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WO2001000819A1 - Remplacements de gene cibles dans une antebacterie par utilisation d'adn lineaire - Google Patents

Remplacements de gene cibles dans une antebacterie par utilisation d'adn lineaire Download PDF

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WO2001000819A1
WO2001000819A1 PCT/US2000/017154 US0017154W WO0100819A1 WO 2001000819 A1 WO2001000819 A1 WO 2001000819A1 US 0017154 W US0017154 W US 0017154W WO 0100819 A1 WO0100819 A1 WO 0100819A1
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gene
recombination
dna
bacteria
proficient
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PCT/US2000/017154
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English (en)
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Du Pont De Nemours And Company E.I.
Inc. Genencor International
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Chauhan, Sarita
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Publication of WO2001000819A1 publication Critical patent/WO2001000819A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

  • the present invention relates to the field of molecular biology and microbiology. More specifically, methods have been developed for the introduction of site-directed alterations in the Escherichia coli genome using linear DNA molecules to generate stable transformants.
  • Genome sequencing efforts have been completed for several prokaryotes including Escherichia coli. At least 46 genomes of different genera of bacteria of industrial, pharmaceutical and agricultural importance, have been completely sequenced and about 70 are in progress
  • Functional genomics seeks to discover gene function once nucleotide sequence information is available.
  • Proteomics the study of protein properties such as expression, post-translational modifications, interactions, etc.
  • metabolomics analysis of metabolite pools
  • the variety of techniques and methods used in this effort include the use of bioinformatics, gene-array chips, mRNA differential display, disease models, protein discovery and expression, and target validation. The ultimate goal of many of these efforts has been to develop high-throughput screens for genes of unknown function.
  • the first and most basic analysis for any gene is to assess the phenotype of the organism when the gene of interest is altered or rendered nonfunctional. The impact of such alterations as well as knockouts on proteome or metabolome can be measured to understand gene function.
  • the "null mutations" are often constructed by gene disruption (also called gene knockouts) by homologous recombination resulting in allelic exchange or gene replacement.
  • Gene disruption vectors are constructed by recombinant DNA techniques. Upon transformation into the organism the DNA construct with disrupted gene integrates at the resident location in the genome by homologous recombination and replaces the functional copy of the gene with the nonfunctional disrupted gene.
  • Gene disruption vectors are constructed from a genomic clone containing the gene of interest, using a vector that does not replicate in the host organism to be mutated.
  • the final disruption construct has 1) a selectable marker inserted in and or around the gene of interest in a manner that eliminates expression of the gene of interest or renders non-functional any protein product that is produced, and 2) additional unmodified genomic DNA flanking the gene disruption site of interest that helps target integration events to the disruption site.
  • Gene disruption vectors have been constructed for microbial and mammalian systems by
  • Yeast and Escherichia coli have previously been used for in vivo cloning for double strand break repair or gap repair (Oldenber et al., Nucleic Acids Research. 25:451-452 (1997); Hua et al., Plasmid 38:91-96 (1997); Oliner et al., Nucleic Acids Research 21:5192-5197 (1993); Bubeck et al., Nucleic Acids Research 21:3601-3602
  • a selectable marker can be amplified by PCR with targeting homology (60 bp on each side) incorporated in the PCR primers, thus avoiding the need for restriction analysis and subcloning with a large isolated genomic clone (Manivasakam et al., Nucleic Acids Research 23:2799-2800 (1995)).
  • This method is the basis of an effort by the yeast researchers worldwide to disrupt all 6000+ genes in this organism. This method is practiced with normal yeast chromosomes as well as with artificial chromosomes. Treco et al. (U.S.
  • the problem to be solved is to provide a convenient method without the limitations of known techniques to create site-directed mutants of Escherichia coli (or other bacteria) by allelic exchange using linear fragments of DNA to identify an altered phenotype that will define the function or effect of the mutated gene.
  • Traditionally the production of Escherichia coli mutants has been accomplished utilizing suicide vectors, which requires cloning of the gene disrupted with a selectable marker in such a vector.
  • Winans et al. J. Bacteriol. 161:1219-1221 (1985) have shown that site-directed mutants of Escherichia coli strains can be obtained using linearized fragments of DNA containing a selectable marker flanked by homologous DNA.
  • recB, recC and or sbcB are examples assert the requirement of specific genetic backgrounds (e.g., recB, recC and or sbcB).
  • the recB and recC mutations inactivate exonuclease, preventing it from degrading the linear DNA, whereas the sbcB mutation restores recombination proficiency in a strain which carries recB and recC mutations.
  • This invention provides a method for targeted DNA replacement in bacteria comprising introducing into a recombination-proficient bacteria a replacement cassette of DNA comprising a 5' recombinant region and a 3' recombinant region, each region independently at least 75% identical to a DNA target region of the bacteria.
  • the replacement cassette may or may not additionally include a functional gene positioned between the two recombinant regions that upon integration into host restores gene functionality.
  • the basis for screening is the phenotypic effect of a selectable marker that results from integration of the replacement cassette in the target sequence. Useful application of this method is to carry out site-directed DNA replacements for generation of desired DNA modifications in bacteria.
  • Figure 1 shows a schematic diagram of E. coli genomic DNA region that harbors yciK gene, the location of yciK ORF with respect to restriction sites relevant to gene constructions in this invention. Location of primers used for amplifying yciK DNA for pyciKCK, pSCyciKl and for knockout confirmations, is shown along with their Seq IDs.
  • Figure 2 shows the plasmid construct pSCyciKl .
  • yciK DNA flanked with engineered Hindl ⁇ . and Xhol sites was cloned into vector pBluescript II SK (+).
  • Figure 3 shows a schematic diagram of HinC ⁇ L fragment of pLoxCatl with relevant restriction sites used in engineering pyciKcat2 and pyciKcat3 and for confirmations of chloramphenicol resistance marker (SEQ ID NO:7) in mutants. Location and direction of cat ORF is shown as an arrow.
  • Figure 4 shows the plasmid construct pyciKcat2.
  • the HinCU fragment from yciK region (See Figure 1) was replaced with the 1.2 kb HinCH-Smal fragment of pLoxCatl .
  • cat gene is in opposite orientation to yciK gene.
  • Figure 5 shows the plasmid construct pyciKcat3 engineered same as above. However, here cat gene is in same direction as yciK gene.
  • Figure 6 is a gel confirming yciK knockouts by genome-specific PCR.
  • Figure 7 is a gel confirming yciK knockouts by Southern hybridization of Sst ⁇ l digested genomic DNA from wild-type and mutual E. coli probed with SEQ ID NO: 18.
  • Figure 8 shows a schematic diagram to describe Overlap-Extension PCR to generate tpiA deletion.
  • Figure 9 shows the plasmid construct pRN 106-2.
  • a 2004 bp overlap extension PCR product with 73% of the tpiA structural gene deleted ( Figure 8) was cloned into pCR-B unt (Invitrogen) to yield the 5517 bp plasmid pRN106-2.
  • the two extreme ends of tpiA are shown with an engineered Seal site in between.
  • Figure 10 shows the plasmid construct pRN 107- 1 , the source of the linearized DNA fragment used to generate the tpiA knockout.
  • the 1.2 kb HinCU fragment from pLoxCatl containing the loxP-CmR-loxR cassette was inserted into the Seal site of pRN106-2 to yield the 6763 bp plasmid pRN107-l.
  • pRN107-l as template and primers SEQ ID NO:l and SEQ ID NO:4
  • the 3246 bp fragment containing tpiA flanking regions and the loxP-CmR-loxP cassette was PCR-amplified and this linear DNA fragment was used to transform KLP23 to generate tpiA mutants by double crossover.
  • Figure 11 shows the design for confirmation of tpiA knockouts by genome-specific PCR. Applicant(s) have provided 19 sequences in conformity with
  • SEQ ID NO:l is the nucleotide sequence of DNA fragment containing yciK gene.
  • SEQ ID NO:2 is the forward primer for amplification of the yciK gene from the Escherichia coli genome.
  • SEQ ID NO: 3 is the reverse primer for amplification of t e yciK gene from the Escherichia coli genome.
  • SEQ ID NO:4 forward primer with Hwdlll site.
  • SEQ ID NO: 5 reverse primer site.
  • SEQ ID NO: 6 is the nucleotide sequence of the yciK fragment flanked with HindfD. and Xhol sites.
  • SEQ ID NO:7 is the Hr ⁇ CII fragment of pLoxCatl.
  • SEQ ID NO:8 is the deduced amino acid sequence encoded byj/c/ATopen reading frame.
  • SEQ ID NO: 9 is the forward primer used in a PCR reaction to confirm yciK knockout.
  • SEQ ID NO: 10 is the reverse primer used in a PCR reaction to confirm yciK knockout.
  • SEQ ID NO: 11 is a primer between sbp and cdh.
  • SEQ ID NO: 12 is a primer within tpiA 3' region.
  • SEQ ID NO: 13 is a primer within tpiA 5' region with an introduced Seal site.
  • SEQ ID NO:14 is a primer within yiiR 5' region.
  • SEQ ID NO: 15 is a primer at 5' end of sbp.
  • SEQ ID NO: 16 is a primer at 3' end of yiiR.
  • SEQ ID NO: 17 is expected PCR fragment when SEQ ID NO:9 and SEQ ID NO: 10 were used as primers with Escherichia coli genomic DNA as template.
  • SEQ ID NO: 18 is a 969bp Sstll fragment of pyciKCK.
  • SEQ ID NO: 19 is the sequence of expected band in Southern experiment when Sstll digested wild-type genomic DNA is probed with 969bp Sstll fragment (SEQ ID NO: 18) of pyciKCK. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a rapid and specific method for the modification or disruption of genes or other nucleic acid fragments.
  • the thus- modified or disrupted genes may then be transformed into their native hosts and screened for an altered phenotype, indicating gene function.
  • the invention creates stable site-directed alterations in E. coli or other gram negative enteric bacterial genome by allelic exchange using linear fragments of DNA to identify an altered phenotype that will define the function or effect of the mutated gene.
  • the invention does not have the limitations of the prior art with regard to time and restriction to specific genetic backgrounds. Additionally the present method has application in the areas of functional genomics, proteomics, metabolomics where gene disruptions to reveal function of unknown genes (and their products) or unknown function of known genes (and their products) or other functional DNA elements is desirable.
  • the present method may be used for metabolic engineering in techniques such as gene silencing by disruption, gene integration or replacement to add genes of new function or to replace a defective gene with a functional gene.
  • the present method may be useful to regulate or modify genes or operons by altering or modifying regulatory sequences such as promoters, ribosomal binding sites, terminators, and enhancers, for example.
  • PCR Polymerase chain reaction
  • ORF Open reading frame
  • IPTG is abbreviated IPTG.
  • SDS sodium dodecyl sulfate
  • LB is abbreviated LB.
  • Gene refers to a nucleic acid fragment that expresses a specific RNA only or a specific RNA and protein, including regulatory sequences preceding (5' noncoding sequences) and following (3' noncoding sequences) the coding sequence.
  • synthetic genes and “synthetic DNA” refer to genes and DNA that can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene constructs. "Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. "Gene constructs" may contain a full gene or less or more than a full gene.
  • gene disruption is used interchangeably with the term “gene knock out” to refer to the process of interfering within a gene such that no functional gene product is expressed.
  • gene replacement refers to a process which replaces a gene with either a functional or a mutated gene such that either no gene product is expressed or a mutant gene product is expressed.
  • DNA replacement means a process in which one fragment of DNA is replaced with another DNA fragment.
  • gene modification means any process where a gene is altered in any way including gene disruption or gene replacement.
  • gene targeting refers to a process where a specific site within a gene or nucleic acid fragment is identified or targeted on the basis of sequence.
  • nucleic acid fragment refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • a nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.
  • a nucleic acid fragment may be a portion of a gene, may be synthetic, or may be a genetic regulatory element.
  • a "target nucleic acid fragment” or “target gene” or “target DNA” is any nucleic acid fragment that is inserted into a modification plasmid that is targeted for modification or disruption.
  • Plasmid refers to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3 1 untranslated sequence into a cell.
  • Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.
  • Expression cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for expression of that gene in a foreign host.
  • selectable marker refers to those genes encoding proteins that may be expressed and/or which convey a phenotype on the host that enables selection.
  • a defective or functional gene may itself act as a selectable marker if its presence leads to a phenotype that can be selected against the background.
  • Background is undesirable colonies that appear during screening of gene replacement events.
  • modification plasmid refers to a specialized plasmid for use in the present invention comprising, at a minimum, a plasmid- specific marker and a replacement cassette.
  • Enteric-specific selectable markers are those genes encoding proteins that may be expressed in Enteric bacteria (e.g., Salmonella sp., Escherichia sp.), which convey a phenotype on the enteric host that enables selection.
  • Enteric bacteria e.g., Salmonella sp., Escherichia sp.
  • Enteric bacteria e.g., Salmonella sp., Escherichia sp.
  • Enteric bacteria are members of the family Enter obacteriaceae, and include such members as Escherichia, Salmonella, and Shigella. They are gram-negative straight rods, 0.3-1.0 X 1.0-6.0 ⁇ m, motile by peritrichous flagella, except for Tatumella, or nonmotile. They grow in the presence and absence of oxygen and grow well on peptone, meat extract, and (usually) MacConkey's media.
  • D-glucose As the sole source of carbon, whereas others require vitamins and or mineral(s). They are chemoorgano-trophic with respiratory and fermentative metabolism but are not halophilic. Acid and often visible gas is produced during fermentation of D-glucose, other carbohydrates, and polyhydroxyl alcohols. They are oxidase negative and, with the exception of Shigella dysenteriae 0 group 1 and Xenorhabdus nematophilus, catalase positive. Nitrate is reduced to nitrite except by some strains of Erwinia and Yersina. The G + C content of DNA is 38-60 mol % (T m , Bd).
  • DNAs from species from species within most genera are at least 20% related to one another and to Escherichia coli, the type species of the family. Notable exceptions are species of Yersina, Proteus, Providenica, Hqfhia and Edwardsiella, whose DNAs are 10-20% related to those of species from other genera. Except for Erwinia chrysanthemi all species tested contain the enterobacterial common antigen (Bergy's Manual of Systematic Bacteriology, D. H. Bergy, et al., Baltimore: Williams and Wilkins, 1984).
  • KO vector or “knock out vector” refer to a modification plasmid lacking the target gene to be modified.
  • knock out plasmid refers to a modification plasmid that contains a gene targeted for disruption.
  • knock out cassette refers to a modification cassette that contains recombination regions designed to insert a modifying nucleic acid within the coding region of the target gene so as to prevent the effective expression of that target gene.
  • a "replacement cassette” refers to a specialized DNA cassette that comprises at a minimum, a selectable marker, a modifying DNA and 5' and 3' recombination regions. Replacement cassettes may optionally also comprise other modifying DNA or RNA sequences, inserted between the flanking recombination regions. Within the context of the present invention, replacement cassettes containing modifying DNA interact with the DNA to be replaced via the mechanism of homologous recombination to permit modification or disruption of the target gene or target nucleic acid.
  • genomic host refers to the cell or host from which the target gene or DNA has been cloned.
  • inserting nucleic acid fragment refers to a DNA (“inserting DNA fragment”) or RNA (“inserting RNA fragment”) molecule residing in a replacement cassette that is useful for the modification or disruption of a target gene or target nucleic acid fragment.
  • the inserting nucleic acid fragment will insert at a site directed by the sequence of the recombination regions on the cassette.
  • Inserting DNA may be either “modifying” or “disrupting”.
  • Modifying DNA or “modifying nucleic acid fragments” will result in the altering of the composition or function of a target gene but will not disrupt the gene.
  • “Disrupting DNA” or “disrupting nucleic acid fragments” will have the effect of disrupting the target gene of interest.
  • Modifying DNA or “disrupting DNA” will include but will not be limited to non-specific DNA or RNA sequences, selectable markers, origins of replication, antisense sequences and regulatory elements.
  • regulatory elements refer to nucleotide sequences located upstream (5'), within, and/or downstream (3') to a coding sequence, which control the transcription and/or expression of the coding sequences, potentially in conjunction with the protein biosynthetic apparatus of the cell. In artificial DNA constructs regulatory elements can also control the transcription and stability of antisense RNA.
  • One specific regulatory element is a "promoter”.
  • Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • recombination region refers to 3' and 5' flanking nucleic acid regions on the replacement cassette. Recombination regions are designed to be either complementary to, or have significant base identity with, the corresponding regions of the target genes or target nucleic acid fragments to be disrupted or modified. Significant identity, for example, may range from about 70% between the bases of the target gene ("target region") and the recombination regions. Recombination regions at the 5' end of the cassette are referred to as “5' recombination regions” and recombination regions at the 3' end of the cassette are referred to as "3' recombination regions". The maximum length of the flanking recombination region is set at approximately 150 bases when using PCR amplification in the technique.
  • adenosine is complementary to thymine and cytosine is complementary to guanine.
  • identity is a relationship between two or more polynucleotide sequences as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polynucleotide sequences as determined by the match between strings of such sequences.
  • identity and similarity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data. Part I (Griffin, A. M., and Griffin, H.
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul et al., Natl. Cent. Biotechnol.
  • nucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a partial sequence or "a portion of a sequence” refers to a sequence of sufficient length to permit homologous recombination according to the conditions of the present method. Typically, "a partial sequence” or “a portion of a sequence” will range from about 15 bp to about 200 bp.
  • coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
  • Suitable regulatory sequences refer to nucleotide sequences located upstream (5' noncoding sequences), within, or downstream (3' noncoding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • operon refers to a group of operably linked genes and regulatory elements that functions as a unit of transcription. An operon starts with a promoter, which binds RNA polymerase and initiates transcription of the operon.
  • expression refers to the transcription and stable accumulation of sense (mR A) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
  • recombination proficient means that a microorganism is capable of integrating extra-chromosomal DNA into its genome via homologous recombination. Additionally, a "recombination proficient" microorganism is also capable of recombining DNA resident on chromosome if sufficient homology exists between another DNA locus.
  • Transformation refers to the transfer of a nucleic acid fragment into the host organism.
  • Stable transformation refers to integrating the nucleic acid into the genome of the cell.
  • “Absolute recombination frequencies” range from 2.3 X 10" 6 to 1.5 X 10" 1 in E. coli depending on the system and length of homology involved. RecA is the most crucial component for the homologous recombination reaction, although recA independent mechanisms also exists. Homologs and analogs of recA-like proteins and other genes linked to homologous recombination have been discovered in a wide range of bacteria and eukaryotes. (Lloyd, R. G. and Low, K. B. (1996) Homologous Recombination. In Escherichia coli and Salmonella Cellular and Molecular Biology, Ed. F.C. Neidhardt, ASM Press, p 2236-2255).
  • absolute recombination frequencies can be calculated to be in the range of 10" 12 to 10' 2 .
  • Linear DNA is the replacement cassette that is isolated from a modification plasmid by restriction digestion or by PCR amplification as described in Examples 1 and 4. Preparation of linear DNA is also described in Example 3.
  • linear DNA has been used for gene replacements in some bacteria such as Bacillus subtilis
  • literature has strongly indicated that gene replacements using linear DNA do not occur in wild type bacteria such as E. coli and Salmonella unless some mutations or special genes are introduced to render these organisms hyper-recombinant.
  • Use of electroporation offering the highest efficiencies of transformation among the presently known methods, demonstrates that linear DNA can be used for gene replacements in wild type E. coli.
  • gene replacements can be performed in any recombination proficient bacteria which carry a functional recombination system.
  • the present method is useful for the rapid disruption or modification of genes or other genetic elements. Genes of unknown function which are disrupted in this fashion are transformed into genomic hosts where screening for an altered phenotype will reveal their function.
  • This method is also useful for replacing defective or mutated genes from bacterial genome with the functional gene provided a selection phenotype exist for the function of the gene.
  • the gene replacement techniques described in examples to follow can also be used for regulation of genes present in operons by replacement of operably linked promoter region with a promoter of desired strength.
  • the method described in this invention to obtain gene replacement can be used in enteric bacteria without specialized host backgrounds or use of suicide vectors.
  • Electrotransformation also constitutes a clear-cut method to obtain gene replacements with linear DNA in wild-type Escherichia coli on plasmid and chromosomal targets. However, any methods (chemical transformation or electrotransformation) that ensure a transformation efficiency high enough to enable the occurrence of double homologous recombination between the target DNA and the replacement cassette will work, in this specific case electrotransformation was used.
  • the method of electrotransformation itself does not guarantee a success in gene replacements if quality of competent cells is not good or if host strains deficient in recombination functions are used.
  • a high efficiency of transformation must occur in recombination proficient bacteria.
  • target gene (or target DNA) locus and the host strain required transformation efficiencies will vary.
  • Use of long PCR primers as described in Example 3 completely eliminates any cloning requirements for gene replacements.
  • the present method is useful for the rapid disruption or modification of genes or other genetic elements.
  • Genes of unknown function disrupted in this fashion may be transformed into genomic hosts where screening for an altered phenotype will reveal their function.
  • PCR reactions were run on GeneAMP PCR System 9700 using Amplitaq or Amplitaq Gold enzymes (PE Applied Biosystems, Foster City, CA). The cycling conditions and reactions were standardized according to manufacture's instructions.
  • EXAMPLE 1 Targeted Gene Replacement by Linear DNA Fragments for Site-directed Mutapenesis of Escherichia coli
  • Example 1 describes the cloning, mutation and integration of a mutant yciK gene into the Escherichia coli genome by homologous recombination using linearized mutant DNA. Isolation of yciK gene:
  • a 1460 bp fragment of DNA (SEQ ID NO:l, Figure 1) containing yciK gene was amplified using pCR-Script Amp Cloning kit from Stratagene (catalog #211188) from genomic DNA of Escherichia coli strain W3110 (Sigma; catalog #D0421).
  • PCR amplification (Mullis and Faloona, Methods E zymol. 155:335-350 (1987)) was performed using ohgonucleotides SEQ ID NO:2 and SEQ ID NO:3 as forward- and reverse primers, respectively ( Figure 1).
  • the PCR product was blunt-ended and ligated into Srfi site of cloning vector pCR-Script Amp SK(+) followed by transformation into XL-1 Blue MRF' Kan supercompetent cells.
  • Transformed cells were plated on Luria-Bertani medium containing ampicillin, X-gal and IPTG. Plasmids were isolated from white, ampicillin-resistant transformants and tested for the presence of the insert by restriction digestion.
  • One such plasmid, pyciKCK was subsequently confirmed for the insert containing yciK gene by sequencing on ABI PRISM (Model 377,
  • the pyciKCK DNA was used as a template with primers SEQ ID NO:4 and SEQ ID NO:5 to generate a DNA fragment (SEQ ID NO:6) containing yciK gene flanked with Hin ⁇ HL restriction sites ( Figure 1) for cloning in Hin ⁇ llVXhol restricted pBluescript II SK(+) vector (Stratagene; catalog #212205).
  • the resultant plamid was named pSCyciKl ( Figure 2).
  • the plasmid construct (pyciKcat2 or pyciKcat3) was digested v ⁇ t Afl ⁇ I and Xmnl, followed by gel purification of a 3.9 kb band of linearized DNA fragments ( Figures 3 and 4) containing mutant yciK gene.
  • Escherichia coli strains FM5 (ATCC 53911) and KLP23 (FM5 glpKrgldA ⁇ ) were electrotransforaied with 0.5 ⁇ g of the above 3.9 kb linear DNA and the resulting transformants were screened for double recombinant phenotype of chloramphenicol resistance (25 ⁇ g/mL) and ampicillin sensitivity (100 ⁇ g/mL) on LB medium. High numbers of transformants (roughly 10 7 -10 9 ) were screened. Genomic DNA from Cm r Amp s colonies was dot blotted on Hybond N + positively charged nylon membrane (Amersham; catalog # RPN2020B).
  • a 982 bp Xbal fragment (bases 58-1039 of SEQ ID NO:7 denote the sense strand) that contains Cm r encoding fragment, was used a probe. All Cm r Amp s colonies tested positive for the presence of DNA encoding chloramphenicol resistance gene under high stringency conditions (0.1 x SSC, 0.1% SDS, 60°C for 15 min). Probe labeling, hybridization and detection for dot blots and subsequent Southern blotting experiments, were performed using ECL random prime labelling and detection systems version II, (Amersham International pic, Buckinghamshire, England).
  • the DNA from mutant colonies yielded a 3.1 kb PCR product compared to 2.1 kb product from wild-type (SEQ ID NO: 17; Figure 6) confirming that yciK gene in the Escherichia coli genome contained an insertion as predicted by the construct design.
  • the mutants were further authenticated by Southern hybridization of SstU digested genomic DNA fragments (Figure 1) probed with 969 bp Sstll fragment of pyciKCK containing yciK DNA (SEQ ID NO: 18). All the mutants yielded a 2.3 kb band compared to the 1.3 kb band (SEQ ID NO: 19) of wild-type strains ( Figure 7).
  • FM204 was a FM5 derivative obtained by integrating linearized DNA from pyciKcat2; the FM5 integrants that resulted from the pyciKcat3 AflRl-Xmnl fragment were named FM301 and FM302.
  • KLP201 and KLP202 were KLP23 derivatives using pyciKcat2 fragments.
  • KLP301 and KLP304 were also derived from KLP23 by using the fragment from pyciKcat3.
  • Linear DNA molecules can also be used to replace mutant genes with the wild-type gene. If a mutant has a phenotype of lack of growth under certain nutrient or environmental conditions, a replacement of wild-type gene can be selected by acquired ability for growth under same conditions. For example, hemB mutants of Escherichia coli and Bradyrhizobium japonicum require exogenously added hemin for growth (Chauhan, S. and O'Brian, M. R. (J. Bacteriol. 175:7222-7227 (1993)).
  • a linear DNA fragment containing functional hemB gene from a homologous or heterologous source is amplified (by PCR or by cloning).
  • the Escherichia coli hemB mutant strain RP523 (Li, J. M. et al., J. Bacteriol, 170:1021-1025 (1988) is electrotransformed with the amplified linearized) DNA fragments that harbor functional hemB gene. Transformants are screened on LB plates for growth. Any colonies that appear are due to a functional hemB gene in the genome. If insertional mutations are being replaced, genomic constructs are further tested by PCR amplification or Southern hybridizations of the region of DNA modified.
  • the transformants may be screened for the absence of the phenotype conferred by the selectable marker.
  • the PCR amplified fragment is sequenced either directly or after cloning in a suitable vector (e.g. pBluescript II SK(+), Stratagene, Cat #212205) for the presence of wild-type sequence. This method is extremely beneficial for restoring specific gene functionality in a host strain that has gone through several sequential mutations in the genome.
  • EXAMPLE 3 One-step PCR Method to Generate Linear DNA Fragments for Site-directed Mutagenesis Oligonucleotide pairs approximately 150 bases in length are synthesized. Up to 30 bp on the 3 '-end of forward primer is identical to a region flanking the 5'-end of a selectable marker and the rest of the bases are identical to the 5' region of the gene to be disrupted. Up to 30 bp on the 3 '-end of reverse primer are complementary to a region flanking the 3'-end of a selectable marker and the rest of the bases are identical to the 3' region of the gene to be disrupted.
  • the above oligonucleotides are used in a PCR reaction to amplify DNA fragments that contain a marker gene flanked on each side with at least 20 bases of sequence homologous to the gene desired to be mutated.
  • the PCR product is used to electrotransform wild-type Escherichia coli cells for generating mutants by homologous recombination which can be tested by methods described in Example 1.
  • EXAMPLE 4 Engineering of Triosephosphate Isomerase Mutant of Escherichia coli KLP23 This example describes the construction of plasmid for triosephosphate isomerase gene replacement in Escherichia coli KLP23 and engineering of triosephosphate isomerase mutant RJ8m by linear DNA transformation.
  • Escherichia coli KLP23 genomic DNA was prepared using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). A 1.0 kb DNA fragment containing cdh and the 3' end of triosephosphate isomerase (tpiA) genes was amplified by PCR (Mullis and Faloona, Methods Enzymol. 155:335-350 (1987)) from KLP23 genomic DNA using primers (SEQ ID NO:l 1 and SEQ ID NO: 12).
  • a 1.0 kb DNA fragment containing the 5' end of tpiA, yiiQ, and the 5' end of yiiR genes was amplified by PCR from KLP23 genomic DNA using primers SEQ ID NO:13 and SEQ ID NO:14.
  • the first 26 bases at 5' end of primer SEQ ID NO:13 are complementary to the primer SEQ ID NO: 12 to enable subsequent overlap extension PCR, followed by a Seal (AGTACT) site.
  • the next 28 bases following S al site at the 3' end of SEQ ID: 13 reside on 5' end of the tpiA gene ( Figure 8).
  • the gene splicing by overlap extension technique was used to generate a 2.0 kb fragment by PCR using the above two PCR fragments as templates and primers SEQ ID NO: 11 and SEQ ID NO: 14.
  • This fragment represented a deletion of 73% of the 768 bp tpiA ORF.
  • this fragment had 1.0 kb flanking regions on either side of the S al cloning site (within the partial tpiA) to allow for chromosomal gene replacement by homologous recombination.
  • the 1.2 kb Hindi fragment (SEQ ID NO:7, Figure 3) containing a chloramphenicol-resistance gene flanked by bacteriophage PI loxP sites (Snaith et al., Gene 166:173-174 (1995)), was used to interrupt the tpiA fragment in plasmid pRN 106-2 by ligating it to Sc ⁇ l-digested plasmid pRN 106-2 to yield the 6.8 kb plasmid pRNl 07- 1 ( Figure 10).
  • Escherichia coli KLP23 was electrotransformed with about 1 ⁇ g of this 3.2 kb linear DNA fragment. Transformants that were chloramphenicol-resistant (12.5 ⁇ g/mL) and kanamycin-sensitive (30 ⁇ g mL) were further screened on M9 minimal media for poor glucose utilization on 1 mM glucose, a phenotype expected for tpiA mutant. An EcoRI digest of genomic DNA from one such mutant, RJ8m, was probed with the intact tpiA gene via Southern analysis (Southern, J. Mol. Biol. 98:503-517 (1975)).

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Abstract

La présente invention concerne une technique rapide et particulière permettant de remplacer un gène ciblé dans une bactérie. Les gènes modifiés ou interrompus ou d'autres fragments d'acide nucléique sont transformés dans leurs hôtes, et ces transformants sont analysés en vue d'intégrations à l'aide d'un marqueur que l'on peut sélectionner pour un phénotype modifié. Plus précisément, cette invention consiste à créer des mutants dirigés d'antébactérie par un échange allèlique de façon à identifier un phénotype modifié qui définira la fonction ou l'effet du gène mutant. Cette invention montre aussi comment on peut utiliser des fragments d'ADN linéaire recevant un gène fonctionnel pour remplacer un gène mutant afin de restaurer la fonctionnalité du gène.
PCT/US2000/017154 1999-06-25 2000-06-21 Remplacements de gene cibles dans une antebacterie par utilisation d'adn lineaire WO2001000819A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003095658A1 (fr) * 2002-05-07 2003-11-20 Novozymes A/S Recombinaison homologue en bacterie pour produire des bibliotheques de polynucleotides
US7005255B2 (en) 2000-04-14 2006-02-28 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7329489B2 (en) 2000-04-14 2008-02-12 Matabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7576319B2 (en) 1999-07-21 2009-08-18 Sionex Corporation Systems for differential ion mobility analysis
US8849577B2 (en) 2006-09-15 2014-09-30 Metabolon, Inc. Methods of identifying biochemical pathways

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990012098A2 (fr) * 1989-04-12 1990-10-18 Cetus Corporation Procedes et sequences d'acides nucleiques pour l'expression de l'operon de synthase de la cellulose
US5530191A (en) * 1994-03-24 1996-06-25 Rutgers, The State University Of New Jersey Method for producing cytoplasmic male sterility in plants and use thereof in production of hybrid seed
WO1998042727A1 (fr) * 1997-03-21 1998-10-01 Sri International Modifications de sequence par recombinaison homologue
US5837528A (en) * 1989-06-22 1998-11-17 Hoffmann La Roche, Inc. Bacterial strains which overproduce riboflavin
WO1999023238A1 (fr) * 1997-10-31 1999-05-14 Amgen Inc. Synthese efficace de vecteurs de ciblage genetique par recombinaison entre phages et plasmides
WO1999066035A2 (fr) * 1998-06-17 1999-12-23 Iconix Pharmaceuticals Inc. Construction de vecteur par recombinaison induite par des cellules hotes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990012098A2 (fr) * 1989-04-12 1990-10-18 Cetus Corporation Procedes et sequences d'acides nucleiques pour l'expression de l'operon de synthase de la cellulose
US5837528A (en) * 1989-06-22 1998-11-17 Hoffmann La Roche, Inc. Bacterial strains which overproduce riboflavin
US5530191A (en) * 1994-03-24 1996-06-25 Rutgers, The State University Of New Jersey Method for producing cytoplasmic male sterility in plants and use thereof in production of hybrid seed
WO1998042727A1 (fr) * 1997-03-21 1998-10-01 Sri International Modifications de sequence par recombinaison homologue
WO1999023238A1 (fr) * 1997-10-31 1999-05-14 Amgen Inc. Synthese efficace de vecteurs de ciblage genetique par recombinaison entre phages et plasmides
WO1999066035A2 (fr) * 1998-06-17 1999-12-23 Iconix Pharmaceuticals Inc. Construction de vecteur par recombinaison induite par des cellules hotes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EL KAROUI MERIEM ET AL: "Gene replacement with linear DNA in electroporated wild-type Escherichia coli.", NUCLEIC ACIDS RESEARCH, vol. 27, no. 5, 1 March 1999 (1999-03-01), pages 1296 - 1299, XP002149590, ISSN: 0305-1048 *
MURPHY, K.C.: "Use of bacteriophage lambda recombination funtions to promote gene replacement in Escherichia coli.", JOURNAL OF BACTERIOLOGY, vol. 180, no. 8, April 1998 (1998-04-01), pages 2063 - 71, XP002149589 *
SHIROZA T ET AL: "Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene, and secrete its active gene product", GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES,GB,ELSEVIER SCIENCE PUBLISHERS, BARKING, vol. 207, no. 2, 30 January 1998 (1998-01-30), pages 119 - 126, XP004108909, ISSN: 0378-1119 *
WHITE A P ET AL: "High efficiency gene replacement in Salmonella enteritidis: chimeric fimbrins containing a T-cell epitope from Leishmania major", VACCINE,GB,BUTTERWORTH SCIENTIFIC. GUILDFORD, vol. 17, no. 17, 23 April 1999 (1999-04-23), pages 2150 - 2161, XP004165000, ISSN: 0264-410X *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7576319B2 (en) 1999-07-21 2009-08-18 Sionex Corporation Systems for differential ion mobility analysis
US7550258B2 (en) 2000-04-14 2009-06-23 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7329489B2 (en) 2000-04-14 2008-02-12 Matabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7550260B2 (en) 2000-04-14 2009-06-23 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7553616B2 (en) 2000-04-14 2009-06-30 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7005255B2 (en) 2000-04-14 2006-02-28 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7635556B2 (en) 2000-04-14 2009-12-22 Cornell Research Foundation, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7682784B2 (en) 2000-04-14 2010-03-23 Cornell Research Foundation, Inc. Methods for drug discovery disease treatment, and diagnosis using metabolomics
US7682783B2 (en) 2000-04-14 2010-03-23 Cornell Research Foundation, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7910301B2 (en) 2000-04-14 2011-03-22 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7947453B2 (en) 2000-04-14 2011-05-24 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
WO2003095658A1 (fr) * 2002-05-07 2003-11-20 Novozymes A/S Recombinaison homologue en bacterie pour produire des bibliotheques de polynucleotides
US8849577B2 (en) 2006-09-15 2014-09-30 Metabolon, Inc. Methods of identifying biochemical pathways

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