) isolating the vector from the host cell.

[0014] In certain embodiments, the host cells are grown in a minimal medium. In certain embodiments, the minimal medium may contain about 100% lactose per volume. In certain embodiments, the minimal medium comprises about 10% to about 4% (w / v) lactose. contains approximately 2% w / v lactose.

[0015] Also provided is (a) an isolated β-galactosidase expression cassette of the present invention, and (b) a la Kits are also provided that include a host cell that includes a deletion in the c operon. The kit further includes a minimal medium containing lactose as the sole carbon source. In certain embodiments, the vector comprises an isolated β-galactosidase expression cassette. In certain embodiments, the host cell comprises a LacZΔM15 deletion. 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 and the following detailed description of preferred embodiments of the present application should be taken in conjunction with the accompanying drawings. The present application will be better understood upon reading the accompanying drawings. It should be understood that there is no limitation to the precise embodiment. [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 in the Background and throughout this specification. or described in US Pat. No. 6,399,413, each of which 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 present specification is in accordance with the context of the present invention. Such discussion is intended to provide a basis for discussion of any or all of these matters. without admitting that any of the information contained herein constitutes prior art to any invention shown or claimed in any patent application. It is not.

[0018] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used herein have the meanings set forth herein.

[0019] As used in this specification and the appended claims, the singular forms "a," "an," and "t" are used interchangeably. It is important to note that "he" includes multiple referents unless the context makes clear otherwise. be.

[0020] Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges, described herein are In all cases, the terms "about" should be understood to be modified. Thus, numerical values ​​typically include ±10% of the stated value. For example, A concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration of 1% to 10 The concentration range of % (w / v) includes 0.9% (w / v) to 11% (w / v). When used herein, the use of a numerical range shall include the range unless the context clearly indicates otherwise. All possible subranges, including integers and fractions of values, and all individual numbers within that range Explicitly included.

[0021] Unless otherwise noted, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the and recognize many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. is intended.

[0022] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having "," "contains," or "containing," or any of these terms Other variations of the invention are intended to include the stated integer or groups of integers, but are not limited to these. is not intended to exclude any other integer or group of integers other than , and is non-exclusive or non-limiting. It will be understood that the present invention is intended to be understood as including, for example, a composition, mixture, process, or the like that includes a series of elements. The methods, articles, or apparatus are not necessarily limited to those elements expressly set forth herein. Any composition, mixture, process, method, article, or apparatus not listed in the In addition, unless expressly stated to the contrary, "or" may be used without the words "or" or "without" as the term. " refers to an inclusive "or" and not an exclusive "or." For example, A condition A or B is true (or exists) and B is false (or does not exist). if A is false (or does not exist) and B is true (or exists), then If both A and B are true (or exist), then it is satisfied by either one of.

[0023] As used herein, the conjunctive term "and / or" between multiple listed elements means Both individual and combined options are understood to be encompassing. For example, When elements are connected by "and / or," the first option may be used without the second option. The first element is applicable. The second option is that the second element can be applied without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is within the meaning and therefore the As used in the specification, it is understood that the term "and / or" is used interchangeably. The simultaneous applicability of two or more of them is also included in the meaning, therefore the term "and / or" It is understood that the requirements are met.

[0024] As used herein, the terms "consists of" or "consist Variations such as "of" or "consisting of" are included in the specification and claims. When used throughout the range, any enumerated integer or group of integers is inclusive, but additional Indicates that the integer or group of integers is not added to the specified method, structure, or composition.

[0025] As used herein, the term "consists essentially of" or "consist essentially of" or "consist essentially of" Variations such as "isting essentially of" are used throughout the specification and claims. When used herein, any enumerated integer or group of integers is inclusive, and optionally, the specified method, structure, or any enumerated integer or integers that do not materially change the basic or novel characteristics of the composition. Indicates inclusion of the set of integers. See MPEP § 2111.03.

[0026] The term "about" is used herein when referring to a size or characteristic of a component of the preferred invention. The terms "approximately," "generally," "substantially," and the like, as will be understood by those of ordinary skill in the art, are used to describe The dimensions / characteristics listed are not strict boundaries or parameters, but are functionally the same or similar. It should also be understood that the above does not exclude slight variations from the above. Such references involving numerical parameters, as used herein, are to be understood as being within the scope of the art. The mathematical and industrial principles used in the manufacture of products (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.) When using fractions such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3

[0027] The terms "identical" or "percent identity" refer to the degree to which two or more nucleic acids or polypeptides peptide sequences (e.g., amino-terminal β-galactosidase peptides and peptide sequences encoding them) In the context of the polynucleotides, the nucleic acids of the isolated vectors described herein, The maximum sequence comparison ratio (SEQ ID NO: 1) is 1, as determined by one of the sequence comparison algorithms below, or by visual inspection. Identical or the same amino acid residues when compared and aligned for large matches or two or more sequences or subsequences having a particular percentage of nucleotides. vinegar.

[0028] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer If necessary, subsequence coordinates are specified and the sequence algorithm program is executed. The sequence comparison algorithm is then run according to the designated program parameters. Based on the data, the percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0029] Optimal alignment of sequences for comparison can be performed using, for example, the method of Smith & Waterman. The local homology algorithm of Adv. Appl. Math. 2:482 (1981); Needleman & Wunsch, J. Mol. Biol. 48:443 (1970) Homology Alignment Algorithm, Pearson & Lipman, Proc. Nat. 'I. Acad. Sci. USA 85:2444 (1988) similarity search method, A computerized implementation of the algorithm by Wisconsin Genetics Software Package,Genetics Computer Group p,575 Science Dr.,Madison,WI GAP,BEST FIT, FASTA, and TFASTA), or visual inspection (generally Current Protocols in Molecular Biology,FMAusu bel et al., eds., Current Protocols, a join t venture between Greene Publishing Asso ciates, Inc. and John Wiley & Sons, Inc.,(1 This can be done by using the 995 Supplement (see Ausubel). .

[0030] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is ltschul et al. (1990) J.Mol.Biol.215:403-4 10 and Altschul et al. (1997) Nucleic Acids R BLAST and BLAST The BLAST analysis software is provided by the National Center for Biotechnology Information. Publicly available through the National Center for Biotechnology Information. The algorithm first identifies words and alphanumerics of the same length in the database sequence. Either they match when interleaved, or they meet some positive threshold score T. By identifying short words of length W in the query sequence, T is the neighborhood word score threshold (Altschul et al., 2011). These initial neighborhood word hits are then used to rank the nucleotide sequences that contain It then serves as a seed to initiate a search to find longer HSPs. Then, word hits are added along each sequence as far as possible to increase the cumulative alignment score. Extend the slit in both directions.

[0031] For nucleotide sequences, the parameter M (the reward for a pair of matching residues) Core (always >0) and N (penalty score for mismatched residues, always <0) are used. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The cumulative alignment score is then reduced by an amount X from its maximum score. When the alignment is performed, the accumulation of one or more negative scoring residues results in When the cumulative score falls 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. For the chido sequence, the default is word length (W) 11, expectation value 10, M=5, Use N=-4 and a comparison of both strands. For amino acid sequences, use the BLASTP program. Gram uses the default word length (W) of 3, expectation (E) of 10, and BLOSUM 62 Using the scoring matrix (Henikoff & Henikoff, Pr (see oc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0032] In addition to calculating percent sequence identity, the BLAST algorithm also Statistical analysis of similarities between columns is also performed (e.g., Karlin & Altschul, Proc. See 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 is the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid may be a nucleic acid that has a minimum match between a test nucleic acid and a reference nucleic acid. The total probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. If the sequence is less than 1, it is considered similar to the reference sequence.

[0033] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. The objective of the present invention is to provide a method for the preparation of a polypeptide that is immunologically cross-reactive with the polypeptide to be administered. A polypeptide is typically substantially identical to a second polypeptide, e.g., two peptides Another indication that two nucleic acid sequences are substantially identical is It is when two molecules hybridize to each other under stringent conditions.

[0034] As used herein, the term "isolated" refers to a material that is free from biological components (e.g., nucleic acids, peptides, proteins, or cells) from other organisms in which these components naturally occur. biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells , and tissues) or are produced separately from these components. It means that the product is either made from or is purified from these components. "Isolated" nucleic acids, peptides, proteins, and cells can be prepared by standard purification methods and those described herein. The nucleic acids, peptides, proteins, and cells purified by the purification methods described herein are included. "Isolated" nucleic acids, peptides, proteins, and cells are part of a composition. and the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell. The term also refers to recombinant expression in a host cell. This includes nucleic acids, peptides, and proteins prepared by the present invention, as well as chemically synthesized nucleic acids. do.

[0035] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid molecule." , also called "nucleotides" or "nucleic acids", and may be unmodified RNA or DNA or modified RNA or Any polyribonucleotide or polydeoxyribonucleotide, which may be NA or DNA. "Polynucleotide" refers to a nucleotide sequence that is a sequence of a nucleic acid molecule, such as a nucleic acid sequence of a nucleic acid molecule, ... or a nucleic acid sequence of a nucleic acid molecule. , single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and Double-stranded RNA, as well as RNA that is a mixture of single-stranded and double-stranded regions, single-stranded or more classical Typically includes DNA and RNA which may be double stranded or a mixture of single and double stranded regions. In addition, "polynucleotide" refers to a hybrid molecule that includes RNA or DNA. Polynucleotides refer to triple-stranded regions that contain both RNA or DNA. , DNA or RNA containing one or more modified bases, and The term "modified" bases also includes DNA or RNA having a backbone that has been modified by any method, e.g. , tritylated bases and unusual bases, e.g., inosine. Various modifications can be made to DNA and R Thus, a "polynucleotide" is a nucleic acid that is typically found in nature. Chemically, enzymatically or metabolically modified forms of the polynucleotides involved, as well as viruses and "Polynucleotides" include chemical forms that have the characteristics of DNA and RNA in cells. also encompasses relatively short strands of nucleic acid, often called oligonucleotides.

[0036] As used herein, the term "vector" refers to a vector that operably couples another nucleic acid segment to another vector. A replicon that can replicate or express a segment by inserting it into a And so.

[0037] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the transcription of a gene into RNA. The translation of the polypeptides into the above polypeptides, including all naturally occurring post-transcriptional and post-translational modifications, is also included. The expressed CAR may be expressed in the cytoplasm of a host cell, in an extracellular space, such as in the growth medium of a cell culture, or in a non-cellular space. It may be present in the environment or may be anchored in a cell membrane.

[0038] As used herein, the term "operably linked" refers to a structural or functional relationship. When positioned, the link between the nucleic acids (e.g., the promoter and the nucleic acid encoding the polypeptide) is For example, segments of a nucleic acid sequence are identical to each other in the same contiguous nucleic acid sequence. and a promoter positioned relative to the coding sequence to promote transcription of the coding sequence. A segment of a nucleic acid sequence may be a structural or functional relationship between the segment and another segment of the nucleic acid sequence, such as a promoter or enhancer. a ribosome binding site or pretag positioned relative to the coding sequence to facilitate translation The expression of a protein (e.g., a preprotein involved in the secretion of the encoded polypeptide) A presequence or secretory leader operably positioned relative to the coding sequence to promote In other embodiments, operably linked nucleic acid sequences are not contiguous, but may be linked together. have a functional relationship to one another as acids or as proteins expressed by them. Enhancers, for example, do not have to be contiguous. Linkage can be achieved by any convenient restriction. by ligation at the site, or by using synthetic oligonucleotide adaptors or can be achieved by using a linker.

[0039] As used herein, the term "promoter" refers to a gene that encodes a gene for a messenger RNA. "promoter" refers to a nucleic acid sequence that allows for the initiation of transcription of a child sequence, such transcription being initiated by a promoter or It is initiated by the binding of RNA polymerase near the promoter.

[0040] As used herein, the term "origin of replication" or "origin of replication" refers to a site of replication of a plasmid. Examples of replication origins include the pBR322 replication origin, the ColE1 replication origin, and the pUC57 origin of replication, pMB1 origin of replication, pSC101 origin of replication, and R6K Replication origins include, but are not limited to, the gamma origin of replication. Replication origins can be high copy or low copy. When present in a vector, a high copy origin of replication may be It can result in a high copy number (e.g., 150 to 200). In the case of a medium copy origin of replication, the 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 This can result in a low copy number (e.g., 1 to 3) of the vector.

[0041] As used herein, the term "dimer resolution element" refers to a nucleic acid sequence (e.g., a vector Promotes the in vivo conversion of multimers of a target molecule (e.g., a target or a plasmid) into monomers in which the target sequence is present. A dimer resolution element refers to a nucleic acid sequence that is a site-specific recombinase target site (e.g., For example, LoxP target site, rfs target site, FRT target site, RP4 res target site, The nucleic acid sequence may include a nucleic acid sequence including a RK2 res target site, and a res target site. The elements are transformed with a site-specific recombinase (e.g., Cre recombinase, ResD recombinase, Recombinase, Flp recombinase, ParA recombinase, Sin recombinase , β recombinase, γδ recombinase, tnpR recombinase, and pSK41 recombinase The isolated vector / nucleic acid dimer may comprise a nucleic acid sequence encoding a nucleotide ... Capable of being degraded by an enzyme acting on a target DNA sequence contained within the dimer resolution element The enzyme recombines the target DNA sequence. Non-limiting examples include host cell or dimer resolution. The enzymes XerC and XerD expressed by any of the vectors containing the elements are Recognizes the target site of the ColE1 dimer resolution element and produces vector / nucleic acid monomers It works with several additional cofactors to ensure that the

[0042] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to molecules composed of amino acids and recognized by those skilled in the art as proteins. In this specification, the conventional one-letter or three-letter codes for amino acid residues are used. The terms "peptide," "polypeptide," and "protein" refer to any length of amino acid sequence. can be used interchangeably herein to refer to a polymer of It may be linear or branched, may contain modified amino acids, and may be intermediated by non-amino acids. The term also refers to compounds that have been modified naturally or by intervention. Amino acid polymers include, for example, disulfide bond formation, glycosylation, lipidation, Acetylation, phosphorylation, or any other manipulation or modification, e.g., covalent coupling with a labeling component. The definition also includes, for example, one or more analogs of an amino acid (e.g., and polypeptides containing amino acids that are known in the art, such as unnatural amino acids. Other modifications known in the art are included.

[0043] The peptide sequences described herein are written according to the usual convention and include the N-terminal region of the peptide. The C-terminal region is on the left and the C-terminal region is on the right. The isomeric forms of the amino acids are known but are not shown separately. Unless explicitly stated, the L-form of the amino acid is shown.

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

[0045] In another general aspect, the invention provides a β-galactosyltransferase operably linked to a promoter. An isolated β-galactomycin-containing nucleic acid sequence encoding the amino terminal fragment of β-galactomycin. Concerning the sidase expression cassette.

[0046] In certain embodiments, the amino terminal fragment of β-galactosidase is In certain embodiments, the amino acid sequence has at least 75% identity to SEQ ID NO:1. , an amino terminal fragment of β-galactosidase that is at least 75% identical to SEQ ID NO:1; 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% amino acid sequence identity. The amino terminal fragment of the tosidase can comprise SEQ ID NO:1.

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

[0048] In certain embodiments, the isolated β-galactosidase expression cassette is a dimer. The dimer resolution element may further comprise a solution element. The site-specific recombinase recognition site may be, for example, a nucleic acid sequence comprising a nucleic acid sequence that includes a site-specific recombinase recognition site. , LoxP site, rfs site, FRT site, RP4 res site, RK2 res site, and res sites. The dimer resolution element may be selected from the group consisting of a site-specific lyco In certain embodiments, the host cell may further comprise a nucleic acid sequence encoding a recombinant enzyme. The site-specific recombinase comprises a nucleic acid sequence encoding the site-specific recombinase. , for example, Cre recombinase, ResD recombinase, Flp recombinase, P arA 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 lE1 dimer resolution element has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, Nucleic acid sequences with 3%, 94%, 95%, 96%, 97%, 98%, or 99% identity In certain embodiments, the ColE1 dimer resolution element may comprise SEQ ID NO:2. 0 nucleic acid sequence.

[0050] In certain embodiments, the isolated vector is an isolated β-galactosyltransferase of the invention. In view of the present disclosure, any vector known to those of skill in the art, e.g. , plasmids, cosmids, artificial chromosomes (e.g., bacterial artificial chromosomes (BACs), yeast artificial chromosomes YACs, and / or P1-derived artificial chromosomes (PACs), transposons, In some embodiments, a recombinant vector, such as a recombinant human vector, can be used. In one embodiment, the vector is a recombinant expression vector, such as a plasmid. Promoters, ribosome binding elements, terminators, enhancers, selection markers, and and a replication origin, which are optional elements for establishing the conventional functions of an expression vector. The promoter may be a constitutive, inducible, or reconfigurable promoter. Several expression vectors capable of delivering nucleic acids to cells are available in the art. It is known that the β-galactosidase peptide is produced by the synthesis of amino-terminal fragments of the β-galactosidase peptide. Conventional cloning techniques, or artificial gene synthesis, can be used herein to can be used to generate recombinant expression vectors according to embodiments of the invention.

[0051] In certain embodiments, the isolated vector is less than about 1.5 kilobases in size. The isolated vectors may 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 1 200 base pairs (approximately 1.2 kilobases), approximately 1300 base pairs (approximately 1.3 kilobases), approximately 1 400 base pairs (approximately 1.4 kilobases) or approximately 1500 base pairs (approximately 1.5 kilobases) in length In certain embodiments, the isolated vector may be less than about 1 kilobase. 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 bases in size. The size of the pair.

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

[0053] Also provided is a method for producing the isolated vector of the invention. The method includes: (a) administering a host cell to the host cell; (b) contacting a host cell with the isolated vector; and (b) producing the host cell under conditions that result in the production of the vector. (c) growing the cell; and (c) isolating the vector from the host cell.

[0054] In certain embodiments, the host cells are grown in a minimal medium. In certain embodiments, the minimal medium may contain about 100% lactose per volume. In certain embodiments, the minimal medium comprises about 10% to about 4% (w / v) lactose. is about 1% to about 4% w / v, about 1% to about 3% w / v, about 1% to about 2% w / v, about 1.5% ~4%w / v, approx.1.5%~3%w / v, approx.1.5%~2%w / v, approx.2%~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 In certain embodiments, the minimal medium contains about 3% to about 4% w / v lactose. The base contains approximately 2% w / v lactose.

[0055] In certain embodiments, the present invention relates to a host cell comprising an isolated vector of the present invention. In view of the present disclosure, any host cell known to those of skill in the art can be used to express the isolated vectors of the present invention. Suitable host cells include those having a LacZΔM15 deletion. These include cells that have a lactose biosynthetic pathway that is not activated but has the rest of the lactose biosynthetic pathway intact. This in the context of the Φ80 integration (i.e., the Φ80lacZΔM15 marker). The strain containing the mutation contained this mutation in the context of an intact lac operon, but It is a suitable host for the LacZ-α complementation plasmid. In the amino-terminal (N-terminal) region of the LacZ gene, which produces β-galactosidase from Other hosts having different deletions in the host cell line may also be suitable hosts. The cell may comprise an E. coli host cell or a yeast host cell.

[0056] Also provided is (a) an isolated β-galactosidase expression cassette of the present invention, and (b) a la and a host cell comprising a deletion in the c operon. In one particular embodiment, the vector comprises an isolated β-galactosidase expression cassette. In certain embodiments, the host cell comprises a LacZΔM15 deletion. The cell may be selected from an E. coli host cell or a yeast host cell.

[0057] In certain embodiments, the kit comprises a minimal medium containing lactose as the only carbon source. In certain embodiments, the minimal medium further comprises about 1% to about 4% by weight (by volume) of In certain embodiments, the minimal medium comprises about 1% to about 4% w / v lactose. / 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%~approx.3%w / v, approx.1.5%~approx.2%w / v, approx.2%~approx.4%w / v, approx.2%~approx. 3% w / v, about 2.5% to about 4% w / v, about 2.5% to about 35% w / v, or about 3% to about In certain embodiments, the minimal medium comprises about 2% w / v lactose. Contains 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 is: a. Contacting a host cell containing a deletion in the lac operon with a nucleic acid construct. Thus, the nucleic acid construct comprises a β-galactosidase gene operably linked to a promoter. An isolated β-galactosidase comprising a nucleic acid sequence encoding an amino terminal fragment of contacting the vector with an expression cassette; and b. under conditions in which only host cells containing the nucleic acid construct are maintained within the host cells; This involves growing the host cell.

[0059] In a second embodiment, the amino terminal fragment of β-galactosidase has at least one sequence similar to SEQ ID NO:1. 2. The method of embodiment 1, wherein the amino acid sequence has at least 75% identity.

[0060] In a third embodiment, the amino terminal fragment of β-galactosidase is the amino acid sequence of SEQ ID NO:1. 3. The method of embodiment 1 or 2, comprising the amino acid sequence

[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. This is the method described above.

[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 according to embodiment 5, wherein the high copy origin of replication is a pUC57 origin of replication. This is the method.

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

[0065] Embodiment 8 is a method for producing an isolated β-galactosidase expression cassette comprising: The method according to any one of embodiments 1 to 7, further comprising:

[0066] Embodiment 9 is a method for preparing a nucleic acid sequence comprising the steps of: 9. The method of embodiment 8, comprising the step of:

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

[0068] Embodiment 11 is a method for producing a method for the production of a nucleic acid molecule comprising the steps of: 10. The method according to embodiment 8 or 9.

[0069] Embodiment 12 is a method for the preparation of a nucleic acid sequence comprising the steps of: The method according to any one of embodiments 8 to 11.

[0070] Embodiment 13 relates to a ColE1 dimer resolution element comprising the nucleic acid sequence of SEQ ID NO: 20. 13. The method according to embodiment 12.

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

[0072] Embodiment 15 is a method for producing an isolated vector comprising the steps of: The method according to any one of embodiments 1 to 14, including

[0073] Embodiment 16 is a method for producing an isolated vector, the method comprising the steps of: 16. The method according to embodiment 15.

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

[0075] Embodiment 18 is a method for producing an isolated vector according to any one of embodiments 15 to 17. The method comprises the steps of: 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 cell is grown in minimal medium. .

[0077] Embodiment 20 is a method according to embodiment 19, wherein the minimal medium comprises lactose as the only carbon source. This is the method described.

[0078] In embodiment 21, the minimal medium comprises about 1% to about 4% by weight (w / v) of lactose. 21. The method of embodiment 20, comprising the step of:

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

[0080] Embodiment 23 is a method for producing a β-galactosidase polypeptide comprising the steps of: An isolated β-galactosidase expression cassette comprising a nucleic acid sequence encoding a terminal fragment thereof. It is.

[0081] Embodiment 24 is a method for producing an amino terminal fragment of β-galactosidase having a sequence similar to SEQ ID NO:1. 24. The isolated nucleic acid of embodiment 23, comprising an amino acid sequence having at least 75% identity thereto. β-galactosidase expression cassette.

[0082] Embodiment 25 is a method for the preparation of a β-galactosidase enzyme comprising the steps of: 25. The isolated β-galactosidase expression vector according to embodiment 23 or 24, comprising an amino acid sequence. It is a cassette.

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

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

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

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

[0087] Embodiment 30 is a method for producing an isolated β-galactosidase expression cassette comprising: 30. The isolated β-galactosidase according to any one of embodiments 23 to 29, further comprising It is an expression cassette for the ribosome entry .

[0088] Embodiment 31 is a method for determining whether the dimer resolution element comprises a site-specific recombinase recognition site. 31. The isolated β-galactosidase expression cassette of embodiment 30, comprising the nucleic acid sequence be.

[0089] Embodiment 32 is a method for the preparation of a nucleic acid sequence comprising the steps of: 32. The isolated β-galactosidase expression vector according to embodiment 30 or 31, further comprising an amino acid sequence. It is a cassette.

[0090] Embodiment 33 is a method for producing a method for the production of a dimer-disassembly element, wherein the dimer-disassembly element is a ColE1 dimer-disassembly element. The isolated β-galactosidase expression cassette according to any one of embodiments 30 to 32. It is.

[0091] Embodiment 34 relates to a ColE1 dimer resolution element comprising the nucleic acid sequence of SEQ ID NO: 20. 34. An isolated β-galactosidase expression cassette as described in embodiment 33.

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

[0093] Embodiment 36 is a method for producing an isolated vector, the method comprising the steps of: 36. The isolated vector of embodiment 35.

[0094] In embodiment 37, the isolated vector comprises SEQ ID NOs: 9-13, 17, and 18. 37. The isolated vector according to embodiment 35 or 36, comprising a nucleic acid sequence selected from the group be.

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

[0096] Embodiment 39 further comprises a minimal medium comprising lactose as the only carbon source. 38. A kit according to claim 38.

[0097] Embodiment 40 is a method for producing a recombinant human β-galactosidase vector comprising the steps of: 40. The kit according to embodiment 38 or 39.

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

[0099] Embodiment 42 is directed to a method for the preparation of a medicament for use in an embodiment of the present invention, wherein the host cell is selected from the group consisting of an E. coli host cell and a yeast host cell. 42. The kit according to embodiment 41, wherein EXAMPLES

[0100] Example 1: Expression of α-galactosidase instead of antibiotic selection in TOP10 cells Plasmid selection via complementation material Cells: One Shot Top10 competent cells (Thermo-Fisher ;Waltham, MA, Catalog No. C404003). NEB 5-α (New E ngland Biolabs, Ipswich, MA, catalog number (C2987). GT115 (InvivoGen, San Diego, CA, Catalog No. GT115 -21). NEB Stable (New England Biolabs, Catalog No. C3040H). Stellar(Takara Bio USA,Mountai) nView, CA, Catalog No. 636766). DH10B (Thermo-Fis her, Catalog No. 18297010). Stbl3 (Thermo-Fisher, Catalog number C737303). Xli-blue (Agilent, Santa Cl ara, CA, catalog number 200236). Plasmid: pUC19 (Thermo-Fisher Scientific, Catalog Log number SD0061;pBluescript II.KS(-)(Agilent (Santa Clara, CA, Catalog No. 212208). Clone P215 ( Row number 9) and P216 (SEQ ID NO: 10). GWIZ-luciferase (Genlant is Corporation;San Diego,CA;P030200);P21 9 (SEQ ID NO: 13, Figure 5). P469-2 (SEQ ID NO: 17, Figure 6). Culture medium: M9 + lactose medium (Teknova, Hollister CA, catalogue No. M1348-04 (plate): 0.3% KH2PO4, 0.6% Na2HP O 4、 0.5% (85mM) NaCl, 0.1% NH4Cl, 2mM MgSO4, 5 0 mg / L L-leucine, 50 mg / L isoleucine; 1 mM thiamine, 2% lactose, and 1.5% agar. M9+glucose medium (Teknova Hollister CA, Cat. No. M 1346-04 (plate): 0.3% KH2PO4, 0.6% Na2HPO 4、 0.5% (85mM) NaCl, 0.1% NH4Cl, 2mM MgSO4, 50mg / liter L-leucine, 50 mg / liter isoleucine, 1 mM thiamine, 1% Glucose, and 1.5% agar. LB-carbenicillin (100) plates (Tekno va, Hollister CA, Catalog No. L1010). LB plates (Tekn ova Hollister CA L1100). LB+60μg / mL X-Gal , 0.1mM IPTG (Teknova Hollister CA L1920). SOC medium (Thermo-Fisher 15544034). LB broth (Thermo mo-Fisher 10855021); D-PBS, pH 7.1, Mg 2+ None, Ca 2+ None (ThermoFisher 14200-075)

[0101] result Plasmids without antibiotic selection markers are useful for the expression of cells in gene therapy applications and therapeutic products. Plasmid backbones of 1 kb or less are desirable for strain development when delivered to animals in vivo. It has also been reported that these methods are useful for avoiding gene silencing. The purpose of the experiment was to develop a small metabolic selection matrix to select for plasmid-containing cells in E. coli. The aim of the study was to explore new strategies for developing lasers.

[0102] A plasmid expressing the α-peptide of β-galactosidase was The LacZΔ15 allele complements the lactose operon, allowing lactose to be secreted exclusively by the It was hypothesized that the cells could be grown on minimal medium with as little as 100% carbon source.

[0103] Plasmids pUC19 and pBluescript II are both β-galactosidases. These plasmids express the α-peptide fusion protein. To test whether the strain was able to complement the lac mutation in β-lactone and grow on minimal medium, did.

[0104] pUC19 and / or pBluescript II were used to characterize the La To test whether it was possible to complement the cZΔ15 mutation, these plates were The smids 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 + 50μL One Shot TO P10 cells. 2) 1 μL (10 pg) of pUC19 plasmid + 50 μL of One Sh ot TOP10 cells. The transformation mixture was incubated on ice for 30 min and then at 42 °C. After the heat shock, the transformation mixture was incubated on ice for 1 min. To the transformation mixture, 450 μL of SOC medium was added and the cells were incubated at 37° C. for 1 hour. The transformation mixture containing the cells was centrifuged and the cells were The cells were centrifuged and resuspended two more times in 500 μL of sterile D-PBS buffer. For each sample, two 1:10 serial dilutions of cells were made in D-PBS. 100 μL of each dilution was spread onto an M9+lactose plate. 200 μL of the first two Dilutions of 100% LB-carbenicillin were also spread onto LB-carbenicillin (100) plates. The plates were incubated at 37°C for 1 h. Incubated overnight.

[0106] After overnight incubation, the cells were plated on LB-carbenicillin (100) plates. There were many colonies from both transformations cultured on plates, and these plates were stored at 4°C. From any of the transformations plated on M9+lactose plates, There were no visible colonies 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 further incubated at 30°C. The plates were incubated for 48 hours. No colonies were observed on these plates even after long incubation periods. could not be seen.

[0107] None of the cloning vectors expressing LacZ-α fusion peptides were TOP10-host Minimal medium containing lactose as the sole carbon source that does not complement the Lac mutation in the main strain Growth in was not possible.

[0108] LacZ- with pUC19 and pBluescript II cloning vectors Expression of the α-peptide fusion protein adequately complemented the lac mutation in the host strains tested. Both vectors were designed for multicloning. The fusion protein is produced by transcribing the fusion protein through the Lac region. It may be suboptimal to complement the ZΔ15 mutation.

[0109] Example 2: LacZ expression plasmid used as a metabolic selection marker in E. coli Sumido. The medium and two La with strong promoters (LacZYA and OmpF, respectively) The cZ-α expression cassette was designed. The OmpF promoter sequence was amplified by Stavropoulo (Stavropoulos and Strathdee, Genomics 7 2(1):99-104(2001)) The LacZYA promoter is a promoter that expresses the lac gene, which is bound by the lac repressor. c operator sequence was derived from the sequence of pBluescript.

[0110] Regarding the open reading frame (ORF) of the LacZα peptide, Reddy (Reddy, Biotechniques 37(6):948-52(2004) The plasmid pUC19 expresses approximately 10 times more β-galactosyltransferase than pBluescript. These plasmids contained the lacZα peptide. However, pBluescript has the same promoter element that drives It has a much longer polylinker than pUC19, and pUC19 lacks the non-lacZ C-terminal residue. Among these differences, the pUC19β-galactosidase activity was enhanced by the It is unclear what causes this. Nishiyama et al. It was found to have the highest β-galactosidase activity in these assays (Ni shiyama et al., Protein Sci. 24(5):599-603 (2015)). The following wild-type LacZα from strain MG1655 was truncated at residue 60: Used area. MTMITDSLAVVLQRRDWENPGVTQLNRLAAHP PFASWRNSEEARTD RPSQQLRSLNGEWR (sequence number 1).

[0111] The terminator sequences were determined according to the method described by Orosz et al. (Eur. J. Biol. rrnBT2 type described by It comes from the minator.

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

[0113] result Plasmids without antibiotic selection markers are useful for the expression of cells in gene therapy applications and therapeutic products. Plasmid backbones of 1 kb or less are desirable for strain development when delivered to animals in vivo. It has also been reported that these methods are useful for avoiding gene silencing. The purpose of the experiment was to develop a small metabolic selection matrix to select for plasmid-containing cells in E. coli. The aim of the study was to explore new strategies for developing lasers.

[0114] A plasmid expressing the α-peptide of β-galactosidase was used to express the α-peptide of β-galactosidase in Top10 cells. The LacZΔ15 allele complements the lactose operon, allowing lactose to be secreted exclusively by the It was hypothesized that the cells could be grown on minimal medium with as little as 100% carbon source.

[0115] In Example 1, pUC19 and pBluescript expressing lacZα fusion peptides were used. Complement TOP10 cells with the pt vector and grow them in minimal medium with lactose These experiments were unsuccessful.

[0116] The lacZα fusion proteins encoded by these vectors were expressed as LacZΔ15 mutants. It is suboptimal in complementing the mutants and is sufficient to allow growth on lactose-containing minimal media. Based on the hypothesis that the lacZα expression vector was not expressed at a sufficiently high level, The ability of these vectors to complement the LacZΔ15 mutation was tested. 10 nanograms (ng) of plasmids P215 and P216, and pBluescr ipt II was transformed into 50 μL of OneShot Top10 cells. The cells were then placed on ice. Incubate with DNA at 4°C for 20 min, heat shock at 42°C for 30 s, and then place on ice. The cells were then returned to incubation at 4°C for 1 min. 450 μL of SOC was added to the cells and the cells were not shaken. The mixture was incubated at 37°C for 1 hour. 250 μL of cells were removed, and the remaining cells were The cells were then returned to the incubator. The extracted cells were washed twice with 500 μL of D-PBS for the final wash. Afterwards, the cells were resuspended in 200 μL of D-PBS. 50 μL of cells were added to LB-carbenicillin (100), M9+glucose, and M9+lactose plates, The plates were incubated at 37°C. After 4.5 hours of heat shock, the plates were removed from the incubator. The remaining cells were washed as above and plated on 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 and were cultured on Top 10 host cells can grow on these plates. Transformants plated on Silin(100) also produced many colonies. The LB-carbenicillin plates were stored at 4°C. The M9+lactose plates were The mixture was kept at 7° C. and incubated for a further 24 hours.

[0118] Transformants were allowed to recover for either 1 hour or 4 hours, both in M9 + Produced numerous colonies when plated on lactose plates. No colonies were present on the uescript II transformants, confirming the results of Example 1. pBluescript II was added to L to allow growth in lactose-minimal medium. We were unable to produce sufficient β-galactosidase through complementation of the acZΔ15 mutation. The plates were stored at 4°C.

[0119] Natural plasmids such as ColE1 are efficient in the E. coli host in the absence of antibiotic selection. pUC series vectors are efficiently maintained in the absence of selection, whereas pUC vectors are efficiently maintained in the absence of selection. (Summers, Molecular Microbiology 29 :1137-1145(1998)). However, the P Considering the very slow growth rate of 215 and P216 transformed cells, If the frequency of plasmid loss is not too high, cell culture in LB in the absence of selection can be performed. It would be much faster and less expensive for plasmid DNA purification to grow. Cells containing the β-galactosidase α complementation plasmid were stained with β-galactosidase, which transformed the cells into blue cells. Color changing XGAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) Grown on LB-IPTG-XGAL plates to hydrolyze the indicator (noside). Using this assay, the The frequency of plasmid loss when these cells were grown without antibiotics was examined.

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

[0121] After obtaining a pure population of cells, the continuous cell culture was grown. Single blue colonies were picked. The culture was incubated for 3 h at 37 °C for 3 min at 37 °C for 1 h at 37 °C and grown in 2 mL of LB medium in a 15 mL tube. Incubated overnight at 7°C.

[0122] Cells from the culture were streaked onto LB-IPTG-XGAL plates and the plates were incubated at 37°C. The plates were incubated at 4°C overnight. The colonies on the restreaked plates were blue. A colony was inoculated into 50 mL of LB in a 250 mL flask and incubated at 37°C with shaking. Incubated overnight.

[0123] 10 of overnight culture -4 Plate 50 μL of the dilution onto LB-IPTG-XGAL plates. Plates were incubated overnight at 37°C. 1 μL of 50 mL culture The culture was incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... for 1 h at 4° C. for 2 h at 4° C. for 1 h at were grown overnight at 4°C.

[0124] After overnight incubation, all colonies on the plate were observed to be blue. 10 of the 50 mL culture from the previous night -4 Add 50 μL of the dilution to LB-IPTG-XGA 1 μL of the 50 mL overnight culture was plated on 50 mL LB plates. (50,000-fold dilution) into fresh cultures. Cultures were grown overnight at 37°C.

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

[0126] The results showed that plasmids P215 and P216 were stable in the absence of selection. These plasmids were 7.2 and 7.3 for P215 and P216, respectively. kb. Dilute from a single colony to 50 mL, then dilute 1:50,000, When grown to confluence twice, the cells were found to still contain the plasmid in most of the cells. 1.25×10 without selection, while keeping 8 Can grow to a volume of 1 litre The transformation efficiency was improved by incubation of cells in SOC medium for 1 hour versus 4 hours after heat shock. The same was true when time was allowed to recover.

[0127] The constructed α-complementing plasmid complemented the LacZΔ15 mutation in Top10 cells. These proteases allow growth on minimal media with lactose as the sole carbon source. The plasmid was 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 In previous experiments, the β-galactosidase α peptide from the P215 and P216 plasmids Expression of the tide is useful as a selectable marker on the plasmid, and can be used to place antibiotic resistance genes. Next, in order to define the smallest possible replicon, To define which regions of the plasmid are essential for plasmid selection and replication in was required.

[0129] result cloning of mCherry and puromycin resistance genes using standard cloning techniques The clone was removed from plasmid P215 to generate plasmid P217 (SEQ ID NO:11) ( Figure 3).

[0130] The ampicillin resistance gene was extracted from plasmid P217 using standard cloning techniques. The ligated DNA was transformed into 50 μL of TOP10 cells. Incubate on ice for 20 minutes, heat shock for 30 seconds, and then incubate on ice for an additional 3 minutes. After incubation, 450 μL of SOC was added to the cells, and the cells were shaken. The cells were then pelleted and resuspended in 1 mL of d-PBS for 1 h at 37 °C. The cells were washed three times. They were plated on M9-lactose plates and incubated at 37°C for 2 days. Colonies were picked from the transformants and incubated with LB-IPTG-XGAL preculture. The resulting colonies were blue for each clone. A clone was picked (clone P218 (SEQ ID NO: 12, FIG. 4)) and DNA sequencing was performed to confirm the desired It was confirmed that the deletion had been created.

[0131] To further reduce the size of the β-galactosidase selection cassette, The transcription terminator (SEQ ID NO: 7) was deleted. This sequence was deleted to maintain the stability of the transcript. In addition to the possibility that was not required, the upstream promoter of the pUC57 / pMB1 origin Read-through transcription of the origin occurs by increasing transcription through the replication primer region of the origin. It has been reported that the copy number can be increased by Cleic Acid Res.12(6):2641-8(1984), Oka et al. al., Mol Gen Genet. 172(2):151-9(1979)).

[0132] Using standard cloning techniques, deletion construct P219 (SEQ ID NO: 13, 5) colonies were obtained, and the deletion was confirmed by DNA sequencing.

[0133] This work defines a minimal β-galactosidase expression cassette / origin of replication cassette. (SEQ ID NO: 18) is 938 bp. It is associated with a larger plasmid backbone. Smaller than 1 kb to avoid DNA silencing in mammalian cells The objective is met (Lu et al., Mol. Ther. 20(11):2111- 9(2012)).

[0134] Example 4: β-galactosidase-α complementation with a firefly luciferase expression cassette Construction of vector In the examples provided above, β-galactosidase was used as a selection marker instead of an antibiotic resistance gene. A plasmid was constructed that uses alpha complementation of the lactosidase mutation. To determine whether DNA replication was still efficient when 100 μg / mL was added to the Using the minimal β-galactosidase expression cassette / origin of replication sequence (SEQ ID NO: 18) , antibiotic selection markers and The origin of replication was replaced.

[0135] Using standard cloning techniques, cloning was performed using the GWIZ-luciferase plasmid (SEQ ID NO: A CMV promoter-luciferase-polyA expression cassette was cloned into P219 from Transformation into One Shot TOP10 cells, M9 + lactose plate Large colonies were obtained by plating on a plate and incubating at 37°C for 2 days. The colonies were restreaked on LB-IPTG-XGAL plates and incubated at 37°C. The mixture was incubated overnight at 4°C.

[0136] Blue colonies from the transformation reaction were amplified using primers CNFOR (SEQ ID NO: 14) and P45 5R2 (SEQ ID NO: 15) was used to screen for inserts. R-positive colonies were picked and inoculated into 6 mL LB cultures grown at 37 °C. The DNA yields isolated from the cultures were measured by measuring their OD using a spectrophotometer. 260 By measuring The results were estimated by (Table 1).

[0137] [Table 1]

[0138] In a 500 mL flask, 200 mL of LB was cultured with a single blue clone of P469-2. Ronnie were seeded and grown for 18 hours at 37°C in a shaker incubator. DNA was purified from this culture using the en HiSpeed ​​MaxiPrep kit. The DNA was purified 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 the origin of replication of GWIZ-luciferase are This clone was successfully replaced with the minimal β-galactosidase / origin of replication defined above. When the clone was grown in LB medium without selective pressure, acceptable plasmid yields were obtained. Achieved.

[0141] Example 5: β-galactosidase-α complementation vector function in various E. coli strains Noh Test β-galactosidase α peptide as a selectable marker instead of an antibiotic resistance gene To identify additional E. coli strains that can be used as One of the plasmids was tested by DNA transfection into eight different strains. I tried it.

[0142] [Table 2]

[0143] result Mix 50 μL of E. coli strain from Table 2 with 1 ng of plasmid DNA in a sterile microcentrifuge tube on ice. The cells were then incubated with P469-2 for 30 min. The cells were then heat shocked at 42°C for 30 s. The cells were incubated on ice for 1 min. 450 μL of SOC medium was added. Add L of NEB-Stable outgrowth medium (supplied by the manufacturer) The cells were incubated at 37°C with shaking for 1 hour. The cells were pelleted and The cells were washed three times with 1 mL of D-PBS. and incubated at 37°C for 3 days.

[0144] As expected, no colonies were detected on plates from Stbl3-transformed cells included as a negative control. No knees were detected. Five of the strains (Top10, GT115, NEB-Stab le, Stellar, and DH10B) had colonies of normal size. The strains (NEB-Alpha and XL1-Blue) had small colonies. The same strains as NEB-alpha (DH5alpha) and XL1-Blue were grown in minimal medium. This was predicted because the strain contains a mutation in the purB gene that slows growth in al.Appl Environ.Micro.76:6307-6309(2010) ).

[0145] Incubate the XL1-blue and NEB-Alpha plates at 37°C for an additional day. did.

[0146] Streaking colonies from M9-lactose plates onto LB-IPTG-XGAL plates Pure colonies were obtained by incubation at 37°C. - Streaking blue colonies (plasmid-containing cells) on XGAL plates (second round) C., which produced mostly blue cells.

[0147] All of the test strains that contained the Φ80dlacZΔM15 marker expressed β-galactosidase Transformed with the α-peptide expression plasmid P469-2 and grown on lactobacillus subtilis as the sole carbon source. The plasmid p53 can be selected on M9 minimal medium carrying the marker lacI on the F episome. q ZΔM Plasmid P469-2 transfectants of strain XL1-blue containing 15 were also The seven commercially available E. coli strains were also selectable on M9-lactose plates. The oli strain was shown to be compatible with the β-galactosidase selectable marker.

[0148] Those skilled in the art may make changes to the above-described embodiments without departing from the broad inventive concept. It will be understood that the invention is therefore not limited to the particular embodiments disclosed. without limitation, and is intended to encompass modifications within the spirit and scope of the invention as defined by this description. is understood to be intended. [Table 3] JPEG2025016484000004.jpg217143JPEG2025016484000005.jpg214143JPEG2025016484000006.j pg219141JPEG2025016484000007.jpg217141JPEG2025016484000008.jpg217142JPEG2025016484 000009.jpg217141JPEG2025016484000010.jpg217141JPEG2025016484000011.jpg217141JPEG20 25016484000012.jpg217142JPEG2025016484000013.jpg217142JPEG2025016484000014.jpg21514 1JPEG2025016484000015.jpg216142JPEG2025016484000016.jpg219141JPEG2025016484000017. jpg214144JPEG2025016484000018.jpg217142JPEG2025016484000019.jpg216142JPEG2025016484 000020.jpg217142JPEG2025016484000021.jpg216140JPEG2025016484000022.jpg217141JPEG20 25016484000023.jpg219144JPEG2025016484000024.jpg217141JPEG2025016484000025.jpg99141

Claims

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 comprises a β-galactosidase operably linked to a promoter. An isolated β-galactosidase comprising a nucleic acid sequence encoding an amino terminal fragment of β-galactosidase. contacting said compound with a tosidase expression cassette; and b. culturing said host cell under conditions such that said nucleic acid construct is maintained within said host cell. To grow The method comprising:

2. The amino terminal fragment of β-galactosidase has a sequence identical to that of SEQ ID NO:1 and a sequence identical to that of SEQ ID NO:

1.

13. The method of claim 1, comprising an amino acid sequence having an identity of 1% to 10%.

3. The amino terminal fragment of β-galactosidase has the amino acid sequence of SEQ ID NO:

1. The method of claim 1 or 2, comprising:

4. The method of any one of claims 1 to 3, wherein the nucleic acid sequence further comprises an origin of replication.

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

6. 6. The method of claim 5, wherein the high copy origin of replication is a pUC57 origin of replication.

7. 7. The method of claim 6, wherein the pUC57 origin of replication comprises the nucleic acid sequence of SEQ ID NO:

19.

8. The isolated β-galactosidase expression cassette further comprises a dimer resolution element. The method according to any one of claims 1 to 7.

9. The dimer resolution element comprises a nucleic acid sequence containing a site-specific recombinase recognition site. The method of claim 8 .

10. The dimer resolution element further comprises a nucleic acid sequence encoding a site-specific recombinase. The method of claim 8 or 9, comprising:

11. 9. The method of claim 8, wherein the host cell comprises a nucleic acid sequence encoding a site-specific recombinase.

9. The method according to claim 9.

12. The dimer resolution element is a ColE1 dimer resolution element.

2. The method according to any one of claims 1 to 11.

13. 12. The ColE1 dimer resolution element comprises the nucleic acid sequence of SEQ ID NO:

20. The method according to

14. The host cell of any one of claims 1 to 13, wherein the host cell comprises a LacZΔ15 deletion. method.

15. an isolated vector comprising the isolated β-galactosidase expression cassette; The method according to any one of claims 1 to 14.

16. 16. The method of claim 15, wherein the isolated vector is less than about 1.5 kilobases in size. Method of posting.

17. The isolated vector is selected from the group consisting of SEQ ID NOs: 9-13, 17, and 18. The method of claim 15 or 16, comprising a nucleic acid sequence comprising

18. A method for producing an isolated vector according to any one of claims 15 to 17, , 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. The method comprising:

19. 20. The method of claim 18, wherein the host cells are grown in minimal medium.

20. 20. The method of claim 19, wherein the minimal medium contains lactose as the sole carbon source.

21. The minimal medium comprises about 1% to about 4% weight per volume (w / v) lactose.

21. The method of claim 20.

22. 22. The method of claim 21 , wherein the minimal medium comprises about 2% w / v lactose.

23. A kit comprising: a. The isolated β-galactosidase expression cassette of any one of claims 1 to 13. t, and b. Host cells containing deletions in the lac operon The kit comprising:

24. 24. The method of claim 23, further comprising a minimal medium comprising lactose as the sole carbon source. Net.

25. 23. A vector comprising the isolated β-galactosidase expression cassette.

25. The kit according to claim 24.

26. 26. The method of claim 23, wherein the host cell comprises the LacZΔ15 deletion. The kit described.

27. The host cell is selected from the group consisting of an E. coli host cell and a yeast host cell.

27. The kit of claim 26.

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