[go: up one dir, main page]

WO1984000381A1 - Manipulations genetiques dans les organismes procaryotiques - Google Patents

Manipulations genetiques dans les organismes procaryotiques Download PDF

Info

Publication number
WO1984000381A1
WO1984000381A1 PCT/US1983/001026 US8301026W WO8400381A1 WO 1984000381 A1 WO1984000381 A1 WO 1984000381A1 US 8301026 W US8301026 W US 8301026W WO 8400381 A1 WO8400381 A1 WO 8400381A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
microorganism
portions
foreign
insertion vehicle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1983/001026
Other languages
English (en)
Inventor
Aladar A Szalay
John G K Williams
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boyce Thompson Institute
Original Assignee
Boyce Thompson Institute for Plant Research Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boyce Thompson Institute for Plant Research Inc filed Critical Boyce Thompson Institute for Plant Research Inc
Priority to JP83502712A priority Critical patent/JPS59501195A/ja
Priority to DE198383902654T priority patent/DE113781T1/de
Publication of WO1984000381A1 publication Critical patent/WO1984000381A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to procaryotic organisms that have foreign DNA stably inserted in a chromosome and a method for their production.
  • chimeric plasmids are designed to replicate autonomously as an extrachromosomal DNA species in a procaryotic host such as E. coli.
  • the chimeric plasmid may either replicate extrachromosomally or it may integrate into yeast chromosomes, although such integration tends to be unstable.
  • Another object of the present invention is to provide a unicellular photosynthetic microorganism which contains at least one stable foreign DNA portion in a chromosome.
  • One aspect of the present invention is directed to a procaryotic microorganism containing at least one stable foreign DNA portion covalently bonded directly to chromsomal DNA wherein said microorganism and its progeny are substantially free of genetic rearrangements involving said foreign DNA.
  • These microorganisms which may be made by a process of the present invention, can contain multiple foreign DNA portions or genes stably incorporated into the chromosomal genome. It is possible to stably encode complete biochemical pathways foreign to a microorganism into the chromosome so that useful chemicals, particularly chemicals other than proteins, can be made in_ vivo by a practical method.
  • the microorganism is a cyanobacterium.
  • the present invention also encompasses pure cultures of the above microorganisms which do not have to be subjected to constant selective pressure in contrast to microorganisms containing foreign genes inserted into plasmids.
  • Another aspect of the present invention provides a method of producing a microorganism having at least one stable foreign DNA portion in its chromosome, said method comprising: (a) providing a DNA insertion vehicle containing first and second DNA portions containing DNA homologous to adjacent portions of a chromosome in said microorganism, said homologous DNA in said first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA portions in said microorganism; and a third DNA portion containing DNA foreign to said microorganism, said third DNA portion located between and covalently bonded to said first and second DNA portions; and (b) introducing said DNA insertion vehicle inside the cell membrane of said microorganism to effect incorporation of the genetic material of said foreign DNA into the chromosomal genome of said microorganisms.
  • the present invention contemplates a circular DNA insertion vehicle which facilitates the stable insertion of foreign DNA into the chromosome of a procaryotic microorganism, said circular DNA insertion vehicle comprising: (a) a first DNA segment comprising first and second DNA portions containing DNA homologous to adjacent portions of a chromosome in a microorganism, said first and second DNA portions oriented in relation to each other in the same manner as said homologous chromosomal DNA portions in said microorganism; and a third DNA portion containing DNA foreign to said microorganism that expresses a selectable phenotype located between and covalently bonded to said first and second DNA portions, and a single restriction site in said first DNA segment for a particular restriction enzyme at a location nonessential to said expressable phenotype between said first and second DNA portions; and (b) a second DNA segment containing a DNA portion that is not homologous to the chromosomal DNA in said microorganism.
  • Figure 1 is a schematic representation of the preferred circular chimeric DNA insertion vehicle of the present invention in its simplest form.
  • Figure 2 is a schematic representation of the circular chimeric DNA molecule from which the vehicle of Figure 1 is made.
  • the present invention is directed to procaryotic microorganisms which have at least one foreign DNA portion stably inserted in the chromosomes.
  • Genetically engineered microorganisms of the prior art generally contain foreign genes either in plasmids or unstably integrated, as in yeast, into the chromosomes. Plasmids can be lost upon reproduction in some daughter cells and unstable foreign genes integrated into' chromosomes are subject to deletion.
  • the foreign DNA in the microorganisms of the present invention is ligated (i.e., covalently bonded) directly to the chromosomal DNA of the microorganism, not to plasmid DNA or other DNA such as viral DNA or transposable elements.
  • the foreign DNA in the present invention is contained within nonessential DNA in the chromosome.
  • a DNA segment or portion is a linear length of DNA.
  • a DNA segment as used herein is a longer length of DNA than a DNA portion.
  • Foreign DNA includes synthetic DNA that does not exist in nature, chromosomal or plasmid DNA that is derived from a genus other than the recipient organism's genus, or a viral DNA length that does not have the ability to naturally infect or transfect the recipient organism. Excluded from foreign DNA, however, is a DNA length that is a transposable element (e.g., transposon or insertion sequence) in the recipient organism.
  • a transposable element e.g., transposon or insertion sequence
  • a transposable element is a length of DNA that interacts with a second DNA molecule (e.g., chromosome of the recipient) by site-specific recombination to produce new linkage relationships in the second DNA molecule, including inversion or deletion of DNA in the second DNA molecule, or addition of the transposable element to the second DNA molecule.
  • a second DNA molecule e.g., chromosome of the recipient
  • a transposable element is a length of DNA that interacts with a second DNA molecule (e.g., chromosome of the recipient) by site-specific recombination to produce new linkage relationships in the second DNA molecule, including inversion or deletion of DNA in the second DNA molecule, or addition of the transposable element to the second DNA molecule.
  • DNA "derived from a genus other than the recipient organism's genus” is DNA found in nature only in organisms of other genera. Even if the particular copy of DNA employed in the present invention was not immediately derived from another genus, the DNA is still "derived from” another genus if it is found in nature only in a genus or genera other than the genus of the recipient organism.
  • Viral DNA that can infect or transfect a recipient is excluded from the term foreign DNA because such viruses can reversibly integrate into the chromosome of the recipient and/or kill the recipient.
  • Transposable elements are excluded from the term foreign DNA because they can cause genetic rearrangement such as inserting replicas at other sites in the recipient, excising themselves from the chromosome causing inversion or deletion mutations.
  • Transposable elements and viral DNA that can infect or transfect the recipients are unstable and, therefore, unsuited to the construction of a microorganism that contains at least one foreign DNA portion in the chromosome. This is particularly true when multiple foreign DNA portions are inserted.
  • the stably inserted foreign DNA of the present invention is actually incorporated into the chromosomal genome of the microorganism.
  • Stably inserted foreign DNA refers to foreign DNA that is not involved in genetic rearrangement in the recipient microorganism or its progeny.
  • the microorganisms of the present invention and their progeny are substantially free of genetic rearrangement involving the foreign DNA.
  • genetic rearrangement involving the foreign DNA is meant recombination between homologous portions of the recipient's chromosome leading to loss of foreign DNA, insertion of replicas of the foreign DNA in the genome of the recipient, or excision of the foreign DNA.
  • Foreign DNA that has been stably inserted in microorganisms of the present invention undergoes a functional change only as a result of mutagenic processes provoked by the repair or replication of DNA.
  • the rate of such mutagenic processes can be defined by the rate at which auxotrophic mutations accumulate in a population of recipient organisms that has been transformed by the foreign DNA.
  • auxotrophic mutations and methods for determining mutation rates see 3. Miller, Experiments in Molecular Genetics 121-82 (Cold Spring Harbor Laboratory 1972).
  • a convenient test for the frequency of an auxotrophic mutation in a recipient is the frequency of chlorate-resistant mutations which can be measured as described in C. MacGregor et al. (1971) 3. Bacteriol. 108: 564-570.
  • the function of foreign DNA stably contained in the chromosome of a microorganism of the present invention is lost from the microorganism at a frequency that is not substantially greater than the highest frequency of a auxotrophic mutation in the organism.
  • the frequency of mutagenesis is substantially lower than the frequency of recombination between homologous chromosomal DNA.
  • the microorganisms of the present invention are procaryotes (i.e., organisms within the kingdom Monera).
  • Procaryotic oganisms lack a nuclear envelope around their chromosome, such as is found in eucaryotic organisms.
  • Procaryotic organisms are generally divided into the phyla schizophyta (all bacteria) and cyanophyta (blue-green algae or cyanobacteria).
  • Typical examples of bacterial genera to which the present invention is applicable include, inter alia, Bacillus, Pseudomonas, Escherichia, Azotobacter, Rhizobium, Rhodopseudomonas, Streptococcus, Haemophilus and Klebsiella.
  • cyanobacteria examples include, inter alia, the genera Aphanocapsa, Anabaena, Nostoc, Oscillatoria, Synechococcus, Gloeocapsa, Agmenellum, Scytonema, Mastigocladus, Arthrosprira and Haplosiphon.
  • the preferred microorganisms of the present invention are cyanobacteria, which are gram-negative procaryotes.
  • Cyanobacteria are photosynthetic unicellular organisms which are either free-living or in symbiotic association with bacteria, fungi, plants, or animals. They are found in lakes, rivers, oceans, mineral hot springs, soil (tropical to arctic) and on rocks and buildings. Some are filamentous organisms and several species can fix nitrogen.
  • Cyanobacteria have the ability to synthesize chemicals from air, water and inorganic salts utilizing the energy of sunlight. In a particularly preferred embodiment of the present invention, therefore, photosynthetic cyanobacteria are contemplated whose chromosomal structure has been altered. Cyanobacteria can produce metabolites, such as carbohydrates, proteins, lipids and nucleic acids, from CO 2 (from the air), water, inorganic salts and light. With a nitrogen fixing cyanobacteria, nitrogen containing salts need not be added because the cyanobacteria can fix N 2 from the air. By introducing into a recipient cyanobacteria foreign DNA portions that encode new biochemical pathways, important and useful products can be produced photosynthetically.
  • the precursor of the foreign biochemical pathway is a metabolite of cyanobacteria
  • the product of the pathway can be produced solely from air, water, inorganic salts and sunlight. Additional precursor may be added, however, to increase the product yield.
  • precursor can be added to cultures of the cyanobacteria. In this case, it is preferred that the precursor be a readily available and inexpensive material such as a waste byproduct of other processes (e.g. lignosulfonates or casein).
  • a DNA insertion vehicle within the cell preferably a circular vehicle comprised of a chimeric plasmid
  • a consideration in selecting a recipient organism is the ease with which it takes up exogenous DNA.
  • Particularly preferred organisms in the present invention are those cyanobacteria which have been shown to have a naturally occurring system for the uptake exogenous DNA such as Gloecapsa alpicola, Agmenellum quadruplicatum and Anacystis nidulans. If a bacterium is employed, bacteria which spontaneously take up exogenous DNA, such as Bacillus subtilis, are preferred.
  • the insertion vehicle employed in present invention can also be introduced into procaryotes, such as E. coli, that take up exogenous DNA only through artificial manipulation. Methods for introducing exogenous DNA into other procaryotes are known in the art.
  • Placement of the foreign DNA into the insertional DNA of the unloaded insertion vehicle may be accomplished using recombinant DNA technology. While the insertion vehicle can be either linear or circular, it is technically difficult to load a linear insertion vehicle with foreign DNA. Loading linear vehicles j ⁇ n vivo is not feasible because linear DNA cannot be maintained due to exonuclease activity in cells. Loading linear Insertion vehicles jn_ vitro can be accomplished but is inefficient because cleavage of the unloaded linear vehicle prior to insertion of the foreign DNA can result in shuffling the orientation of the insertional DNA fragments upon rejoining. From the standpoint of efficiency, these problems are obviated if circular vehicles, such as depicted in Figures 1 and 2, are employed.
  • the transformation vehicle desirably contains one or more unique restriction cleavage sites, or "loading sites,” located in or near the central portion of the insertional DNA (i.e., sites in the insertional DNA for an endonuclease that cleaves at no other sites).
  • a "loaded" circular DNA insertion vehicle employed in the production of microorganisms of the present invention in its simplest form is comprised of three components as is shown in Figure 1.
  • the loaded insertion vehicle is made from an unloaded insertion vehicle or plasmid as depicted in Figure 2.
  • the structure of the unloaded vehicle is substantially the same as that of the loaded vehicle described in Figure 1, excepting that there is no foreign DNA portion B and there is a restriction site 1 at the junction of insertional DNA portions A and A'.
  • Portions A and A' are termed insertional DNA.
  • the insertional DNA is homologous to a stable region of DNA in a chromosome of the recipient organism.
  • Portions A and A' are homologous to adjacent chromosomal DNA regions in the recipient and are ideally derived from a single fragment of chromosomal DNA from the recipient organism that has been cleaved at a centrally located restriction site (the loading site). "Adjacent" regions in the chromosomal DNA of the recipient
  • f OMP means that either (1) chromosomal DNA regions that are immediately adjacent to each other, or (2) chromosomal DNA regions that are separated only by nonessential DNA.
  • Portions A and A' are, relative to each other, in the same orientation in the insertion vehicle as are the homologues of A and A' in the chromosome of the recipient organism.
  • the ends of the homologous regions in the chromosome closest to each other correspond to the end portions of A and A 1 in contact with DNA portion B in Figure I (or restriction site 1 in Figure
  • the insertional DNA should have as long a length as practical on either side of the loading site. For example, it was found that when foreign DNA portions were loaded randomly into insertional DNA segments having an average length between 80,000 and 120,000 base pairs in a linear insertion vehicle so that the great majority of foreign DNA was located thousands of base pairs from the ends of the insertion vehicles. There was a 400-fold increase in transformation efficiency (based on transformations per foreign DNA segment) with these linear insertion vehicles over an insertional DNA segment 4,100 base pairs long with a single loading site 500 base pairs from one end in a circular insertion vehicle. Insertional DNA should generally have at least 100 base pairs on either side of the loading site to obtain reasonable transformation efficiency. The length of the insertional DNA has not been found to affect the stability of the inserted foreign DNA.
  • Foreign DNA portion B also referred to herein as interrupting DNA
  • interrupting DNA is ligated between DNA portions A and A'.
  • Useful foreign DNA which can be inserted into the chromosome of the recipient organism include genes which encode the production of proteins (e.g., an enzyme in a metabolic pathway) or regions that regulate gene expression (e.g., promoters and operators). Transformation efficiency has been found to drop as the length of the interrupting (foreign) DNA increases. For example, when the interrupting DNA is 1,300 base pairs long, there is a 7-fold increase in transformation efficiency over foreign DNA 4,000 base pairs long, all other conditions being equal. Although it is possible to insert quite long foreign DNA portions (e.g., up to at least 20,000 base pairs), it is desirable to use the shortest DNA length that will encode the desired information.
  • DNA segment C is ligated to the opposite ends of DNA portions A and A 1 . Flanking DNA is so-called because it flanks and does not interrupt the insertional DNA. Flanking DNA is derived ideally from a plasmid compatable with and having the ability to replicate in a microorganism other than the recipient organism.
  • the flanking DNA segment is required only in circular insertion vehicles. Since a linear vehicle only need be comprised of homologous DNA portions A and A' and interrupting DNA portion B.
  • the flanking DNA is derived from a plasmid which is compatable with and replicates in a second microorganism
  • the flanking DNA is preferably derived from an entire plasmid which has been cleaved at a single restriction site.
  • a functioning repiicon in the flanking portion allows the transformation vehicle to be cloned in a microorganism other than the recipient organism in large quantities.
  • No repiicon in the insertion vehicle should be functional in the recipient organism since it is the function of the insertion vehicle to transform the chromosome and not to replicate automonously in the recipient organism.
  • the loaded transformation vehicle is introduced inside the cell membrane of the recipient. If the recipient organism naturally takes up exogenous DNA, the loaded transformation vehicle need only be introduced into a culture of the recipient organism. If the recipient organism does not naturally take up exogenous DNA, the loaded plasmid can be introduced into the cell by conventional methods known in the art (e.g., conjugation or treatment with CaCl 2 ). See M. Suzuki & A. Szalay, (1979) Meth. Enzy ol. 68: 331-341.
  • the stably inserted transformant will usually have to be distinguished from two other types of unstable transfor ants.
  • the flanking DNA and the insertional DNA are added (i.e., an additive recombinant event) to the chromosome. If the flanking DNA expresses a selectable phenotype, this type of transformant is readily distinguished.
  • the entire vehicle interrupting, insertional and flanking DNA
  • both the interrupting (foreign) DNA and flanking DNA express selectable phenotypes, this type of transformant is also readily isolated.
  • Pure cultures of the recipient organism with stably inserted foreign DNA can be obtained by selecting those organisms which have been stably transformed. Selection methods, well known in the art, include testing colonies grown from individual cells for product or other expressable phenotype, and colony hybridization. See M. Grunstein et al., (1975) Proc. Natl. Acad. Sci USA 72: 3962-3965.
  • pure culture is meant a culture of a species of microorganisms containing foreign DNA stably inserted into the chromosome that is substantially free of the same microorganism without foreign DNA in the chromosome.
  • Applicants have demonstrated that when a transformation vehicle according to the present invention is employed, foreign DNA can be stably inserted into the chromosomes. Once inside the cell membrane, the insertional DNA portions undergo recombination with the chromosome of the recipient organism at the site of homology between the insertional DNA and the chromosomal DNA. Since the foreign DNA is ligated between the adjacent portions of insertional DNA, the foreign DNA is carried into the chromosomal DNA of the recipient organism. Foreign DNA so inserted has been found to be a stable component of the chromosomal genome.
  • OMPI eucaryotic organisms by introducing viable loaded insertion vehicles of the present invention inside the nuclear membrane of eucaryotes.
  • the recombination event involves recombination between two molecules of DNA, identical in a portion of the molecules except for the presence in one of the molecules of a heterologous insertion. Recombination results in the transfer of the latter from the first DNA molecule to the second.
  • biosynthetic pathways By stably inserting several foreign genes into the chromosomes of a microorganism, multistep biosynthetic pathways can be introduced which synthesize nonproteins. After identifying and isolating all the genes necessary in a desired biochemical pathway, the method of the present invention can be straightforwardly applied to produce a microorganism that will produce the desired product.
  • Biological systems exist which produce chemicals of industrial importance, such as butanol, acetone, polysacchar ⁇ des, carotenoids, hydrocarbons and molecular hydrogen. Genes for other biological systems can be isolated or synthesized. An n_ vivo method of producing such chemicals, particularly photosynthetically, is obviously of great value.
  • butanol-acetone fermentation which produces butanol and acetone from glucose
  • Clostridium acetobutylicum See G. Gottschalk, Bacterial Metabolism 182 (Springer Verlag 1979). Stable introduction of those genes into a cyanobacterium provides a blue-green algae with a capability of producing butanol and acetone. All microorganisms produce glycerol. See, e.g. ? A. Newman, Glycerol (CRC Press 1968); L. Stryer, Biochemistry 292 (Freeman 1975).
  • Glycerol production can be enhanced in a selected microorganism by introducing foreign DNA that affects the expression of the genes responsible for glycerol production.
  • Production of hydrogen from water by a cyanobacteria is possible by mutating ferredoxin-NADP oxidoreductase, so that electrons produced by photosynthesis accumulate on ferrodxin, and by inserting a foreign gene encoding a hydrogenase into the mutated cyanobacteria. See, e.g., 3.R. Benemann et al., (1982) Proc. Nati. Acad. Sci. USA JQi 2317-2320.
  • a selectable gene can be spliced into the insertional DNA such that a unique loading site is located either within a nonessential region of the selectable marker, or at the molecular juncture between the selectable marker and one of the insertional DNA portions.
  • the unloaded vehicle has the same configuration as in Figure 2, except that an additional foreign gene that expresses a selectable phenotype is located between restriction site 1 and either DNA portion A or A'.
  • an additional foreign gene that expresses a selectable phenotype is located between restriction site 1 and either DNA portion A or A'.
  • insertion vehicles can be constru ⁇ ted by the following, relatively simple procedure.
  • insertional DNA is isolated from the chromosome of the recipient organism by cleavage with a restriction enzyme.
  • DNA fragments of the desired length preferably 5000 to about 10,000 base pairs long, are isolated by electrophoresis.
  • the desired fragments are cloned into a restriction site of a plasmid (which becomes the flanking DNA) from another microorganism (host organism).
  • the plasmid from the host organism desirably expresses a selectable phenotype in the host and the recipient.
  • the recombinant plasmids are used to transform the host organism and transformants are isolated according to phenotypes encoded on the plasmid. Milligram quantities of the mixture of recombinant plasmids are isolated from the host organism.
  • Acceptable insertion vehicles are those having centrally-located loading sites that are not within insertional DNA that is homologous to an essential gene in the recipient organism.
  • One method of isolating acceptable insertion vehicles is to construct restriction cleavage maps of a number of the recombinant plasmids. This approach is unnecessarily laborious.
  • OMPI A simpler approach is to allow the recipient organism itself to isolate useful insertion vehicles.
  • the mixture of recombinant plasmids is cleaved with a restriction enzyme that has sites only within the insertional DNA.
  • the enzyme should not have restriction sites within the flanking DNA or at the junctions between the insertional DNA and the flanking DNA.
  • interrupting (foreign) DNA is ligated into the cleaved plasmids.
  • the interrupting DNA desirably contains a gene which expresses a selectable phenotype distinguishable from the selection marker in the flanking DNA.
  • the plasmids containing interrupting DNA are then used to transform a culture of recipient microorganisms. Isolated transformants which exhibit the selectable phenotype of only the interrupting DNA have obviously been transformed by a chimeric DNA molecule that is active as an insertion vehicle. The insertion vehicle itself, of course, is destroyed in the process of integration of the interrupting DNA into the recipient chromosome.
  • chromosomal DNA is isolated from several of the transformants. Cleaving the recovered chromosomal DNA from the transformants with the restriction enzyme originally used to isolate* chromosomal DNA from the recipient organism (to be employed as insertional DNA) and then ligating the cleaved chromosomal DNA to cleaved plasmid DNA (flanking DNA) that contains a selection marker will yield a mixture that contains acceptable loaded transformation vehicles admixed with ligation products.
  • acceptable insertion vehicles which have a loading site in a nonessential region and which have been demonstrated to be effective can be isolated.
  • the loaded insertion vehicles can be unloaded by cleavage of the junctions between the insertional DNA and the interrupting DNA with the restriction enzyme described above which has restriction sites only within the insertional DNA.
  • DNA molecules with the blunted ends are then ligated in a solution which has a very low concentration of DNA and a very high concentration of T4 DNA ligase to join linear molecules into a circular form. This mixture is then used to transform a host microorganism and several transformants are chosen for further examination.
  • Transformants containing acceptable insertion vehicles are identified by isolating plasmid DNA, digesting it with restriction enzymes, and subjecting the linear digestion products to electrophoresis to identify vehicles that have been cleaved at a single site.
  • A. nidulans strain (A. nidulans R-2 isolated by S. V. Shestakov et al., (1970) Molec. Gen. Genet. 107: 372-375) containing a gene within the chromosome capable of complimenting the thi-1 mutation in E. coli was lysed and DNA was isolated by dye-bouyant density centrifugation. Plasmids pBR322 and pACYC184 were isolated by dye-bouyant density centrifugation of lysozyme-sarkosyl lysates of appropriate E. coli strains.
  • Plasmid pBR322 is a self-replicating E. coli plasmid which contains a selection marker which encodes resistance to ampicillin and is employed as the flanking DNA.
  • the replicons in pBR322 and pACYC184 are not functional in A. nidulans. Plasmid pACYC184, which encodes resistance to chloramphenicol, is employed as the foreign or interrupting DNA.
  • a mixture of broken plasmids and chromosomal DNA from A. nidulans, at a concentration of 50 micrograms of DNA per milliliter was digested with 5au3A resrtriction endonuclease under conditions specified by Bethesda Research Laboratories, Inc. (BRL).
  • the enzyme concentration, 6 units per milliliter, was chosen to provide incomplete digestion after a 30 minute incubation at 37°C.
  • To stop the reaction .080 ml of 0.25 M EDTA was added to the 2 ml reaction mixture.
  • the partially digested DNA was fractionated by electrophoresis through a 0.7% agarose gel and DNA fragments 5000 base pairs and larger were recovered from the gel by electroelution as described by Yang et al, (1979) Meth. Enzymoi. 68: 176-182.
  • the purified A. nidulans DNA fragments were ligated to pBR322 DNA cleaved beforehand with Ba Hl and treated with bacterial alkaline phosphatase.
  • the 0.220 ml ligation mixture contained pBR322 DNA at a concentration of 15 ug ml and A. nidulans DNA at 8.6 ug/ml.
  • the ligation mixture was incubated at 14 ⁇ C for 12 hours under ligation conditions specified by Boehringer-Mannheim. The reaction was terminated by addition of 0.011 ml of .25 M EDTA.
  • the ligated DNA was used to transform E. coli HB101 according to the method of Bolivar and Backman, (1979) Meth.
  • a mixed culture of the transformants is designated as a "gene library" of A. nidulans DNA in E. coli.
  • E. coli HB101 which contains the gene library plasmids, has a thi- 1 mutation that blocks the biosynthesis of thiamine.
  • a chimeric plasmid designated pKW1006 thT 1" was identified.
  • Plasmid pKW1006 thi contained only one cleavage site for BamHl located at one of the junctions between flanking DNA pBR322 and the cloned insertional DNA fragment from A. nidulans. To eliminate this cleavage site, the plasmid was digested exhaustively with BamHl and was used to transform E. coli HB101 to ampicillin resistance. From one of the transformants, a new plasmid, pKW1034 thi , was recovered which was identical to pKW1006 thi* except for a deletion of about 700 base pairs bracketing the BamHl cleavage site.
  • BRL Haelll
  • the reaction was terminated by the addition of 0.300 ml of 0.25 M EDTA.
  • the reaction mixture was extracted once with phenol, the volume was reduced to about 0.5 ml by extraction with n-butanol and the DNA was dialyzed against TE (10 mM Tris-HCl pH 7.5, 0 ⁇ mM EDTA). Only 8% of the plasmids were cleaved by Haelll as determined by agarose gel electrophoresis. The partial digest, therefore, presumably consisted of a population of full length linear molecules having termini at various Haelll cleavage sites. BamHl linkers (CGGATCCG; BioLogicals) were added to the Haelll partial digest.
  • the 0.36 ml reaction mixture contained 9 nM pKW1034 thi* DNA, 220 nM BamHl linker, 100 units of T4 DNA ligase per milliliter (Boehringer- Mannheim) and other ingredients as specified by Boehringer-Mannheim for ligation. Incubation was carried out at 15°C for 15 hours. The reaction was terminated by the addition of .040 ml of .25 M EDTA, extracted once with phenol and the DNA was dialyzed against TE (described above). Eight micrograms of the dialyzed DNA was digested with BamHl (250 units per milliliter) in a volume of .200 ml under conditions specified by Boehringer-Mannheim. The reaction mixture was incubated at 37°C for 4.5 hours, followed by 10 minutes at 65°C. Then .020 ml of .25 M EDTA was added and the DNA was dialyzed against TE as described above.
  • pACYC184 was ligated.
  • the pACYC184 DNA (foreign or interrupting
  • OMPI _ DNA had been cleaved beforehand with BamHl and treated with bacterial alkaline phosphatase.
  • a plasmid, designated pKW1039 thi " was recovered from the transformants resistant to both chloramphenlcol and ampicillin.
  • the pKW1039 thif plasmid contained pACYC184 in the interrupting position and pBR322 in the flanking position.
  • the plasmid was unable to compliment the E. coli thi-1 mutation, presumably because the complimenting function was destroyed by the insertion of pACYC184 into the insertional DNA derived from A. nidulans DNA
  • plasmid pKW1039 thT is suitable for inserting pACYC184 plasmid fragment into A. nidulans, it may be desirable at times to insert only a portion of a foreign plasmid fragment.
  • pACYC184 was digested exhaustively with Haell and the clevage products were fractioned by electrophoresis through a 1.4% agarose gel. The largest fragment, which was 1,270 base pairs long and contained the chloramphenicol resistance gene, was purified according to the method of M. Albring et al., (1982) Anal. Biochem (in press) which is described below.
  • a Haell digest of pACYC184 DNA (150 ug of DNA) was fractionated by electrophoresis at 2 volts per cm for 15 hours through a 64 percent agarose gel (Low Melting Point agarose; BRL). The gel was stained with ethidium bromide, the gel portion (5 ml) containing the slowest- migrating band of DNA 0.3 kb) was excised, and the agarose containing the DNA was dissolved by stirring for 15 min in 20 mi of 50% urea (w/w) plus 5 g of urea crystals. All operations were performed at room temperature in siliconized glassware and centrifugations were at 2000 x g for 5 min.
  • the DNA was extracted from the agarose by adding l € ml of DHA solution (40 ml. of n-butanol; 0.8 mi of glacial acetic acid; 4.6 ml of 2,2'-diethyldihexylamine from Eastman Kodak). The mixture was stirred vigorously for 5 min. The emulsion was centrifuged, the butanol phase (top) containing the DNA was recovered and saved, and the aqueous phase was extracted again
  • OMPI _ with 11 ml of DHA solution.
  • the second butanol phase was pooled with the first to give a volume of ca 30 ml.
  • the pool butanol phases were extracted with 6 ml of 1.25 M ammonium acetate for 5 min, the emulsion was centrifuged, the bottom aqueous phase was saved, and the upper phase was extracted again with 6 ml of 1.25 M ammonium acetate.
  • the aqueous phases were pooled and were concentrated to about 0.4 mi by repeated extraction with n-butanol.
  • the 0.4 ml sample was dialyzed against TE (described above).
  • reaction mixture (.454 ml) containing 20 mM Tris- HC1, 10 mM MgCl,, 1 mM 2-mercaptoethanol, 10 micromolar each of dATP, dGTP, dCTP, dTTP, and 18 units of E. coli DNA polymerase I large fragment (New England Bio. Labs) at a pH of 7.5 and a temperature of 37°C for 4 hours, followed by 1 hour at 15°C.
  • a reaction mixture (.454 ml) containing 20 mM Tris- HC1, 10 mM MgCl,, 1 mM 2-mercaptoethanol, 10 micromolar each of dATP, dGTP, dCTP, dTTP, and 18 units of E. coli DNA polymerase I large fragment (New England Bio. Labs) at a pH of 7.5 and a temperature of 37°C for 4 hours, followed by 1 hour at 15°C.
  • reaction volume was increased to .478 ml by the addition of .013 ml of 20 mM ATP, .0045 ml 1 M dithiothreithol, .004 ml of 14 micromolar BamHl linkers, and 45 units of T4 DNA ligase.
  • This mixture was incubated at 15°C for 4 hours and terminated by the addition of .027 ml of .25 M EDTA and heating to 65°C and holding that temperature for 10 minutes.
  • the reaction mixture containing DNA (.505 ml) was mixed with .037 ml of water, .0056 ml of 1 M MgCI 2 , .012 ml of 1 M Tris-HCl (pH 7.0), .012 ml of 5 M NaCl, .0021 ml of 14.3 M 2-mercaptoethanol, .006 ml of bovine serum albumin (20 g per ml held at 75°C for 30 minutes) and 120 units of BamHl.
  • This mixture was incubated at 37°C for 14 hours and terminated by the addition of .040 ml of .25 M EDTA followed by holding the mixture at 65°C for 10 minutes and dialyzing it against TE (described above).
  • the reaction products are DNA fragments encoding resistance to chloramphenlcol and having single-stranded termini complementary to cleavage sites recognized by BamHl.
  • Plasmid pKW1039 thi " at a concentration of 30 ug/ml was digested exhaustively with BamHl, diluted ten-fold and ligated with T4 DNA ligase to remove pACYC184 from the interrupting position. This ligated DNA was used to transform E. coli HB101 and one transformant resistant to ampicillin and sensitive to chloramphenlcol was recovered. Plasmid DNA isolated from this transformant was designated pKWI048 thT.
  • the plasmid pKW1048 thi " contains a single BamHl cleavage site at the interrupting position defined in the parental plasmid pKW1039 thi " .
  • Plasmid pKW1048 thi " was cleaved with BamHl, extracted with phenol and dialyzed against TE (described above).
  • the cleaved pKW1048 thi " plasmid (3.6 ug/ml) was ligated to the 1,270 base pair fragment of pACYC184 0.6 ug/ml) in a volume of .200 ml.
  • the ligated DNA was used to transform E. coli HB101 and from among the transformants, a new plasmid designated pKW1065 thi " was recovered that specified resistance to both ampicillin and chloramphenicol.
  • Plasmid pKW1065 thi contained pBR322 in the flanking position and a 1,270 base pair fragment encoding chloramphenicol resistance in the interrupting position. The new plasmid was unable to complement the E. coli thi-1 mutation.
  • a more simplified method of constructing the above loaded insertion vehicle for A. nidulans is as follows.
  • A. nidulans chromosomal DNA can be cleaved with Bgill and fragments between 5,000 and 10,000 nucleotides long isolated by electrophoresis. These fragments can then be cloned into the BamHl site of the plasmid pBR322.
  • a partial homology between the cleavage sites of Bglll and BamHl makes it possible to ligate together DNA fragments produced by these endonucleases.
  • the hybrid junctions are not cleaved by either of the endonucleases.
  • the recombinant plasmid can then be used to transform a strain of E. coli to ampicillin resistance.
  • Milligram quantities of the plasmid DNA can then be isolated from a mixed culture of the transformed cells.
  • the mixed plasmids are screened for those having loading sites located in the insertional DNA using the recipient organism (A. nidulans) to screen.
  • the mixture of recombinant plasmids would be cleaved with BamHl. This enzyme will only cleave within the insertional DNA.
  • Plasmid pBR322 in the recombinant plasmid has no BamHl restriction sites; the junctions between the insertional and flanking DNA are not cleavable by BamHl.
  • the mixture of cleaved DNA can be ligated to the purified 1,270 nucleotide base pair fragment that encodes chloramphenicol resistance.
  • This fragment can be conveniently isolated by electrophoresis of a BamHl digest of the plasmid pKW1065 employed in Example 1.
  • the mixture of ligated DNA will contain loaded insertion vehicles.
  • the entire mixture can then be used to transform a strain of E. coli.
  • Transformants resistant to both ampicillin and chloramphenicol will contain various recombinant plasmids in which the 1,270 base pair fragment is linked to insertional DNA.
  • a preparative quantity of the mixture of recombinant plasmids can then be isolated from the resistant E. coli cultures.
  • This entire mixture of plasmids can then be used to transform a culture of A. nidulans.
  • Type I transformants those resistant to chloramphenicol but sensitive to ampicillin, isolated.
  • Chromosomal DNA can be recovered from several different transformants and cleaved with BgHI. The cleaved DNA can then be ligated to pBR322 cleaved with BamHl. This ligated DNA can then be used to transform a culture of E. coli. Transformants resistant to both chloramphenicol and ampicillin will contain useful insertion vehicles.
  • This example describes the transformation of A. nidulans with plasmid pKW1065 thi " produced by the method of Example 1.
  • the cell pellet was suspended in 20 ml of fresh BG-11 medium at a concentration of I x 10 cells per milliliter.
  • OMPI OMPI To 1.2 ml of competent cells from the fresh suspension in a 10 x 13 mm clear glass test tube was added ⁇ 2 ug of pKW1065 thi " (from Example 1) in .006 ml of TE (as described in Example 1). The transformation mixture was incubated at 37°C for 10 hours under the lighting conditions described above. The test tube was agitated intermitentiy to prevent settling of the cells. Aliquots of the mixture were spread onto the surface of membrane filters (Nuclepore Membra- Fil filters, .45 um pore size, cut to 8 cm diameter) resting on solid BG-11 medium in plastic petri plates.
  • membrane filters Nuclepore Membra- Fil filters, .45 um pore size, cut to 8 cm diameter
  • the solid medium was prepared by mixing equal volume of autoclaved BG-li liquid (2x concentrated) and autoclaved Difco Bacto Agar (3% in water). Membrane filters were first sterilized by autoclaving in water. The cells on the filters were incubated under the growth conditions described above for 20 hours and then the filters were transferred to solid medium containing either chloramphenicol (5 ug/ml) or ampicillin (0.2 ug ml), or both. The plates were then incubated for 10 days and the numbers of each type of transformant were tabulated. It was found that the cells had a plating efficiency of about 40%.
  • Type I transformants were the most common type and were resistant to chloramphenicol only. Of the total cells in the transformation mixture, one cell in one thousand was a type I transformant. Out of each 266 transformants, however, 250 transformants were of type I, 15 were of type II and 1 was of type III. As determined by Southern hybridization analysis, E. Southern, (1975) 3. Mol. Biol. 98: 503-517, type I transformants contain a stably inserted single copy of the interrupting (foreign) DNA segment which is integrated in the recipient chromosome at a site homologous to the position of interrupting DNA in pKW1065 thi " .
  • Type II transformants are those in which the flanking DNA has been inserted in the chromosome of the recipient organism.
  • Type II transformants demonstrate a resistance to ampicillin, but are sensitive to chlorampheticol.
  • Type III transformants are resistant to both chloramphenicol and ampicillin.
  • a type III transformant arises by the addition to the chromosome of at least one copy of the loaded plasmid, including the foreign DNA, the flanking DNA and the insertional DNA.
  • Plasmid pKW1065 thi " was cleaved with the restriction enzyme Hindlll.
  • the plasmid contained one Hindlll site located within one portion of the insertional DNA and another site approximately 300 base pairs into the flanking DNA from the end of the other insertional DNA portion.
  • the cleaved DNA was used to transform a culture of A. nidulans in substantially the same manner as in Example 1.
  • a 3-fold decrease in the number of stable type I transformants was observed vis ⁇ a-vis transformations with uncleaved pKW1065 thi " as in Example 1. No type II or III transformants were observed. Since modifications will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the appended claims.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

Micro-organisme procaryotique et son procédé de production, le micro-organisme contenant au moins une partie d'ADN stable dans le chromosome. Les micro-organismes ci-décrits ainsi que leurs descendants sont exempts de restructuration génétique comprenant l'ADN étranger. Dans un mode préférentiel de réalisation on utilise des cyanobactéries. Les micro-organismes sont produits en introduisant dans la cellule un véhicule d'insertion contenant de l'ADN étranger lié entre deux parties d'ADN homologues à des parties adjacentes du chromosome du récepteur.
PCT/US1983/001026 1982-07-09 1983-07-06 Manipulations genetiques dans les organismes procaryotiques Ceased WO1984000381A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP83502712A JPS59501195A (ja) 1982-07-09 1983-07-06 原核生物における遺伝子工学
DE198383902654T DE113781T1 (de) 1982-07-09 1983-07-06 Genetische transformation in prokaryotischem organismus.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US39659582A 1982-07-09 1982-07-09

Publications (1)

Publication Number Publication Date
WO1984000381A1 true WO1984000381A1 (fr) 1984-02-02

Family

ID=23567885

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1983/001026 Ceased WO1984000381A1 (fr) 1982-07-09 1983-07-06 Manipulations genetiques dans les organismes procaryotiques

Country Status (5)

Country Link
EP (1) EP0113781A4 (fr)
JP (1) JPS59501195A (fr)
CA (1) CA1214737A (fr)
DE (1) DE113781T1 (fr)
WO (1) WO1984000381A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2563533A1 (fr) * 1984-04-27 1985-10-31 Centre Nat Rech Scient Procede d'amplification de l'expression d'un gene determine chez bacillus subtilis et souches obtenues
EP0174096A3 (fr) * 1984-08-02 1987-11-04 Biotechnica International, Inc. Vecteurs pour l'introduction d'ADN dans les chromosomes d'une bactérie ou pour la suppression d'ADN de celle-ci et pour la production de protéine
EP0195078A4 (fr) * 1984-09-21 1988-01-25 Genex Corp Souches de bacillus ayant des niveaux reduits de protease extracellulaire.
US5362734A (en) * 1989-10-14 1994-11-08 American Home Products Corporation Certain benzo-quinolizine compounds and derivatives thereof
US5624829A (en) * 1984-07-03 1997-04-29 Gist-Brocades, B.V. Transformed industrial bacillus strains and methods for making and using them
US8343509B2 (en) 2008-01-11 2013-01-01 Genelux Corporation Methods and compositions for detection of bacteria and treatment of diseases and disorders
US8609385B2 (en) 2007-05-01 2013-12-17 Zuvachem, Inc. Methods for the direct conversion of carbon dioxide into a hydrocarbon using a metabolically engineered photosynthetic microorganism

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0074808A3 (fr) * 1981-09-16 1984-07-04 University Patents, Inc. Méthode recombinante et matériaux
JPS59205983A (ja) * 1983-04-28 1984-11-21 ジエネツクス・コ−ポレイシヨン 異種遺伝子を原核微生物で発現させる方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DUBNAU, the Molecular Biology of the Bacilli Vol I Academic Press pp. 147-178 1982. *
Journal of Bact. Vol. 133 pp. 1246-1253 March 1978. *
MOLEC. Gen. Genet. 177, 459-467 1980. *
PNAS USA No. 8 pp. 3664-3668, August 1978. *
See also references of EP0113781A4 *
WALTON, Recombinant DNA pp. 83-91 Elsevier Scientific Publishing Co. 1981. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6083718A (en) * 1983-07-06 2000-07-04 Gist-Brocades, N.V. Transformed industrial bacillus strains and methods for making and using them
FR2563533A1 (fr) * 1984-04-27 1985-10-31 Centre Nat Rech Scient Procede d'amplification de l'expression d'un gene determine chez bacillus subtilis et souches obtenues
EP0166628A1 (fr) * 1984-04-27 1986-01-02 Etablissement Public dit: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) Procédé d'amplification de l'expression d'un gène déterminé chez bacillus subtilis et souches obtenues
US4959316A (en) * 1984-04-27 1990-09-25 Centre National De La Recherche Scientifique (Cnrs) Process for amplifying the expression of a specific gene in bacillus subtilis, and strains obtained
US5624829A (en) * 1984-07-03 1997-04-29 Gist-Brocades, B.V. Transformed industrial bacillus strains and methods for making and using them
EP0174096A3 (fr) * 1984-08-02 1987-11-04 Biotechnica International, Inc. Vecteurs pour l'introduction d'ADN dans les chromosomes d'une bactérie ou pour la suppression d'ADN de celle-ci et pour la production de protéine
EP0195078A4 (fr) * 1984-09-21 1988-01-25 Genex Corp Souches de bacillus ayant des niveaux reduits de protease extracellulaire.
US4828994A (en) * 1984-09-21 1989-05-09 Genex Corporation Bacillus strains with reduced extracellular protease levels
US5362734A (en) * 1989-10-14 1994-11-08 American Home Products Corporation Certain benzo-quinolizine compounds and derivatives thereof
US8609385B2 (en) 2007-05-01 2013-12-17 Zuvachem, Inc. Methods for the direct conversion of carbon dioxide into a hydrocarbon using a metabolically engineered photosynthetic microorganism
US8343509B2 (en) 2008-01-11 2013-01-01 Genelux Corporation Methods and compositions for detection of bacteria and treatment of diseases and disorders
US8357486B2 (en) 2008-01-11 2013-01-22 Genelux Corporation Methods and compositions for detection of bacteria and treatment of diseases and disorders

Also Published As

Publication number Publication date
CA1214737A (fr) 1986-12-02
JPS59501195A (ja) 1984-07-12
EP0113781A4 (fr) 1986-05-14
EP0113781A1 (fr) 1984-07-25
DE113781T1 (de) 1985-06-20

Similar Documents

Publication Publication Date Title
US4778759A (en) Genetic engineering in cyanobacteria
CA1183090A (fr) Microorganismes transformes genetiquement pour la production d'enzymes amylolytiques et procede de preparation
EP2270180B1 (fr) Plasmides de vecteur à clonage d'ADN et leurs procédés d'utilisation
DE3883041T2 (de) Stabile Genamplifizierung in prokaryotischer chromosonaler DNA.
AU2005248371B2 (en) Methods for dynamic vector assembly of DNA cloning vector plasmids
US12146135B2 (en) Method for editing filamentous fungal genome through direct introduction of genome-editing protein
EP0057976B1 (fr) Procédé pour cloner le gène codant pour une alpha-amylase thermostable dans escherichia coli et bacillus subtilis
Kuhlemeier et al. Cloning of nitrate reductase genes from the cyanobacterium Anacystis nidulans
JP4801151B2 (ja) 不活性化された乳酸デヒドロゲナーゼ遺伝子を有する改変微生物
Potter et al. DNA recombination: in vivo and in vitro studies
Douchi et al. Nuclear and chloroplast genome engineering of a productive non-model alga Desmodesmus armatus: Insights into unusual and selective acquisition mechanisms for foreign DNA
EP0077196A2 (fr) Microorganismes produisant des acides aminés aromatiques
CA1214737A (fr) Utilisation du genie genetique chez les procaryotes
CN112746075B (zh) 诱导型内源CRISPR-Cas系统的构建方法及其在多基因同时编辑中的应用
CA1150169A (fr) Anneaux de plasmides comme vehicule de clonage
US4612287A (en) Plasmids containing a gene coding for a thermostable pullulanase and pullulanase-producing strains of Escherichia coli and Bacillus subtilis containing the plasmids
Wenko et al. Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase from Glycine max
CN115747243B (zh) 植物病原细菌基因组编辑工具的开发及应用
CN113166741A (zh) Dna文库的多重确定性组装
US4419450A (en) Plasmid cloning vector for Bacillus subtilis
CN110669788B (zh) 一种紫球藻叶绿体表达系统及其应用
US20040235120A1 (en) Method for producing vitamin b12
US4876202A (en) Chimeric plasmids
McCLUNG et al. A genetic locus essential for formate-dependent growth of Bradyrhizobium japonicum
EP0164117B1 (fr) Mutant asporogène amylase-négatif de Bacillus subtilis utilisable comme hôte dans un système hôte-vecteur

Legal Events

Date Code Title Description
AK Designated states

Designated state(s): CH DE GB JP NL

AL Designated countries for regional patents

Designated state(s): CH DE FR GB NL

WWE Wipo information: entry into national phase

Ref document number: 1983902654

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1983902654

Country of ref document: EP

EL Fr: translation of claims filed
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1983902654

Country of ref document: EP