KR101638690B1 - Suicide vector for producing murtant strain of multidrug-resistant strain - Google Patents

Abstract

The present invention relates to a suicide vector which is not replicated in a multidrug-resistant strain but can be used for producing a mutant strain of a multidrug-resistant strain, a kit for producing a mutant strain of a multidrug-resistant strain containing the suicide vector, A method for producing a mutant strain in which a gene is deleted, and a mutant strain produced by the above method and having deletion of a target gene of a multidrug-resistant strain. Using the kit for producing a mutant strain of a suicide vector or a multidrug resistant strain for producing a mutant strain of a multidrug-resistant strain provided by the present invention, even when only a limited antibiotic resistance gene that can be used for studying various multidrug resistant strains can be used, Since a mutant strain in which one or more target genes or target polynucleotides are deleted from the genomic DNA can be produced, it can be widely used in various fields such as genome and physiological activity of various multidrug-resistant strains.

Description

A suicide vector for producing a mutant strain of a multidrug-

The present invention relates to a suicide vector for producing a mutant strain of a multidrug-resistant strain. More particularly, the present invention relates to a suicide vector which is not replicated in a multidrug resistant strain but can be used for producing a mutant strain of a multidrug resistant strain, Resistant mutant strain, a method for producing a mutant strain in which a target gene of a multidrug-resistant strain has been deleted using the suicide vector, and a mutant strain produced by the method and a target gene of the multidrug-resistant strain have been deleted.

Antibiotics have been frequently used in the livestock and fishery industries for the prevention or growth of diseases, or for treatment purposes. However, the overuse of antibiotics not only increases the incidence of antibiotic-resistant bacteria resistant to antibiotics, but also increases the risk that the antibiotic-resistant bacteria can spread to humans through fish food, Researches are being actively carried out.

On the other hand, in addition to the strains induced by the above-mentioned antibiotics, resistant strains resistant to various antibiotics naturally exist in nature. For example, strains of Acinetobacter sp., Which are known to belong to the multidrug-resistant Gram-negative strains, are widely present in natural environments such as soil and water. In particular, in strains of the immune system, It is known as a typical hospital infection causing complications such as dermatitis, pneumonia, meningitis, sepsis, endocarditis, intraperitoneal infection, surgical site infection and urinary tract infection. Twenty-seven strains identified as strains of the genus Ashinoto bacterium that have been studied to date are known, and about 11 strains are known to be distinguished by the hybridization method. Of these, A. calcoaceticus (genospecies type 1), A. baumannii (genospecies type 2), A. pittii (genospecies type 3) and Ashineto A. nosocomialis (genospecies 13 TU) is closely related genetically, so it is difficult to distinguish the above strains as a general method of classification based on phenotypes. Sometimes, these strains are collected and the ACB complex strain ( A. calcoaceticus - A. baumannii complex, ACB complex).

Studies have been actively undertaken to develop a method for distinguishing more difficult-to-distinguish multidrug resistant strains from simpler strains. For example, in Korean Patent Laid-Open Publication No. 2014-19219, a primer capable of amplifying a region showing a genetic difference of a strain of the genus Ashithobacter, a kind of multidrug-resistant strain, or a probe capable of detecting the region, A method for identifying a strain belonging to the genus Ashentubara is disclosed. In addition, since it is known that the above-mentioned multidrug-resistant strains can be secondarily infected in hospitals, studies are actively conducted to develop a method for rapidly treating various symptoms caused by such infections. For example, WO 2010/064261 discloses a method for treating an infectious disease of Acinetobacter baumannii, which is a kind of multidrug-resistant strain, using a combination preparation of carbapenum and aztreonam; Korean Patent Registration No. 820960 A method of treating a strain of multidrug-resistant Staphylococcus aureus, Streptomyces cinnamonensis, which exhibits antibacterial activity, is disclosed.

Nevertheless, studies on multidrug-resistant strains which are resistant to the antibiotics or exhibit resistance to various naturally occurring antibiotics have not been sufficiently conducted, and thus, the infection of the multidrug- The problem that the treatment success rate is low is not solved. In order to solve such a problem, studies to characterize the functions and genes of the genes expressed in the multidrug-resistant strains should be preceded. However, because of their resistance to antibiotics, the genetic and physiological Research is not actively progressing. Therefore, there is an urgent need to develop a methodology for easily producing a mutant strain of a multidrug-resistant strain and studying the genome of the resistant strain more specifically.

Under these circumstances, the present inventors have made extensive efforts to develop a method for easily producing a multidrug-resistant mutant strain using a limited antibiotic resistance gene that can be used in the study of a multidrug-resistant strain. As a result, , It was confirmed that mutant strain of the desired multidrug-resistant strain can be easily produced even by using only the limited antibiotic resistance gene which can be used for the study of multidrug-resistant strain, and the present invention has been completed.

It is an object of the present invention to provide a suicide vector for producing a mutant strain of a multidrug-resistant strain.

Another object of the present invention is to provide a kit for producing a mutant strain of a multidrug-resistant strain containing the suicide vector.

It is still another object of the present invention to provide a method for producing a mutant strain of a multidrug-resistant strain using the suicide vector.

Another object of the present invention is to provide a mutant strain produced by the above method, wherein a target gene of a multidrug-resistant strain has been deleted.

The present inventors have conducted various studies to develop a method for efficiently producing a mutant strain of a multidrug-resistant strain using a limited antibiotic resistance gene that can be used for studying a multidrug-resistant strain exhibiting resistance to various antibiotics, .

Generally, a suicide vector means a plasmid or a phage that is not replicated in a specific host. The present inventors have found that a suicide vector is a means for mutating all or part of a target gene contained in the genomic DNA of a multidrug-resistant strain by deletion or substitution , A suicide vector not replicated in the multidrug-resistant strain was constructed. The suicide vector produced by the present inventors includes a polyclonal region into which a foreign gene can be inserted as in the case of other conventional vectors, and the polyclonal region can be inserted into the suicide vector without using a complex restriction enzyme The restriction endonuclease cleavage site can be introduced so that the restriction endonuclease can be introduced. Because of this polyclonal site, DNA insert fragments produced by conventional PCR methods and having smooth ends can be easily introduced into the suicide vectors without treatment with special restriction enzymes.

On the other hand, the present inventors have succeeded in introducing a DNA insertion fragment into the prepared suicide vector to prepare a recombinant vector, and introducing the recombinant vector into the specific host, the single stranded DNA fragment is introduced into the host Into the genome of the transformant.

In one embodiment of the present invention, the present invention provides a suicide vector for producing a mutant strain of a multidrug-resistant strain.

The inventors of the present invention have found that the use of a multidrug-resistant strain (hereinafter, referred to as " target polynucleotide ") for use as a means for mutating all or part of a target gene contained in genomic DNA of a multidrug-resistant strain . The suicide vector was constructed to include R6K ori, mob RP4 gene, cat gene, sacB gene, and polyclonal region in sequence. The polyclonal site contained in the suicide vector includes various restriction enzyme cleavage sites capable of introducing a smooth end into the suicide vector so as to easily introduce the DNA insert into the suicide vector without using complex restriction enzymes. Therefore, a DNA-inserted fragment having a smooth end prepared by a conventional PCR method can be easily introduced into the suicide vector without treating with a special restriction enzyme. In addition, the sacB gene expresses sucrase, which is known as an enzyme that degrades sucrose. The expressed sucrase decomposes sucrose in an environment containing excessive sucrose to produce a toxic product , It is possible to induce a single cross-over recombination in the genomic DNA of the host cell.

When a recombinant vector into which a DNA-inserted fragment is introduced into a suicide vector containing such components is introduced into a multidrug-resistant strain, the 5'-region fragment or the 3'-region fragment contained in the DNA- A transformant in which the recombination is induced and the whole of the recombinant vector is inserted into the genomic DNA of the multidrug-resistant strain can be produced. Successful production of the transformant can be confirmed by the expression of a marker region fragment contained in the DNA-inserted fragment. Then, the transformant thus prepared is inoculated into a culture medium containing an excess amount of sucrose and cultured, whereby the sucrose is degraded by sucrase expressed from the sacB gene contained in the suicide vector, and toxic products , A second single cross-over recombination can be induced in the genomic DNA into which the recombinant vector is inserted. When the second single cross-over recombination is induced, the inserted recombinant vector may be removed from the genomic DNA to return to the wild-type multidrug-resistant strain, or a mutant strain of the multidrug-resistant strain in which the target gene or the target polynucleotide has been removed may be produced. Whether or not the mutant is produced can be confirmed by comparing the function of the finally produced strain or the genomic DNA size.

Therefore, when a recombinant vector into which a DNA-inserted fragment is introduced into a suicide vector that is not replicated in the prepared multidrug-resistant strain is introduced into the multidrug-resistant strain to obtain a transformant and cultured in a medium containing sucrose, Mutant strains in which a single cross-over recombination is induced in the genome of a multidrug-resistant strain, whereby a part of the genomic DNA of the multidrug-resistant strain is deleted or substituted, can be produced.

The suicide vectors provided in the present invention can be used to prepare mutant strains of multidrug-resistant strains, and it is advantageous to continuously produce various mutant strains by repeatedly using the same marker region fragments in the production of the mutant strains.

The multidrug-resistant strains used in the present invention are resistant to various antibiotics, so that the antibiotic resistance marker genes that can be used in the production of the mutant strains are limited. When the conventional mutant strains are used, the antibiotic resistance marker genes used are mutants Of the genomic DNA of the present invention, so that it is necessary to use another antibiotic resistance marker gene in order to produce additional mutants, so that mutant strains of multidrug-resistant strains are limited.

Unlike the conventional mutant strains, antibiotic resistance marker genes are used in the intermediate process using the suicide vectors provided in the present invention, but the antimicrobial resistance marker gene is not left in the genomic DNA of the finally prepared mutant strains , A second mutant can be produced using the same antibiotic resistance marker gene. Further, in the same manner using the same marker region fragment, successive mutants such as the production of a third mutant using the second mutant and the production of a fourth mutant using the third mutant can be produced.

Since the suicide vector provided by the present invention does not show specificity for a specific strain, it can be used to prepare a mutant as well as a multidrug-resistant strain as well as a multidrug resistant strain. However, when a strain that does not show multidrug resistance is used, a mutant strain can be produced more effectively when a conventional vector is used. On the other hand, in the case of a multidrug-resistant strain, when the suicide vector provided in the present invention is used It is possible to produce a mutant strain more effectively. Therefore, the suicide vector of the present invention can be used without limitation for the production of a mutant strains thereof, as long as it is a strain exhibiting multidrug resistance, and can enjoy the advantages provided by the suicide vector of the present invention.

The term " multidrug-resistant strain " of the present invention means a strain showing resistance to various antibiotics, as well as a strain showing resistance to antibiotics inherently due to an artificial antibiotic treatment, as well as a strain Lt; / RTI >

In the present invention, the multidrug-resistant bacterium is not particularly limited, but preferably it can be a gram negative strain showing multidrug resistance, more preferably a gram negative bacterium showing multidrug resistance, ( Acinetobacter , < RTI ID = 0.0 > Acinetobacter < / RTI > baumannii , A. baumannii ). For example, in the present invention, examples of the multidrug-resistant strains include Acinetobacter baumannii , A. baumannii ) strains were used.

Terms of the present invention, "Acinetobacter baumannii strains (Acinetobacter baumannii , A. baumannii ) strain "refers to MRAB (multidrug-resistant Acinetobacter baumannii ), and refers to an infectious pathogen resistant to carbapenem , aminoglycoside and fluroquinolone antibiotics. It is known that the strain is easily infected with immunosuppression, chronic lung disease, diabetes mellitus, and the like.

The term "suicide vector " of the present invention is a kind of introduction vector which serves as a delivery medium for delivering a DNA-inserted fragment to a genomic DNA of a host strain, and is not replicated in a specific host strain, Lt; RTI ID = 0.0 > plasmid < / RTI > or phage.

In the present invention, the suicide vector may be interpreted as a suicide vector for producing a mutant strain of a multidrug-resistant strain, wherein the suicide vector exhibits a property of not replicating in a multidrug-resistant strain. As a constituent for this, the R6K ori, the mob RP4 gene, the cat gene, the sacB gene and the polyclonal region may be sequentially included, and the polyclonal region may be cleaved by restriction enzymes generating various smooth ends And the like. The restriction endonuclease producing the blunt end is not particularly limited, but PstI, FspI, NotI, BglII and the like can be used alone or in combination.

In addition, the R6K ori, mob RP4 gene, cat gene, sacB gene, polyclonal region, and the like included in the suicide vector include not only the wild type sequence but also a polynucleotide including a sequence showing homology thereto, Can be used, and can be, for example, a polynucleotide exhibiting a similarity of preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 98% or more.

For example, the suicide vector provided in the present invention may be a suicide vector having the cleaved map shown in Fig. 1B, a suicide vector containing the polynucleotide of SEQ ID NO: 1.

The term "DNA insert fragment " of the present invention means a gene or polynucleotide that can be introduced into a target strain to cause mutation and thereby mutate the genetic traits of the target strain.

In the present invention, the DNA-inserted fragment is inserted into the genomic DNA of the host-resistant multidrug-resistant strain to introduce a polynucleotide configured to induce a single cross-over recombination, and a suicide vector And may include a marker for confirming the introduction.

Specifically, the DNA-inserted fragment includes a nucleotide sequence (hereinafter referred to as a '5'-region fragment') upstream of a target gene or a target polynucleotide located in the genomic DNA of the multidrug-resistant strain, a downstream region of a target gene or a target polynucleotide (Hereinafter, referred to as a '3'-region fragment') and a nucleotide sequence of a gene encoding an antibiotic marker protein having resistance to an antibiotic against which the multidrug-resistant strain does not exhibit resistance (hereinafter referred to as 'marker region fragment' , Wherein the 5'- and 3'-region fragments can induce a single cross-over recombination in the genomic DNA of a multidrug-resistant strain, wherein the marker region fragment is a suicide vector As a marker for confirming whether or not the introduction of the marker is possible.

The term "nucleotide sequence adjacent to the upstream of the target polynucleotide" of the present invention means a DNA sequence having the base sequence or the same base sequence linked to 5 'of the target polynucleotide in the genomic DNA of the multidrug-resistant strain, The term "base sequence adjacent to the downstream of the target polynucleotide" means a base sequence or a DNA fragment having the same base sequence linked to the 3 'of the target polynucleotide in the genomic DNA of the multidrug-resistant strain.

The term "antibiotic marker protein" of the present invention means a protein which is resistant to an antibiotic and can be used as a marker in transformation.

In the present invention, the gene encoding the antibiotic marker protein is used as a marker region fragment of the DNA-inserted fragment, and the recombinant vector prepared by introducing the DNA-inserted fragment into the suicide vector is introduced into the multidrug-resistant strain, Once inserted, they are independently expressed until they are removed from the genomic DNA. The gene encoding the antibiotic marker protein may be a gene coding for any antibiotic marker protein which has been used in the past unless the gene encoding the antibiotic marker protein is implicitly contained in the multidrug resistant strain. .

For example, in the present invention, a strain of Asnithobacter baumannii was used as a multidrug-resistant strain. Antibiotic marker proteins that can be used for the strains of Asnithobacter baumannii include kanamycin resistance gene nptI, tetracycline resistance genes tetM, tetC , tetR can be used singly or in combination.

According to one embodiment of the present invention, the nucleotide sequence (5'-region fragment) adjacent to the upstream of the target gene located in the genomic DNA of the strain of Asnithobacter baumannii, which is a kind of multidrug resistant strain, a base adjacent to the downstream of the target gene A DNA insert fragment designed to sequentially include a sequence (3'-region fragment) and a nucleotide sequence (marker region fragment) of an nptI gene coding for a protein exhibiting resistance to kanamycin that does not exhibit resistance to Asnithobacter baumannii Were synthesized and used.

The term "target gene" of the present invention refers to a gene that is also referred to as an " inherent gene ", which is included in the genomic DNA of the desired multidrug-resistant strain and is used to induce mutation using the target gene.

In the present invention, the target gene may be interpreted as a gene that is included in the genomic DNA of a multidrug-resistant strain to be subjected to deletion or substitution, and the target gene may be appropriate depending on the multidrug-resistant strain.

For example, in the present invention, a strain of Asnithobacter baumanni was used as a multidrug-resistant strain. As the target gene of the strain of Asnithobacter baumannii, basD, bauA, rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD, rhbC3, etc., alone or in combination.

The suicide vectors provided in the present invention can be used to prepare mutant strains of various types of multidrug-resistant strains by designing the DNA insert fragments that can be inserted into their polyclonal regions to be compatible with different purposes.

As a first use example, the suicide vector provided in the present invention can be used together with the DNA insert fragment to prepare a mutant strain in which a target gene or a target polynucleotide contained in the genomic DNA of a multidrug-resistant strain has been deleted.

Hereinafter, the genomic DNA (hereinafter, referred to as "-AXB-") of a multidrug-resistant strain containing the 5'-region fragment (A), the target gene (X) (Hereinafter referred to as '-AB-nptI-sacB-') comprising a region segment (A), a 3'-region segment (B), a marker region segment (nptI), and a sacB gene The method for producing the mutant strain of the multidrug-resistant strain provided by the present invention will be described in more detail.

When the recombinant vector (-AB-nptI-sacB-) is introduced into a multidrug-resistant strain, it is introduced into the genomic DNA (-AXB-) through primary single cross-over recombination and transformed (-AB-nptI- AXB- or -AXB-nptI-sacB-AB-). Subsequently, when the transformant is inoculated into a culture medium containing an excessive amount of sucrose and cultured, the sucrose is degraded by sucrase expressed from the sacB gene contained in the transformant, and toxic products , A second single cross-over recombination can be induced in the genomic DNA into which the recombinant vector is inserted.

When a second single cross-over recombination is induced in the 5'-region fragment (A) of the transformant (-AB-nptI-sacB-AXB-), wild-type genome DNA (-AXB-) and DNA fragment nptI-sacB-A) is generated; When the second single cross-over recombination is induced in the 3'-region fragment (B) of the transformant (-AB-nptI-sacB-AXB-), genomic DNA (-AB-) (-nptI-sacB-AXB) is generated; When the second-order single cross-over recombination is induced in the 5'-region fragment (A) of the transformant (-AXB-nptI-sacB-AB-), genomic DNA (-AB-) (-XB-nptI-sacB-A) is generated; When a second single cross-over recombination is induced in the 3'-region fragment (B) of the transformant (-AXB-nptI-sacB-AB-), wild-type genome DNA (-AXB-) and DNA fragment (-nptI- sacB - AB) is generated.

Since the resulting DNA fragment is cleaved and removed by various nuclease in cells, a wild-type strain having a wild-type DNA or a mutant strain having genomic DNA in which a target gene has been deleted can be obtained as a result. Since the target gene is deleted and the size of the genomic DNA is reduced compared with the wild-type strain, the mutant strain can be easily distinguished by the function expression of the target gene or the size analysis of the genomic DNA using electrophoresis .

The suicide vector of the present invention can be applied to a variety of mutant strains by repeatedly using the same marker region fragment as described above. Thus, the suicide vector of the present invention can be applied to a variety of target genes or target polynucleotides Wherein the multiple target genes or target polynucleotides can be sequentially deleted one by one to produce a mutant strain in which all target genes or target polynucleotides have been deleted.

For example, in a multidrug-resistant strain containing genomic DNA (-MNOPQ-) in which genes M, N, O, P and Q are sequentially included, a recombinant vector (-MO-nptI-sacB -) was added and cultured in a sucrose medium to prepare a first mutant strain containing genomic DNA (-MOPQ-) in which gene N was deleted; A recombinant vector (-MP-nptI-sacB-) for deletion of the gene O was added to the first mutant and cultured in a sucrose medium to prepare a second mutant strain containing the genomic DNA (-MPQ-) in which the gene O was deleted ; A recombinant vector (-MQ-nptI-sacB-) for deletion of the gene P is added to the second mutant and cultured in a sucrose medium to prepare a third mutant strain containing the genomic DNA (-MQ-) in which the gene P has been deleted . At this time, the same antibiotic resistance marker gene (nptI) can be repeatedly used.

As a second use example, the suicide vector provided in the present invention can be used together with the DNA insertion fragment to prepare a mutant strain in which a part of the target gene contained in the genomic DNA of the multidrug-resistant strain is deleted.

Hereinafter, a genomic DNA of a multidrug-resistant strain comprising a target gene containing a 5'-region fragment (a), a target polynucleotide (x) and a 3'-region fragment (b) a recombinant vector derived from a suicide vector (referred to as '-axb-') and a 5'-region fragment (a), a 3'-region fragment (b), a marker region fragment (nptI), a sacB gene (Hereinafter referred to as "-ab-nptI-sacB-") is used to specifically describe a method for producing a mutant strain in which a target polynucleotide of a multidrug-resistant strain has been deleted.

When the recombinant vector (-ab-nptI-sacB-) is introduced into a multidrug-resistant strain, it is introduced into the genomic DNA (-axb-) through primary single cross-over recombination and transformants (-ab-nptI- axb- or -axb-nptI-sacB-ab-). Subsequently, when the transformant is inoculated into a culture medium containing an excessive amount of sucrose and cultured, the sucrose is degraded by sucrase expressed from the sacB gene contained in the transformant, and toxic products , A second single cross-over recombination can be induced in the genomic DNA into which the recombinant vector is inserted.

When a second single cross-over recombination is induced in the 5'-region fragment (a) of the transformant (-ab-nptI-sacB-axb-), wild-type genomic DNA (-axb-) and DNA fragment -sacB-a) is generated; When a second single cross-over recombination is induced in the 3'-region fragment (b) of the transformant (-ab-nptI-sacB-axb-), genomic DNA (-ab-) with the target polynucleotide deleted and DNA A fragment (-nptI-sacB-axb) is generated; When a second single cross-over recombination is induced in the 5'-region fragment (a) of the transformant (-axb-nptI-sacB-ab-), genomic DNA (-ab-) A fragment (xb-nptI-sacB-a) is generated; When a second single cross-over recombination is induced in the 3'-region fragment (b) of the transformant (-axb-nptI-sacB-ab-), wild-type genome DNA (-axb-) and DNA fragment (-nptI- sacB - ab) is generated.

Since the resulting DNA fragment is cleaved and removed by various nuclease in cells, a wild-type strain having a wild-type DNA or a mutant strain having genomic DNA in which a target gene has been deleted can be obtained as a result. Since the target polynucleotide is deleted and the function of the target gene is inhibited or the size of the genomic DNA is reduced compared to the wild-type strain, the mutant strain can be expressed by the expression of the target gene function or the size analysis of the genome DNA through electrophoresis Mutants can be easily distinguished.

As a third use example, the suicide vector provided in the present invention can be used together with the DNA insert fragment to prepare a mutant strain in which the target gene contained in the genomic DNA of the multidrug-resistant strain is substituted.

Hereinafter, the genomic DNA (hereinafter, referred to as "-AXB-") of a multidrug-resistant strain containing the 5'-region fragment (A), the target gene (X) Derived recombinant vector (hereinafter referred to as '-AEB-1') comprising a region fragment (A), a substitution gene (E), a 3'-region fragment (B), a marker region fragment (nptI), a sacB gene quot; nptI-sacB- "), a method for producing a mutant strain in which a target gene of a multidrug-resistant strain is substituted will be described in detail.

When the recombinant vector (-AEB-nptI-sacB-) is introduced into a multidrug-resistant strain, it is introduced into the genomic DNA (-AXB-) through primary single cross-over recombination and transformed (-AEB-nptI- AXB- or -AXB-nptI-sacB-AEB-). Subsequently, when the transformant is inoculated into a culture medium containing an excessive amount of sucrose and cultured, the sucrose is degraded by sucrase expressed from the sacB gene contained in the transformant, and toxic products , A second single cross-over recombination can be induced in the genomic DNA into which the recombinant vector is inserted.

When a second single cross-over recombination is induced in the 5'-region fragment (A) of the transformant (-AEB-nptI-sacB-AXB-), wild-type genome DNA (-AXB-) and DNA fragment (-EB- nptI-sacB-A) is generated; When a second single cross-over recombination is induced in the 3'-region fragment (B) of the transformant (-AEB-nptI-sacB-AXB-), genomic DNA (-AEB-) (-nptI-sacB-AXB) is generated; When a second-order single cross-over recombination is induced in the 5'-region fragment (A) of the transformant (-AXB-nptI-sacB-AEB-), genomic DNA (-AEB-) (-XB-nptI-sacB-A) is generated; When a second-order single cross-over recombination is induced in the 3'-region fragment (B) of the transformant (-AXB-nptI-sacB-AEB-), wild-type genome DNA (-AXB-) and DNA fragment (-nptI- sacB - AEB) is generated.

The resulting DNA fragment is decomposed and removed by various nuclease agents in the cell. As a result, a wild-type strain having a wild-type DNA or a mutant strain having a genomic DNA in which a target gene has been substituted can be obtained. Since the target gene is substituted for the mutant in comparison with the wild-type strain, the mutant can be easily distinguished through a method such as the expression of the function of the target gene or the substitution gene.

As a fourth use example, the suicide vector provided in the present invention can be used together with the DNA insertion fragment to prepare a mutant strain in which some polynucleotides (target polynucleotide) of the target gene contained in the genomic DNA of the multidrug-resistant strain are substituted have.

Hereinafter, a genomic DNA of a multidrug-resistant strain comprising a target gene containing a 5'-region fragment (a), a target polynucleotide (x) and a 3'-region fragment (b) (a), a substituted polynucleotide (e), a 3'-region fragment (b), a marker region fragment (nptI), and a sacB gene (sacB) Hereinafter, a method for producing a mutant strain in which a target polynucleotide of a multidrug-resistant strain is substituted using a suicide vector-derived recombinant vector (hereinafter referred to as '-aeb-nptI-sacB-') will be described in detail.

When the recombinant vector (-aeb-nptI-sacB-) is introduced into a multidrug-resistant strain, it is introduced into the genomic DNA (-axb-) through primary single cross-over recombination to produce a transformant (-aeb-nptI- axb- or -axb-nptI-sacB-aeb-). Subsequently, when the transformant is inoculated into a culture medium containing an excessive amount of sucrose and cultured, the sucrose is degraded by sucrase expressed from the sacB gene contained in the transformant, and toxic products , A second single cross-over recombination can be induced in the genomic DNA into which the recombinant vector is inserted.

When a second single cross-over recombination is induced in the 5'-region fragment (a) of the transformant (-aeb-nptI-sacB-axb-), wild-type genomic DNA (-axb-) and DNA fragment (eb- -sacB-a) is generated; When a second single cross-over recombination is induced in the 3'-region fragment (b) of the transformant (-aeb-nptI-sacB-axb-), the genomic DNA (-aeb-) substituted with the target polynucleotide and DNA A fragment (-nptI-sacB-axb) is generated; When a second single cross-over recombination is induced in the 5'-region fragment (a) of the transformant (-axb-B-nptI-sacB-aeb-), the target polynucleotide is substituted with genomic DNA (-aeb-) And a DNA fragment (xb-nptI-sacB-a) are generated; When a second single cross-over recombination is induced in the 3'-region fragment (b) of the transformant (-axb-nptI-sacB-aeb-), wild-type genome DNA (-axb-) and DNA fragment (-nptI- sacB - aeb) is generated.

The resulting DNA fragment is decomposed and removed by various nuclease in the cell. As a result, a wild-type strain having a wild-type DNA or a mutant strain having a genomic DNA in which a target polynucleotide has been substituted can be obtained. Since a part of the target gene is substituted for the mutant in comparison with the wild-type strain, the mutant strain can be easily distinguished through a method such as the function expression of the target gene.

As a fifth use example, the suicide vector provided in the present invention can be used together with the DNA insertion fragment to produce a mutant strain in which two different target genes contained in the multidrug-resistant strain are simultaneously deleted.

Hereinafter, the genome of a multidrug-resistant strain comprising the 5'-region fragment (A), the first target gene (X), the intermediate fragment (B), the second target gene (Y) and the 3'- DNA (hereinafter referred to as "-AXBYC-"); A first recombinant vector derived from a suicide vector (hereinafter referred to as '-AB-nptI-sacB') containing a 5'-region fragment (A), an intermediate fragment (B), a first marker region fragment (nptI) - '); And a second recombinant vector derived from a suicide vector (hereinafter referred to as '-BC-tetR-R') including a middle fragment (B), a 3'-region fragment (C), a second marker region fragment (tetR) sacB- '), a method for producing a mutant strain in which two different target genes of a multidrug-resistant strain are simultaneously deleted is described in detail.

As an example, when the first recombinant vector (-AB-nptI-sacB-) and the second recombinant vector (-BC-tetR-sacB-) are introduced into the multidrug-resistant strain, the first recombinant vector Can be introduced into the 5'-region fragment (A) of the genomic DNA (-AXBYC-) to produce a first transformant (-AB-nptI-sacB-AXBYC-), followed by a second single cross- A second recombinant vector is introduced into the first intermediate fragment (B) of the first transformant to prepare a second transformant (-ABC-tetR-sacB-B-nptI-sacB-AXBYC-) have. Subsequently, the secondary transformant is inoculated into a culture medium containing an excessive amount of sucrose and cultured to cause a tertiary single-crossed recombination in the 3'-region fragment (C) of the secondary transformant, Genomic DNA (-ABC-) in which the target gene (X) and the second target gene (Y) are deleted and a DNA fragment (-tetR-sacB-B-nptI-sacB-AXBYC) The fragment is cleaved and removed by various nuclease in the cell. As a result, a mutant strain having the genomic DNA in which the first target gene (X) and the second target gene (Y) are deleted can be produced.

As another example, when the first recombinant vector (-AB-nptI-sacB-) and the second recombinant vector (-BC-tetR-sacB-) are introduced into the multidrug-resistant strain, the second recombinant Vector can be introduced into the 3'-region fragment (C) of genomic DNA (-AXBYC-) to produce a first transformant (-AXBYC-tetR-sacB-BC-), and a second single cross- , A first recombinant vector is introduced into the second intermediate fragment (B) of the first transformant to prepare a second transformant (-AXBYC-tetR-sacB-B-nptI-sacB-ABC-) . Subsequently, the secondary transformant is inoculated into a culture medium containing an excess amount of sucrose and cultured to cause a tertiary single cross-over recombination in the 5'-region fragment (A) of the secondary transformant, Genomic DNA (-ABC-) in which the target gene (X) and the second target gene (Y) are deleted and a DNA fragment (-XBYC-tetR-sacB-B-nptI-sacB-A) DNA fragments are cleaved and removed by various nuclease in the cells. As a result, a mutant strain having the genomic DNA in which the first target gene (X) and the second target gene (Y) are deleted can be produced.

As another example, when the first recombinant vector (-AB-nptI-sacB-) and the second recombinant vector (-BC-tetR-sacB-) are introduced into a multidrug-resistant strain, A second recombinant vector can be introduced into the intermediate fragment (B) of the first recombinant vector to produce a third recombinant vector (-ABC-tetR-sacB-B-nptI-sacB-) (-ABC-tetR-sacB-B-nptI-sacB-AXBYC-) was introduced into the 5'-region fragment (A) of genomic DNA (-AXBYC-) through the third recombinant vector . Subsequently, the secondary transformant is inoculated into a culture medium containing an excessive amount of sucrose and cultured to cause a tertiary single-crossed recombination in the 3'-region fragment (C) of the secondary transformant, Genomic DNA (-ABC-) in which the target gene (X) and the second target gene (Y) are deleted and a DNA fragment (-tetR-sacB-B-nptI-sacB-AXBYC) The fragment is cleaved and removed by various nuclease in the cell. As a result, a mutant strain having the genomic DNA in which the first target gene (X) and the second target gene (Y) are deleted can be produced.

Since the mutant strains prepared by each of the above methods are deficient in two target genes as compared to the wild type strains, mutants can be easily distinguished through the expression of function of each of the target genes or the size analysis of genomic DNA through electrophoresis .

According to one embodiment of the present invention, a pHKD01 vector, which is a non-replicating suicide vector in A. baumannii , a kind of multidrug-resistant strain, is prepared (FIGS. 1A and 1B) (PHKD02 to pHK13) capable of deleting various target genes (basD, bauA, rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD or rhbC3) A).

As another embodiment for achieving the object of the present invention, the present invention provides a kit for producing a mutant strain of a multidrug-resistant strain comprising a suicide vector for producing a mutant strain of the multidrug-resistant strain.

The kit of the present invention can be used for introducing a DNA insert into a genomic DNA of a multidrug-resistant strain, but is not limited to the suicide vector, but may include one or more other components suitable for producing a mutant strain of a multidrug-resistant strain Compositions, solutions, or devices.

As a specific example, in order to confirm whether the DNA-inserted fragment has been introduced into a multidrug-resistant strain, a multidrug-resistant strain, which can be used as a marker region fragment of the DNA-inserted fragment, has antibiotic resistance A gene encoding a marker protein may be included. (PstI, FspI, NotI, BglII, etc.), a test tube or other appropriate container, a reaction buffer (for example, a primer for amplifying a gene encoding the antibiotic marker protein, enzymes such as deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, etc., And may be combined and included.

As another embodiment for achieving the object of the present invention, the present invention provides a method for producing a mutant strain of a multidrug-resistant strain using the suicide vector.

Specifically, the method for producing a mutant strain of a multidrug-resistant strain of the present invention comprises the steps of (a) introducing a DNA insert fragment (a) into a polyclonal region of a suicide vector sequentially containing R6K ori, mob RP4 gene, cat gene, sacB gene, Obtaining the introduced recombinant vector; (b) introducing the obtained recombinant vector into a multidrug-resistant strain to obtain a transformant; And (c) culturing the transformant in a medium containing sucrose to select a mutant strain in which the target gene has been deleted.

As described above, when the suicide vector of the present invention is used, various mutant strains can be continuously produced by repeatedly using the same marker region fragment. Therefore, the mutant strains can be produced by repeating the entire process with different target genes , A continuous mutant strain in which one or more genes among the various genes contained in the multidrug-resistant strain are sequentially deleted can be produced.

That is, the step (a) to (c) may be repeated to further delete other target genes present in the genomic DNA of the mutant strain in which one target gene has been deleted from the multidrug-resistant strain have. Comparing the method comprising repeating the steps (a) to (c) compared to the method comprising the steps (a) to (c), the multi- Is a mutation of a multidrug-resistant strain that has been deleted and that the DNA insert used is designed to delete another target gene.

That is, since the other target gene is an object to be further deleted from the genomic DNA of the desired wild-type or mutated multidrug-resistant strain, the DNA insert fragment can be obtained by analyzing the base sequence of another target gene, The 5 '-region fragment and the 3'-region fragment may be designed so as to be compatible with each other, and may be a DNA fragment synthesized to include a marker region fragment together therewith. In addition, the marker region fragment included in the DNA insert may be the same as or different from the previously used marker region fragment.

For example, in the present invention, strains of Asnithobacter baumanni were used as multidrug-resistant strains, and the strains of basD, bauA, rimL, piuB, cirA, menG, fhuF, rhbC1, a first mutant strain in which the basD gene has been deleted is prepared by applying the method for producing the mutant strains of the multidrug-resistant strain to the basD gene among genes such as rhbC2, araJ, iucD, and rhbC3; Among the genes such as bauA, rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD and rhbC3 contained in the genomic DNA of the first mutant, the bauA gene was used to prepare mutant strains of the above- To produce a second mutant in which the basD and bauA genes have been deleted; Among the genes such as rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD and rhbC3 contained in the genomic DNA of the secondary mutant, the method of producing the mutant strains of the multidrug-resistant strain is applied to the rimL gene To produce a third mutant in which the basD, bauA and rimL genes were deleted; Among the genes such as piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD and rhbC3 contained in the genomic DNA of the third mutant strain, piuB gene was applied to the mutant strains of the above- the fourth strain of mutant strain in which the basD, bauA, rimL and piuB genes have been deleted can be produced in succession to produce the Ashinotobacter baumanni mutant.

Hereinafter, the method for producing the mutant strain of the multidrug-resistant strain provided by the present invention will be described in more detail by dividing each step.

First, in the method for producing a mutant strain of a multidrug-resistant strain provided by the present invention, step (a) is a step of obtaining a recombinant vector into which the DNA-inserted fragment is introduced into a suicide vector not replicated in a multidrug-resistant strain.

The step (a) comprises the step of preparing a recombinant vector capable of mutating a target gene present in the genomic DNA of the multidrug-resistant strain, wherein the suicide vector is selected from the group consisting of R6K ori, mob The RP4 gene, the cat gene, the sacB gene, and the polyclonal region, and the DNA insertion fragment sequentially includes a 5'-region fragment, a 3'-region fragment, and a marker region fragment, as described above. Since the 5'-region fragment and the 3'-region fragment are respectively adjacent to the upstream and downstream of the target gene, the 5'-region fragment and the 3'-region fragment are determined by the nucleotide sequence of the target gene Wherein the marker region fragment can be a gene encoding an antibiotic marker protein having resistance to an antibiotic that does not exhibit resistance by the multidrug-resistant strain.

According to one embodiment of the present invention, an Acetobacter baumanni strain is used as a multidrug resistant strain, and a 5'-region fragment (A), a 3'-region fragment (B) and a marker region fragment (nptI) (-AB-nptI-) was introduced into a multi-cloning site (MCS) of suicide vector (-R6K-mobRP4-cat-sacB-MCS-) to obtain a recombinant vector (-R6K-mobRP4 -cat-sacB-AB-nptI-).

Second, in the method for producing a mutant strain of a multidrug-resistant strain provided by the present invention, step (b) is a step of introducing the obtained recombinant vector into a multidrug-resistant strain to obtain a transformant.

The step (b) is a step of obtaining a transformant into which a recombinant vector is introduced. When the recombinant vector obtained is introduced into a multidrug-resistant strain containing the target gene in the genomic DNA, -Region fragment) or a downstream site (3'-region fragment), so that the recombinant vector is introduced upstream or downstream of the target gene contained in the genomic DNA of the multidrug-resistant strain, resulting in a recombinant vector To obtain a transformant. In addition, the transformant can be selected by culturing in a medium containing an antibiotic exhibiting resistance by a protein expressed in a fragment of a marker region contained in the recombinant vector.

According to one embodiment of the present invention, an Acetobacter baumannii strain is used as a multidrug-resistant strain, and a target gene (X), its upstream region (5'-region fragment, A) (-R6K-mobRP4-cat-sacB-AB-nptI-) is introduced into the genomic DNA (-AXB-) of an Escherichia bacterium var. (-AB-nptl-R6K-mobRP4-cat-sacB-AXB-) or genomic DNA (-AXB-AXB-) which undergoes a single cross-over recombination on the downstream of the target gene by performing a single cross- -nptI-R6K-mobRP4-cat-sacB-AB-) can be obtained.

Since the obtained transformant was introduced with a marker region fragment (nptI) in the genomic DNA, the recombinant vector exhibited resistance to kanamycin by the protein expressed by the nptI but the recombinant vector was not introduced into genomic DNA, Since the recombinant vector is not replicated in the strain of Asnithobacter baumannii due to the nature of the suicide vector, the strains of Asnithobacter baumannii are diluted and erased as the strain grows. As a result, there is no protein expressed from nptI , Indicating no resistance to kanamycin. Therefore, when the strain of Asnithobacter baumannii into which the recombinant vector is introduced is cultured in a medium containing kanamycin, the transformant can be obtained by selecting.

Third, in the method for producing a mutant strain of a multidrug-resistant strain provided by the present invention, the step (c) is a step of culturing the transformant in a medium containing sucrose to select a mutant strain in which the target gene has been deleted.

The step (c) is a step of producing a mutant by deleting the target gene by a single cross-over recombination in the genomic DNA of the obtained transformant. This step is a step of transforming sucrase and sucrose expressed in the sacB gene contained in the suicide vector ≪ / RTI > That is, when the transformant obtained is inoculated and cultured in a culture medium containing an excessive amount of sucrose, the sucrose is decomposed by sucrase expressed in sacB inserted into the genomic DNA of the transformant, Toxic products are generated as degradation products of sucrose, which damage the transformation and create an environment in which single cross-over recombination can occur within the genomic DNA. Due to the recombination vector introduced into the genomic DNA, the 5'-region fragment and the 3'-region fragment adjacent to the target gene are overlapped in the genomic DNA of the transformant, so that an environment in which the single cross-over recombination can occur , Single cross-over recombination may occur in the 5'-region fragment or the 3'-region fragment. When a single cross-over recombination occurs in the 5'-region fragment or the 3'-region fragment, the recombinant vector introduced into the genomic DNA is deleted as it is and is recovered to the genomic DNA of the wild-type multidrug-resistant strain or the genomic DNA of the multidrug-resistant strain A mutant strain in which the target gene has been deleted can be produced. Since the target gene is deleted from the genomic DNA, the mutant strain of the prepared multidrug-resistant strain can be easily distinguished from the wild type strain by comparing the size of the genomic DNA or checking the function of the target gene.

Therefore, selection of the mutant strain in which the target gene is deleted can be performed by (c1) culturing the transformant cultured in the medium containing sucrose in a medium containing an antibiotic resistant to the multidrug-resistant strain, Selecting; And (c2) obtaining a genomic DNA from the selected strain and comparing the size of the genomic DNA with the size of the genomic DNA of the wild-type multidrug-resistant strain, thereby selecting a strain of reduced size of the genomic DNA; Or (c1 ') sucrose in a culture medium containing an antibiotic in which the multidrug-resistant strain does not exhibit resistance, and selecting a non-viable strain; And (c2 ') measuring the activity of the target gene expressed in the selected strain, and selecting a strain showing a level that is relatively lower than the activity of the target gene measured in the wild-type multidrug-resistant strain.

According to one embodiment of the present invention, an Acetobacter baumanni strain is used as a multidrug-resistant strain, and a genomic DNA (-AB-nptI-R6K-mobRP4-cat-sacB- Domain fragment (A) and 3'-region fragment (B) are present in each of the 5'-AXB- or -AXB-nptI-R6K-mobRP4- Recombination may be induced.

First, when a second single cross-over recombination is induced in the 5'-region fragment (A) of the first genomic DNA (-AB-nptI-R6K-mobRP4-cat-sacB-AXB-), genomic DNA (-AXB-) And the DNA fragment (-B-nptI-R6K-mobRP4-cat-sacB-A) is generated. Since the DNA fragment is degraded by intracellular nucleases, only the genomic DNA is left. As a result, It is restored to the genomic DNA (-AXB-) of the strain Ashtenobacter baumannii. In addition, when a second single cross-over recombination is induced in the 3'-region fragment (B) of the first genomic DNA (-AB-nptI-R6K-mobRP4-cat-sacB- AXB-), genomic DNA (-AB- ) And a DNA fragment (-nptI-R6K-mobRP4-cat-sacB-AXB) are generated. For the same reason, only the genomic DNA remains, resulting in the deletion of the target gene (X) Genomic DNA (-AB-) is newly formed.

Next, when a second single cross-over recombination is induced in the 5'-region fragment (A) of the second genomic DNA (-AXB-nptI-R6K-mobRP4-cat-sacB-AB-), genomic DNA (-AB- ) And a DNA fragment (-XB-nptI-R6K-mobRP4-cat-sacB-A) are generated. For the same reason, only genomic DNA remains, resulting in the deletion of the target gene (X) The mutant genomic DNA (-AB-) is newly formed. In addition, when a second single cross-over recombination is induced in the 3'-region fragment (B) of the second genomic DNA (-AXB-nptI-R6K-mobRP4-cat-sacB-AB-), genomic DNA (-AXB- ) And a DNA fragment (-nptI-R6K-mobRP4-cat-sacB-AB) are generated. For the same reason, only the genomic DNA is left. As a result, the genomic DNA of the wild-type Asnithobacter baumannii strain (-AXB- ).

Since the second single cross-over recombination occurs stochastically, the second single cross-over recombination may not be induced even if an environment in which single cross-over recombination occurs may be generated in some cases. Whether or not the second single cross-over recombination is performed can be determined by culturing the strain cultured in the sucrose medium in a medium containing an antibiotic exhibiting resistance by a protein expressed in the marker region fragment contained in the recombinant vector, Can be selected. That is, in any case, the second single cross-over recombination can not survive in the medium containing the antibiotic because the marker region fragment does not remain in the genomic DNA, while the second single cross-over recombination is performed Strains can survive in the same medium, so that the strain subjected to the second single cross-over recombination can be selected using this difference.

Therefore, when the transformant is cultured in a sucrose medium, a single cross-over recombination is further generated to produce a mutant strain of a wild-type multidrug-resistant strain or a multidrug-resistant strain. The mutant strain of the multidrug- Since the target gene has been deleted, it can be easily distinguished from the wild-type strain by screening for the function of the target gene or comparing the sizes of the genomic DNA.

According to one embodiment of the present invention, a pHKD01 vector, which is a non-replicating suicide vector, is prepared from the Acetobacter baumannii strain, which is a kind of multidrug-resistant strain (Fig. 1) (pHKD02 to pHK13) capable of deleting the bovine, bauA, rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD or rhbC3 Using the homologous recombination vector, various mutant strains (HDK01 to HDK12) in which the target gene was deleted in the A. baumannii strain (FIG. 3B and FIG. 4B) were prepared and the transcript of the prepared mutant strain (Fig. 3C and Fig. 5). As a result, the expression level of the target gene was quantitatively analyzed by real-time PCR. As a result, it was confirmed that the target gene was not expressed in the mutant strain (Fig.

As another embodiment for accomplishing the object of the present invention, the present invention provides a mutant strain produced by the above method, in which a target gene of a multidrug-resistant strain is deleted from genomic DNA.

The term " mutant strain of a multidrug-resistant strain "of the present invention means a multidrug-resistant strain in which a target gene is mutated in a multidrug-resistant strain.

In the present invention, the mutant strain of the multidrug-resistant strain shows a genotype in which only the target gene is removed from the wild-type multidrug-resistant strain, and no gene externally introduced remains in the genomic DNA. Therefore, It can be used as a multidrug-resistant strain into which a recombinant vector is introduced. That is, a mutant strain in which the first target gene has been deleted can be prepared using the method provided in the present invention, and a mutant strain in which the second target gene has been deleted can be produced using the mutant strains again by the method provided in the present invention .

According to one embodiment of the present invention, another recombinant vector produced by using the suicide vector of the present invention in a mutant strain (HKD02) in which basD is deleted in the strain, using an Acetobacter baumanni strain as a multidrug-resistant strain, As a result, a mutant strain in which the basD gene as well as the cirA gene was further deleted was prepared (Fig. 6).

Using the kit for producing a mutant strain of a suicide vector or a multidrug resistant strain for producing a mutant strain of a multidrug-resistant strain provided by the present invention, even when only a limited antibiotic resistance gene that can be used for studying various multidrug resistant strains can be used, Since a mutant strain in which one or more target genes or target polynucleotides are deleted from the genomic DNA can be produced, it can be widely used in various fields such as genome and physiological activity of various multidrug-resistant strains.

1A is a schematic diagram showing a method for producing a pHKD01 vector, which is a suicide vector provided in the present invention.
1B shows a cleavage map of the pHKD01 vector.
FIG. 2 is a schematic diagram showing the flow of a method of deleting target genes from the strain of Ashtonobacter baumanni using the pHKD01 vector.
FIG. 3A shows the result of electrophoresis of three amplified polynucleotides (5'-region fragment, 3'-region fragment and nptI gene) and overlap extension PCR on basD or bauA as a target gene This is an electrophoresis image of a smooth end-portion DNA fragment.
FIG. 3B is a photograph of a genomic DNA of an Asnithobacter baumannii mutant (HKD01 or HKD02) in which a target gene (basD or bauA) has been deleted.
FIG. 3C is a graph showing the results of quantitative analysis of the expression of the target gene (basD or bauA) in the Ashtonobacter baumannii mutant (HKD01 or HKD02).
FIG. 4A is a schematic diagram showing the relative positions of various target genes (rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD, or rhbC3) in the genomic DNA of an Acinetobacter baumannii strain.
FIG. 4B shows an electrophoresis image of the genomic DNA of an Asnithobacter baumannii mutant (HKD03 to HKD12) lacking the target gene (rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD or rhbC3) to be.
5 is a graph showing the results of quantitative analysis of the expression of target genes (rimL, piuB, cirA, menG, fhuF, rhbC1, rhbC2, araJ, iucD, or rhbC3) in the Ashtonobacter baumanni mutant HKD03 to HKD12 .
Fig. 6 is a photograph of genomic DNA of an Asnithobacter baumannii mutant (HKD13) in which two target genes (bauA and cirA) are deleted.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Ashton Bauber Baumanni  Suicidal vectors not replicated in the strain ( pHKD01  Vector)

Resistant strains of the present invention were prepared using the Acetobacter baumanni strain, which is one of the multidrug resistant strains.

An annealed oligonucleotide cloning method was applied to construct a vector (pHKD01 vector) capable of blunt end cloning which does not require restriction enzyme sites to be considered as follows.

First, a complementary oligonucleotide having a restriction enzyme cleavage site sequence for the first smooth end was prepared. At this time, the oligonucleotide includes TGCGCA, which is a cleavage site sequence of FspI, which is a restriction enzyme that cleaves at the smooth end. In addition, the complementary oligonucleotides were constructed such that, when annealed to each other, the 5 'and 3' ends of the DNA fragments could be directly linked to the DNA truncated by the PstI restriction enzyme.

Next, 2 쨉 g of each oligonucleotide thus prepared was added to 50 의 of reaction buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 7.5) and reacted at 95 캜 for 5 minutes and at 25 캜 for 60 minutes to obtain double stranded oligonucleotides , And introduced into a pDS132 vector digested with restriction enzyme PstI to produce a suicide vector pHKD01 (SEQ ID NO: 1) which is not replicated in the multidrug-resistant Acinetobacter baumanni strain (Fig. 1).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a method for producing a pHKD01 vector, which is a suicide vector provided by the present invention, and a cleavage map of the pHKD01 vector prepared.

On the other hand, the prepared pHKD01 vector can be used to delete a target gene in an Acinetobacter baumannii strain (Fig. 2). FIG. 2 is a schematic diagram showing the flow of a method of deleting target genes from the strain of Ashtonobacter baumanni using the pHKD01 vector.

As shown in FIG. 2, a blunt-ended DNA fragment containing the deleted target gene (the 5'-terminal portion (Gene A region) of the target gene, the 3'-terminal portion (Gene B region) of the target gene and the kanamycin resistance gene Gene A-Gene B-nptI) was obtained by amplifying the homologous recombinant vector (sacB-Gene B-nptI) by amplifying it by the PCR method and introducing the obtained smooth end DNA fragment into the pHKD01 vector digested with restriction enzyme FspI Then, the homologous recombination vector obtained can be introduced into an Acinetobacter baumannii strain to produce a transformant. The transformant is selected using a medium containing kanamycin. When the first single cross-over recombination occurs in the 5'-terminal region (Gene A region) of the target gene, an expression vector (sacB-Gene A-Gene B-nptI ) Is crossed with a normal target gene (Gene A-Gene X-Gene B) contained in the chromosome of the strain of Acinetobacter baumanni, and the expression vector is inserted into the inside of the target gene existing on the chromosome of the strain of Asnithobacter baumannii (Gene A-Gene B-nptI-sacB-Gene A-Gene X-Gene B). When the transformant is cultured in a culture medium containing sucrose, the sucrose is decomposed by sucrase expressed in the sacB gene introduced into the chromosome, thereby forming a toxic by-product. As a result, the homologous recombinant vector (Gene X region) derived from a chromosome and a portion derived from an expression vector (sacB region) derived from a chromosome, when a second single-cross homologous recombination occurs in the 3'-terminal portion (Gene B region) Region and nptl region) is removed from the chromosome and finally a chromosome (Gene A-Gene B) in which a normal target gene (Gene X region) is deleted can be produced. The Asnithobacter baumannii mutant can be screened using a medium containing kanamycin. Since the Asnithobacter baumannii mutant does not contain nptI which shows resistance to kanamycin, another target gene can be deleted from the Asnithobacter baumannii mutant using the nptI.

Example  2: Ashton Bauber Baumanni  Using a suicide vector that is not replicated in the strain, a mutant strain in which the target gene is deleted is produced

Using the suicide vector pHKD01 vector prepared in Example 1, it was expected that various genes could be deleted in the strain of Ashtonobacter baumanni.

Example  2-1: basD  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

First, a polynucleotide of the 5'-region fragment of the basD gene was obtained by carrying out PCR using the genomic DNA of the strain of Ashinobacter baumanni as a template and the S1 / S2 primer for amplifying the 5'-end of the basD gene Respectively.

S1 primer (BASD01F):

5'-GAAGCAATTGAGCGGTTCAGG-3 '(SEQ ID NO: 2)

S2 primer (BASD01R):

5'-AAATGATGAAAGTTCAAAATACGAATTGTTAATCATTTCCAATTTTGCTGT-3 '(SEQ ID NO: 3)

Next, PCR was carried out using S3 / S4 primer for amplifying the 3'-terminal of the basD gene, using the genomic DNA of the strain of Asnithobacter baumanni as a template to obtain a polynucleotide of the 3'-region fragment of the basD gene .

S3 primer (BASD02F):

5'-TTCGTATTTTGAACTTTCATCATTTTGG-3 '(SEQ ID NO: 4)

S4 primer (BASD02R):

5'-GCAACACCTTCTTCACGAGGCAGACTGTGCAAACAGGTTAAACGCAG-3 '(SEQ ID NO: 5)

In addition, PCR was carried out using U1 / U2 primer for amplifying the nptI gene with pUC4K as a template to obtain polynucleotide of nptI gene.

U1: 5'-GTCTGCCTCGTGAAGAAGGTG-3 '(SEQ ID NO: 6)

U2: 5'-GATCCGTCGACCTGCAGG-3 '(SEQ ID NO: 7)

The resulting three polynucleotides (5'-region fragment, 3'-region fragment and nptI gene) were electrophoresed and their sizes were measured (FIG. 3A). FIG. 3A shows the 5'-terminal of the target gene amplified by the PCR method. 3 ' -terminal and polynucleotide of nptI gene, and polynucleotides thereof, respectively, and performing an overlap extension PCR using the polynucleotide as a template.

Using the above-obtained three polynucleotides (5'-region fragment, 3'-region fragment and nptI gene) as a template, overlap extension PCR was performed using the S1 / U2 primer, and each of the polynucleotides was sequenced (Figure 3 A). ≪ tb >< TABLE >

On the other hand, the pHKD01 vector produced in Example 1 was digested with restriction enzyme FspI, and the thus obtained smooth end DNA fragment was introduced into the cleavage site to obtain a recombinant vector (pHKD02).

The obtained recombinant vector (pHKD02: pHKD01 with ΔbasD :: nptI; Cmr, Kmr) was introduced into an Acinetobacter baumanni strain (ATCC 19606), and the introduced strain was transformed with LB containing kanamycin (30 μg / The transformants were selected by culturing on a plate medium. The selected transformants were cultured in a plate medium containing 10% (w / v) sucrose to obtain respective colonies, and each of the colonies obtained was cultured on LB plate medium and kanamycin (30 μg / ml) The colonies which did not grow in the LB plate medium containing kanamycin but grown in the LB plate medium were cultured in the LB plate medium contained, and then the colonies of the Ashinobacter baumannii mutant (HKD01: ATCC 19606 with ΔbasD) (FIG. 3B). FIG. 3B is a photograph of the genomic DNA of an Asnithobacter baumannii mutant in which the target gene has been deleted. As shown in Fig. 3B, it was confirmed that the size of the genomic DNA was decreased in the case of the Ashinotobacter baumanni mutant as compared with the wild type Ashinotobacter baumanni strain. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-2: bauA  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that BAUA01F / BAUA01R / BAUA02F / BAUA02R were used as S1 / S2 / S3 / S4 primers and pHKD03 (pHKD01 with ΔbauA :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD02: ATCC 19606 with ΔbauA) in which the bauA gene was deleted was prepared. At this time, electrophoresis results of the amplified three polynucleotides (5'-region fragment, 3'-region fragment and nptI gene) and electrophoresis results of the smoothed end DNA fragment obtained by carrying out overlap extension PCR were shown in FIG. 3 And the result of electrophoresis of the genomic DNA derived from the Ashtonobacter baumannii mutant is shown in Fig. 3B.

BAUA01F:

5'-GTCGATTTGGAGAAAACGTAAGC-3 '(SEQ ID NO: 8)

BAUA01R:

5'-CATAAATTTTCACTATTTTGCTATGATAAAAAAACCCGCAATCGC-3 '(SEQ ID NO: 9)

BAUA02F:

5'-CATAGCAAAATAGTGAAAATTTATGCTC-3 '(SEQ ID NO: 10)

BAUA02R:

5'-GCAACACCTTCTTCACGAGGCAGACATCATAGCGGCAGCTGTCAC-3 '(SEQ ID NO: 11)

As shown in Fig. 3B, it was confirmed that the size of the genomic DNA was decreased in the case of the Ashinotobacter baumanni mutant as compared with the wild type Ashinotobacter baumanni strain. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-3: rimL  Gene-deficient Ashton Bauber Baumanni Production of mutant strains

Except that RIML01F / RIML01R / RIML02F / RIML02R were used as S1 / S2 / S3 / S4 primers and pHKD04 (pHKD01 with ΔrimL :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD03: ATCC 19606 with? BauA) in which the rimL gene was deleted was prepared (Fig. 4). At this time, the relative position of the target gene rimL gene in the genomic DNA of the strain of Ashinobacterium Vaumani is shown in Fig. 4A. The result of electrophoresis of genomic DNA of the Ashinotobacter baumannii mutant (HKD03) B of Fig.

RIML01F:

5'-ACTTTGCTTCTATCTGGATGCG-3 '(SEQ ID NO: 12)

RIML01R:

5'-CTTGCAGGTCTACCCATACCAGAAACGGTCCTTGCTATGACATAACC-3 '(SEQ ID NO: 13)

RIML02F:

5'-TTTCTGGTATGGGTAGACCTGC-3 '(SEQ ID NO: 14)

RIML02R:

5'-GCAACACCTTCTTCACGAGGCAGACGGGATAAATTGATCAAGATCGG-3 '(SEQ ID NO: 15)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-4: piuB  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that PIUB01F / PIUB01F / PIUB01R / PIUB02F / PIUB02R was used as S1 / S2 / S3 / S4 primer and pHKD05 (pHKD01 with ΔpiuB :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD04: ATCC 19606 with ΔpiuB) in which the piuB gene was deleted was prepared (FIG. 4). At this time, the relative position of the target gene piuB gene in the genome DNA of the strain of Ashinobacterium var. Mariana is shown in Fig. 4A. The result of electrophoresis of the genome DNA of the Ashinotobacter baumanni mutant (HKD04) B of Fig.

PIUB01F:

5'-GGCTTAAATCTGGGATTGACTGG-3 '(SEQ ID NO: 16)

PIUB01R:

5'-CCCAAAATAATGATGCAAAGCTCATTTAAGCCACTCCCCATTTAGCTA-3 '(SEQ ID NO: 17)

PIUB02F:

5'-ATGAGCTTTGCATCATTATTTTGG-3 '(SEQ ID NO: 18)

PIUB02R:

5'-GCAACACCTTCTTCACGAGGCAGACGCACATGTCTTCTACTACAGCCG-3 '(SEQ ID NO: 19)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-5: cirA  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that CIRA01F / CIRA01F / CIRA01R / CIRA02F / CIRA02R were used as S1 / S2 / S3 / S4 primers and pHKD06 (pHKD01 with ΔcirA :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD05: ATCC 19606 with ΔcirA) lacking the cirA gene was prepared (FIG. 4). The relative position of the target gene, cirA gene, in the genomic DNA of the strain of Aspergillus bacterium var. Marii is shown in Fig. 4A. The result of electrophoresis of genomic DNA of the Asnithobacter baumannii mutant (HKD05) B of Fig.

CIRA01F:

5'-AGTTAGCGAAAATAGGTCGCGTACC-3 '(SEQ ID NO: 20)

CIRA01R:

5'-ACAGGCATCTCTTTCGTTAGTTGCATTCTCCCTAGCCCAAATGTTACTCAAC-3 '(SEQ ID NO: 21)

CIRA02F:

5'-TGCAACTAACGAAAGAGATGCCTG-3 '(SEQ ID NO: 22)

CIRA02R:

5'-GCAACACCTTCTTCACGAGGCAGACAACAGTCCAGTAAAGGCCATCACC-3 '(SEQ ID NO: 23)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-6: menG  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that MENG01F / MENG01R / MENG02F / MENG02R was used as the S1 / S2 / S3 / S4 primer and pHKD07 (pHKD01 with ΔmenG :: nptI; Cmr, Kmr) was used as the homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD06: ATCC 19606 with? MenG) in which the menG gene was deleted was prepared (Fig. 4). The relative position of the target gene, the menG gene, in the genomic DNA of the Acinetobacter baumannii strain is shown in Fig. 4A. The result of electrophoresis of the genomic DNA of the Asnithobacter baumannii mutant (HKD06) B of Fig.

MENG01F:

5'-TGGTCTGTACTTGCTCATGCGT-3 '(SEQ ID NO: 24)

MENG01R:

5'-CGAACATTAATGAGAATGATTTTACTTTAAATTTTCCGATTTCTTTTAAACGTAAG-3 '(SEQ ID NO: 25)

MENG02F:

5'-TAAAAATCATTCTCATTAATGTTCGTATT-3 '(SEQ ID NO: 26)

MENG02R:

5'-GCAACACCTTCTTCACGAGGCAGACGAGTAATCAGGTCCGTAGTCAGTATCC-3 '(SEQ ID NO: 27)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-7: fhuF  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that FHUF01F / FHUF01R / FHUF02F / FHUF02R was used as the S1 / S2 / S3 / S4 primer and pHKD08 (pHKD01 with ΔfhuF :: nptI; Cmr, Kmr) was used as the homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD07: ATCC 19606 with ΔfhuF) in which the fhuF gene was deleted was prepared (FIG. 4). The relative position of the target gene, fhuF gene, in the genomic DNA of the strain of Aspergillus baumannii is shown in Fig. 4A. The result of electrophoresis of the genome DNA of the genus Asnithobacter baumannii mutant (HKD07) B of Fig.

FHUF01F:

5'-CCGATAGGAATTAAAGCTCTTGG-3 '(SEQ ID NO: 28)

FHUF01R:

5'-CTTTAGAAGTATCAATTTGATTCATCGTCAGATATCACTAACAATTTAAGGC-3 '(SEQ ID NO: 29)

FHUF02F:

5'-ATGAATCAAATTGATACTTCTAAAGCACT-3 '(SEQ ID NO: 30)

FHUF02R:

5'-GCAACACTTCTTCACGAGGCAGACAGCAACTTTACGACCTGCCG-3 '(SEQ ID NO: 31)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-8: rhbC1  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that RHBC101F / RHBC101R / RHBC102F / RHBC102R were used as S1 / S2 / S3 / S4 primers and pHKD09 (pHKD01 with ΔrhbC1 :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD08: ATCC 19606 with? RhbC1) lacking the rhbC1 gene was prepared (Fig. 4). At this time, the relative position of the target gene rhbC1 gene in the genome DNA of the strain of Ashinobacterium Vaumani is shown in Fig. 4A. The genomic DNA of the Ashinotobacter baumanni mutant (HKD08) B of Fig.

RHBC101F:

5'-GCCCAACAATCTCTACGCAC-3 '(SEQ ID NO: 32)

RHBC101R:

5'-AAGACATATTCATCGTCAGATATCAAGATAAATCTCACTTCACCTGCAAT-3 '(SEQ ID NO: 33)

RHBC102F:

5'-TGATATCTGACGATGAATATGTCTTTTAA-3 '(SEQ ID NO: 34)

RHBC102R:

5'-GCAACACTTCTTCACGAGGCAGACGACTCAATCCGAACCGTACG-3 '(SEQ ID NO: 35)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-9: rhbC2  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that RHBC201F / RHBC201R / RHBC202F / RHBC202R were used as S1 / S2 / S3 / S4 primers and pHKD10 (pHKD01 with ΔrhbC2 :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD09: ATCC 19606 with ΔrhbC2) in which the rhbC2 gene was deleted (FIG. 4) was prepared. At this time, the relative position of the target gene rhbC2 gene in the genome DNA of the strain of Ashinobacterium var. Mariana is shown in Fig. 4A. The result of electrophoresis of the genome DNA of the Ashinotobacter baumanni mutant (HKD09) B of Fig.

RHBC201F:

5'-CCATGATCATGCTAACCTTAGCAG-3 '(SEQ ID NO: 36)

RHBC201R:

5'-GCTGAAGTGCATTCATAGATAAATCTTTTATTAATCCTATTTCTTTAATTGGTCG-3 '(SEQ ID NO: 37)

RHBC202F:

5'-GATTTATCTATGAATGCACTTCAGCC-3 '(SEQ ID NO: 38)

RHBC202R:

5'-GCAACACCTTCTTCACGAGGCAGACCCAAGAGCTTTAATTCCTATCGG-3 '(SEQ ID NO: 39)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-10: araj  Gene-deficient Ashton Bauber Baumanni Production of mutant strains

ARAJ01F / ARAJ01R / ARAJ02F / ARAJ02R were used as S1 / S2 / S3 / S4 primers and pHKD11 (pHKD01 with ΔaraJ :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD10: ATCC 19606 with ΔaraJ) in which the araJ gene was deleted was prepared (FIG. 4). At this time, the relative position of the araJ gene as a target gene in the genomic DNA of the strain of Ashinobacterium var. Marijuana is shown in Fig. 4A. The result of electrophoresis of the genome DNA of the Ashinotobacter baumannii mutant (HKD10) B of Fig.

ARAJ01F:

5'-GGGTACAACTCCACATTTACCAG-3 '(SEQ ID NO: 40)

ARAJ01R:

5'-TGTAATTGTCCCATTTTTATTAATCATCCTCTGTTTAGCTTATTCAATATGC-3 '(SEQ ID NO: 41)

ARAJ02F:

5'-GATTAATAAAAATGGGACAATTACATCC-3 '(SEQ ID NO: 42)

ARAJ02R:

5'-GCAACACCTTCTTCACGAGGCAGACGCAACCAGTCTGTAATAATTGGC-3 '(SEQ ID NO: 43)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-11: iucD  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

Except that IUCD01F / IUCD01R / IUCD02F / IUCD02R were used as S1 / S2 / S3 / S4 primers and pHKD12 (pHKD01 with ΔiucD :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD11: ATCC 19606 with? IucD) lacking the iucD gene was prepared (Fig. 4). At this time, the relative position of the target gene iucD gene in the genomic DNA of the strain of Ashinobacterium bumani is shown in Fig. 4A. The result of electrophoresis of the genomic DNA of the Ashinotobacter baumannii mutant (HKD11) B of Fig.

IUCD01F:

5'-GATATTGGTTTACGTGGATGGC-3 '(SEQ ID NO: 44)

IUCD01R:

5'-TCCATACACATATCCTCTGTTTAGCTCAAGCATAACTCACATCCTTCTGT-3 '(SEQ ID NO: 45)

IUCD02F:

5'-GCTAAACAGAGGATATGTGTATGGAAC-3 '(SEQ ID NO: 46)

IUCD02R:

5'-GCAACACTTCTTCACGAGGCAGACTCATTAGCGCAAAACCAGTCC-3 '(SEQ ID NO: 47)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  2-12: rhbC3  Gene-deficient Ashton Bauber Baumanni  Production of mutant strains

RHb301F / RHBC301R / RHBC302F / RHBC302R were used as S1 / S2 / S3 / S4 primers and pHKD13 (pHKD01 with ΔrhbC3 :: nptI; Cmr, Kmr) was used as a homologous recombination vector. -1, an Asnithobacter baumannii mutant (HKD12: ATCC 19606 with ΔrhbC3) in which the rhbC3 gene was deleted (FIG. 4) was prepared. At this time, the relative position of the target gene rhbC3 gene in the genomic DNA of the strain of Ashinobacterium Vaumani is shown in Fig. 4A. The result of electrophoresis of the genome DNA of the Ashinotobacter Vaumani mutant (HKD12) B of Fig.

RHBC301F:

5'-ATTGTTTTTTGTTCGGTGTTCG-3 '(SEQ ID NO: 48)

RHBC301R:

5'-GTCCAATTCCAATAAAATCAAGCATCTGACTACATCCTATTCAGAATCTTAAGC-3 '(SEQ ID NO: 49)

RHBC302F:

5'-ATGCTTGATTTTATTGGAATTGGA-3 '(SEQ ID NO: 50)

RHBC302R:

5'-GCAACACCTTCTTCACGAGGCAGACTGCTGCAATAACACAATCAGCC-3 '(SEQ ID NO: 51)

As shown in Fig. 4B, it was confirmed that the genome DNA was reduced in size compared to the wild-type Acinetobacter baumannii strains of the Ashinotobacter baumanni mutant. From these results, it was found that the target gene was deleted in the Asnithobacter baumannii mutant provided in the present invention.

Example  3: Real time PCR  analysis

Real-time PCR analysis was carried out to confirm whether the target gene was expressed in each of the strains of Asmanobacter baumannii prepared in Example 2 above.

Example  3-1: Ashton Bauber Baumanni  Mutation HKD01 ) Real time PCR  analysis

(WT) and an Asnithobacter baumannii mutant (HKD01) in which the basD gene had been deleted were inoculated in an LB medium containing 0.15 mM iron capturing material (2,2-dipyridyl) The culture was continued until the absorbance reached 0.7, and a transcript was obtained from each cultured cell after the completion of the culture. Real-time PCR using the cDNA synthesized from the transcript as a template and S5 / S6 primer was performed to quantitatively analyze the expression level of the target gene (FIG. 3C). At this time, quantitative analysis of the expression level was carried out by measuring the expression level of the target gene and normalizing the measured value to the expression level of the internal control 16S rRNA.

S5 primer (BASD03F):

5'-GACTGACCCAAACACCTCAA-3 '(SEQ ID NO: 52)

S6 primer (BASD03R):

5'-GGAAATGCTTCTTGGGCATAATC-3 '(SEQ ID NO: 53)

As shown in FIG. 3C, the expression level of the basD gene, the target gene, was increased (the expression level was increased 53 times (log ₁₀ 1.72)) in the wild-type Asnithobacter baumanni strain (WT) It was confirmed that the basD gene, the target gene, was not expressed in the Asnithobacter baumannii mutant (HKD01) in which the basD gene was deleted.

From the above results, it was found that the basD gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashinotobacter baumanni mutant strain (HKD01).

Example  3-2: Ashton Bauber Baumanni  Mutation HKD02 ) Real time PCR  analysis

The same method as in Example 3-1 was used, except that HKD02 in which the bauA gene was deleted as the Aspinabacter baumannii mutant and BAUA03F / BAUA03R was used as the S5 / S6 primers, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD02) (Fig. 3C).

BAUA03F:

5'-CACGTGGTGTTAACGTCTCTAC-3 '(SEQ ID NO: 54)

BAUA03R:

5'-CACGAATCATTGCGCCTTTATC-3 '(SEQ ID NO: 55)

As shown in FIG. 3C, the bauA gene, which is a target gene, is expressed at a high level (the expression level is increased 91 times (log ₁₀ 1.95)) in the wild type Ashtonobacter baumanni strain (WT) , and that the target gene was not expressed at all in the Asnithobacter baumanni mutant strain (HKD02) in which the bauA gene was deleted.

From the above results, it was found that the bauA gene, which is an essential gene for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD02).

Example  3-3: Ashton Bauber Baumanni  Mutation HKD03 ) Real time PCR  analysis

The same procedure as in Example 3-1 was carried out except that HKD03 in which the rimL gene was deleted as the Ashinatoottera baumannii mutant and RIML03F / RIML03R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene rimL gene in the Baumani mutant (HKD03) (Fig. 5).

RIML03F:

5'-GCTACTCATTACGTCAGGTTCAG-3 '(SEQ ID NO: 56)

RIML03R:

5'-CCACTGAGGAATAACGTGTGG-3 '(SEQ ID NO: 57)

As shown in FIG. 5, the expression level of the target gene, rimL gene (expression level: 198 times (log ₁₀ 2.30)) was increased in the wild type Ashtonobacter baumanni strain (WT) under iron deficiency condition, whereas the rimL gene (HKD03) showed that the target gene, rimL gene, was not expressed at all.

From the above results, it was found that the rimL gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-4: Ashton Bauber Baumanni  Mutation HKD04 ) Real time PCR  analysis

The same procedure as in Example 3-1 was carried out except that HKD04 in which the piuB gene was deleted as the Ashinatootberger baumannii mutant and PIUB03F / PIUB03R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD04) (Fig. 5).

PIUB03F:

5'-AAAGATGGCGAGTCACAGTAG-3 '(SEQ ID NO: 58)

PIUB03R:

5'-CCCAATAGCTCTCCGGTATTAG-3 '(SEQ ID NO: 59)

As shown in FIG. 5, the expression level of the target gene, piuB gene (expression level 8 times (log ₁₀ 0.89)) was increased in the wild-type Asnithobacter baumanni strain (WT) under iron deficiency conditions, whereas the piuB gene (HKD04) showed that the target gene, piuB gene, was not expressed at all.

From the above results, it was found that the piuB gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-5: Ashton Bauber Baumanni  Mutation HKD05 ) Real time PCR  analysis

The same method as in Example 3-1 was used except that HKD05 in which the cirA gene had been deleted as the Ashinatto bacterium mutant and CIRA03F / CIRA03R was used as the S5 / S6 primers, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD05) (Fig. 5).

CIRA03F:

5'-GTAGTAACTGCGACTCGTACAC-3 '(SEQ ID NO: 60)

CIRA03R:

5'-GCCGTAGCTTGCTGGATAA-3 '(SEQ ID NO: 61)

5, the expression of the target gene, cirA gene (expression level 26 times (log ₁₀ 1.41)) was increased in the wild-type Asnithobacter baumanni strain (WT) under iron deficiency conditions, whereas the cirA gene (HKD05) showed that the target gene, cirA gene, was not expressed at all.

From the above results, it was found that the cirA gene, which is an essential target gene for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-6: Ashton Bauber Baumanni  Mutation HKD06 ) Real time PCR  analysis

The same method as in Example 3-1 was used, except that HKD06 in which the menG gene was deleted as the Ashinabotobacter baumanni mutant was used and MENG03F / MENG03R was used as the S5 / S6 primer in each case, Real-time PCR analysis confirmed the deletion of the target gene in the Baumani mutant (HKD06) (Fig. 5).

MENG03F:

5'-CGTTGTGGTCTTAGGTGCTATT-3 '(SEQ ID NO: 62)

MENG03R:

5'-CCCTTCATTGCGAGTGGTTA-3 '(SEQ ID NO: 63)

As shown in FIG. 5, in the wild type Ashtonobacter baumannii strain (WT) under the condition of iron deficiency, the expression level of the target gene, menG gene was increased to 3 times (log ₁₀ 0.46) (HKD06) showed no expression of the target gene, menG, at all.

From the above results, it was found that the menG gene, which is an essential target gene for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-7: Ashton Bauber Baumanni  Mutation HKD07 ) Real time PCR  analysis

The same procedure as in Example 3-1 was carried out except that HKD07 in which the fhuF gene was deleted as the Ashinatoottera baumannii mutant and FHUF03F / FHUF03R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD07) (Fig. 5).

FHUF03F:

5'-GTGCGTTTATGGAATGGATGG-3 '(SEQ ID NO: 64)

FHUF03R:

5'-AGGGACAATTACGGCATAAGG-3 '(SEQ ID NO: 65)

As shown in FIG. 5, in the wild-type Asnithobacter baumanni strain (WT) under the condition of iron deficiency, the expression level of the target gene, fhuF gene was increased to 10 times (log ₁₀ 1.00) (HKD07) showed that the target gene, fhuF gene, was not expressed at all.

From the above results, it was found that the fhuF gene, which is an essential gene for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-8: Ashton Bauber Baumanni  Mutation HKD08 ) Real time PCR  analysis

The same procedure as in Example 3-1 was carried out except that HKD08 in which the rhbC1 gene was deleted as the Ashinatootberta baumannii mutant was used and RHBC103F / RHBC103R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD08) (Fig. 5).

RHBC103F:

5'-CAAAGCTTGGCACATAGATTCC-3 '(SEQ ID NO: 66)

RHBC103R:

5'-CTTGCTGACTGAGACCTTCTT-3 '(SEQ ID NO: 67)

As shown in FIG. 5, while under conditions of iron deficient in the wild type Acinetobacter baumannii strain (WT) in the target gene of rhbC1 gene expression (expression amount is 104 times (log ₁₀ 2.01) increase) at a high level, rhbC1 gene (HKD08) showed that the target gene, rhbC1 gene, was not expressed at all.

From the above results, it was found that the rhnC1 gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-9: Ashton Bauber Baumanni  Mutation HKD09 ) Real time PCR  analysis

The same procedure as in Example 3-1 was carried out except that HKD09 in which the rhbC2 gene was deleted as the Ashinatto bacterium mutant and RHBC203F / RHBC203R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD09) (Fig. 5).

RHBC203F:

5'-GGCAAAGTCAGGATCGCTATT-3 '(SEQ ID NO: 68)

RHBC203R:

5'-ACGTGAGCTTGATGTGTTAGTT-3 '(SEQ ID NO: 69)

As shown in Fig. 5, in the wild type Ashtonobacter baumanni strain (WT) under the condition of iron deficiency, the expression level of the target gene rhbC2 gene was increased (the expression level was increased 18 times (log ₁₀ 1.25)), whereas the rhbC2 gene (HKD09) showed that the target gene, rhbC2 gene, was not expressed at all.

From the above results, it was found that the rhbC2 gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-10: Ashton Bauber Baumanni  Mutation HKD10 ) Real time PCR  analysis

The same procedure as in Example 3-1 was carried out except that HKD10 in which the araJ gene had been deleted as the Aspinobacter baumannii mutant and ARAJ03F / ARAJ03R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD10) (Fig. 5).

ARAJ03F:

5'-CATGGCTTAGGAATGGGACTT-3 '(SEQ ID NO: 70)

ARAJ03R:

5'-GGCCGCTTGAGTAACTAGATG-3 '(SEQ ID NO: 71)

As shown in FIG. 5, the expression level of the araJ gene (expression level 27 times (log ₁₀ 1.41) increased) in the wild-type Asnithobacter baumanni strain (WT) under iron deficiency conditions, whereas the araJ gene (HKD10) showed that the target gene, araJ gene, was not expressed at all.

From the above results, it was found that in the case of the Ashinotobacter baumanni mutant strain (HKD03), the araJ gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted.

Example  3-11: Ashton Bauber Baumanni  Mutation HKD11 ) Real time PCR  analysis

The same method as in Example 3-1 was carried out except that HKD11 in which the iucD gene was deleted as the Ashinatootberger baumani mutant and IUCD03F / IUCD03R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the baumannii mutant (HKD11) (Fig. 5).

IUCD03F:

5'-TGATGTGGAGGTAGTGTGTAATG-3 '(SEQ ID NO: 72)

IUCD03R:

5'-GCTTTCTGGTAAATGTGGAGTTG-3 '(SEQ ID NO: 73)

As shown in FIG. 5, the expression level of the target gene iucD gene (expression level 30 times (log ₁₀ 1.45)) was increased in the wild-type Asnithobacter baumanni strain (WT) under iron deficiency conditions, whereas the iucD gene (HKD11) showed that the target gene, iucD gene, was not expressed at all in the Asnithobacter baumannii mutant (HKD11).

From the above results, it was found that the iucD gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  3-12: Ashton Bauber Baumanni  Mutation HKD12 ) Real time PCR  analysis

Using the same method as in Example 3-1, except that HKD12 in which the rhbC3 gene had been deleted as the Aspinabata baumannii mutant was used and RHBC303F / RHBC303R was used as the S5 / S6 primer, respectively, Real-time PCR analysis confirmed the deletion of the target gene in the Baumani mutant (HKD12) (Fig. 5).

RHBC303F:

5'-GAGCAGCCAATTTCAGATCAAG-3 '(SEQ ID NO: 74)

RHBC303R:

5'-GAGCTTGCCAAGGATGTAGT-3 '(SEQ ID NO: 75)

As shown in FIG. 5, in the case of the wild type Ashtonobacter baumanni strain (WT) under the condition of iron deficiency, the rhbC3 gene as a target gene was expressed at a high level (the expression level was increased by 102 times (log ₁₀ 2.00)), whereas the rhbC3 gene (HKD12) showed that the target gene, rhbC3 gene, was not expressed at all.

From the above results, it was found that the rhbC3 gene, which is a target gene essential for obtaining iron at the time of iron deficiency, was deleted in the Ashtonobacter baumannii mutant (HKD03).

Example  4: Ashton Bauber From Baumanni  Production of a mutant strain in which two target genes have been deleted using a non-replicating suicide vector

The homologous recombinant vector pHKD06 used in Example 2-5 was introduced into the strain of Asnithobacter baumani (HKD02) in which the bauA gene prepared in Example 2-2 was deleted and cultured in the same manner as in Example 2-1, (HKD13) was obtained in which the cirA gene was further deleted from the Ashinotobacter baumanni strain (HKD02). In order to confirm that the bacterium bauA gene and the cirA gene have been deleted in the wild type Ashtonobacter baumanni strain (ATCC 19606), the Ashinotobacter baumanni strain (HKD13) (HKD13) were electrophoresed (FIG. 6).

6 is a photograph showing the result of electrophoresis of the genomic DNA of the strain Ashinotobacter baumanni (HKD13), which is a mutant strain in which the bauA gene and the cirA gene are simultaneously deleted in the wild-type Asnithobacter baumanni strain (ATCC 19606).

As shown in FIG. 6, it was found that the strain Ashnootobacter baumanni (HKD13) produced was a mutant strain in which the bauA gene and the cirA gene were simultaneously deleted.

<110> Industry-Academic Cooperation Foundation, Dankook University <120> Suicide vector for producing murtant strain of          다 다르그 발진 형 <130> KPA140482-KR <160> 75 <170> Kopatentin 2.0 <210> 1 <211> 5315 <212> DNA <213> Artificial Sequence <220> <223> pHKD01 <400> 1 ccatgtcagc cgttaagtgt tcctgtgtca ctcaaaattg ctttgagagg ctctaagggc 60 ttctcagtgc gttacatccc tggcttgttg tccacaaccg ttaaacctta aaagctttaa 120 aagccttata tattcttttt tttcttataa aacttaaaac cttagaggct atttaagttg 180 ctgatttata ttaattttat tgttcaaaca tgagagctta gtacgtgaaa catgagagct 240 tagtacgtta gccatgagag cttagtacgt tagccatgag ggtttagttc gttaaacatg 300 agagcttagt acgttaaaca tgagagctta gtacgtgaaa catgagagct tagtacgtac 360 tatcaacagg ttgaactgct gatcttcaga tcctctacgc cggacgcatc gtggccggat 420 ccagccgacc aggctttcca cgcccgcgtg ccgctccatg tcgttcgcgc ggttctcgga 480 aacgcgctgc cgcgtttcgt gattgtcacg ctcaagcccg tagtcccgtt cgagcgtcgc 540 gcagaggtca gcgagggcgc ggtaggcccg atacggctca tggatggtgt ttcgggtcgg 600 gtgaatcttg ttgatggcga tatggatgtg caggttgtcg gtgtcgtgat gcacggcact 660 gcgcgctga tgctcggcga agccaagccc agcgcagatg cggtcctcaa tcgcgcgcaa 720 cgtctccgcg tcgggcttct ctcccgcgcg gaagctaacc agcaggtgat aggtcttgtc 780 ggcctcggaa cgggtgttgc cgtgctgggt cgccatcacc tcggccatga cagcgggcag 840 gt; gtcggtgatg tacttcacca gctccgcgaa gtcgctcttc ttgatggagc gcatggggac 960 gtgcttggca atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt catggctctg 1020 ccctcgggcg gaccacgccc atcatgacct tgccaagctc gtcctgcttc tcttcgatct 1080 tcgccagcag ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga 1140 gccagagttt cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc 1200 ggacgtgctc atagtccacg acgcccgtga ttttgtagcc ctggccgacg gccagcaggt 1260 aggccgacag gctcatgccg gccgccgccg ccttttcctc aatcgctctt cgttcgtctg 1320 gaaggcagta caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca 1380 tccgcttgcc ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc 1440 cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg 1500 ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac 1560 gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg 1620 ctataatgac cccgaagcag ggttatgcag cggaaaagcg ctgcttccct gctgttttgt 1680 ggaatatcta ccgactggaa acaggcaaat gcaggaaatt actgaactga ggggacaggc 1740 gagagacgat gccaaagagc tacaccgacg agctggccga gtgggttgaa tcccgcgcgg 1800 ccaagaagcg ccggcgtgat gaggctgcgg ttgcgttcct ggcggtgagg gcggatgtcg 1860 aggcggcgtt agcgtccggc tatgcgctcg tcaccatttg ggagcacatg cgggaaacgg 1920 ggaaggtcaa gttctcctac gagacgttcc gctcgcacgc caggcggcac atcaaggcca 1980 agcccgccga tgtgcccgca ccgcaggcca aggctgcgga acccgcgccg gcacccaaga 2040 cgccggagcc acggcggccg aagcaggggg gcaaggctga aaagccggcc cccgctgcgg 2100 ccccgaccgg cttcaccttc aacccaacac cggacaaaaa ggatcctcta cgccggacgc 2160 atcgtggccg gcatcaccgg cgccacaagt gcggttgctg gcgcctatat cgccgacatc 2220 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt 2280 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgct gcctcgcgcg 2340 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 2400 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 2460 gtgtcggggc gcagccatga cccgggaatt acgccccgcc ctgccactca tcgcagtact 2520 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct 2580 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa 2640 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc 2700 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt 2760 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt 2820 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag 2880 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat 2940 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt 3000 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt 3060 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg 3120 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgcc ctatgggatt 3180 cacctttatg ttgataagaa ataaaagaaa atgccaatag gatatcggca ttttcttttg 3240 cgtttttatt tgttaactgt taattgtcct tgttcaagga tgctgtcttt gacaacagat 3300 gttttcttgc ctttgatgtt cagcaggaag cttggcgcaa acgttgattg tttgtctgcg 3360 tagaatcctc tgtttgtcat atagcttgta atcacgacat tgtttccttt cgcttgaggt 3420 acagcgaagt gtgagtaagt aaaggttaca tcgttaggat caagatccat ttttaacaca 3480 aggccagttt tgttcagcgg cttgtatggg ccagttaaag aattagaaac ataaccaagc 3540 atgtaaatat cgttagacgt aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg 3600 aacaggtacc atttgccgtt cattttaaag acgttcgcgc gttcaatttc atctgttact 3660 gtgttagatg caatcagcgg tttcatcact tttttcagtg tgtaatcatc gtttagctca 3720 atcataccga gagcgccgtt tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt 3780 ttttgacttt cttgacggaa gaatgatgtg cttttgccat agtatgcttt gttaaataaa 3840 gattcttcgc cttggtagcc atcttcagtt ccagtgtttg cttcaaatac taagtatttg 3900 tggcctttat cttctacgta gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag 3960 ttgccttcat cgatgaactg ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt 4020 gatttataat cctctacacc gttgatgttc aaagagctgt ctgatgctga tacgttaact 4080 tgtgcagttg tcagtgtttg tttgccgtaa tgtttaccgg agaaatcagt gtagaataaa 4140 cggatttttc cgtcagatgt aaatgtggct gaacctgacc attcttgtgt ttggtctttt 4200 aggatagaat catttgcatc gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc 4260 cagctgtcaa tagaagtttc gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc 4320 gcatttttag gatctccggc taatgcaaag acgatgtggt agccgtgata gtttgcgaca 4380 gtgccgtcag cgttttgtaa tggccagctg tcccaaacgt ccaggccttt tgcagaagag 4440 atatttttaa ttgtggacga atcgaattca ggaacttgat atttttcatt tttttgctgt 4500 tcagggattt gcagcatatc atggcgtgta atatgggaaa tgccgtatgt ttccttatat 4560 ggcttttggt tcgtttcttt cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta 4620 gtaaaggtta atactgttgc ttgttttgca aactttttga tgttcatcgt tcatgtctcc 4680 ttttttatgt actgtgttag cggtctgctt cttccagccc tcctgtttga agatggcaag 4740 ttagttacgc acaataaaaa aagacctaaa atatgtaagg ggtgacgcca aagtatacac 4800 tttgcccttt acacatttta ggtcttgcct gctttatcag taacaaaccc gcgcgattta 4860 cttttcgacc tcattctatt agactctcgt ttggattgca actggtctat tttcctcttt 4920 tgtttgatag aaaatcataa aaggatttgc agactacggg cctaaagaac taaaaaatct 4980 atctgtttct tttcattctc tgtatttttt atagtttctg ttgcatgggc ataaagttgc 5040 ctttttaatc acaattcaga aaatatcata atatctcatt tcactaaata atagtgaacg 5100 gcaggtatat gtgatgggtt aaaaaggatc gatcctctag agtcgacctg cagttttctg 5160 cagtgcgcag cggccgcaga tctcctcgaa gactgcaggt cgacggatcc caagcttctt 5220 ctagaggtac cgcatgcgat atcgagctct cccgggaatt ccacaaattg ttatccgctc 5280 acaattccac atgtggaatt ccacatgtgg aattc 5315 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gaagcaattg agcggttcag g 21 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaatgatgaa agttcaaaat acgaattgtt aatcatttcc aattttgctg t 51 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttcgtatttt gaactttcat cattttgg 28 <210> 5 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcaacacctt cttcacgagg cagactgtgc aaacaggtta aacgcag 47 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtctgcctcg tgaagaaggt g 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gatccgtcga cctgcagg 18 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gtcgatttgg agaaaacgta agc 23 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cataaatttt cactattttg ctatgataaa aaaacccgca atcgc 45 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 catagcaaaa tagtgaaaat ttatgctc 28 <210> 11 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gcaacacctt cttcacgagg cagacatcat agcggcagct gtcac 45 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 actttgcttc tatctggatg cg 22 <210> 13 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cttgcaggtc tacccatacc agaaacggtc cttgctatga cataacc 47 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tttctggtat gggtagacct gc 22 <210> 15 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcaacacctt cttcacgagg cagacgggat aaattgatca agatcgg 47 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggcttaaatc tgggattgac tgg 23 <210> 17 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cccaaaataa tgatgcaaag ctcatttaag ccactcccca tttagcta 48 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 atgagctttg catcattatt ttgg 24 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcaacacctt cttcacgagg cagacgcaca tgtcttctac tacagccg 48 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 agttagcgaa aataggtcgc gtacc 25 <210> 21 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 acaggcatct ctttcgttag ttgcattctc cctagcccaa atgttactca ac 52 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tgcaactaac gaaagagatg cctg 24 <210> 23 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gcaacacctt cttcacgagg cagacaacag tccagtaaag gccatcacc 49 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 tggtctgtac ttgctcatgc gt 22 <210> 25 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 cgaacattaa tgagaatgat ttttacttta aattttccga tttcttttaa acgtaag 57 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 taaaaatcat tctcattaat gttcgtatt 29 <210> 27 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gcaacacctt cttcacgagg cagacgagta atcaggtccg tagtcagtat cc 52 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ccgataggaa ttaaagctct tgg 23 <210> 29 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ctttagaagt atcaatttga ttcatcgtca gatatcacta acaatttaag gc 52 <210> 30 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 atgaatcaaa ttgatacttc taaagcact 29 <210> 31 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 gcaacacctt cttcacgagg cagacagcaa ctttacgacc tgccg 45 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gcccaacaat ctctacgcac 20 <210> 33 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 aagacatatt catcgtcaga tatcaagata aatctcactt cacctgcaat 50 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tgatatctga cgatgaatat gtcttttaa 29 <210> 35 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gcaacacctt cttcacgagg cagacgactc aatccgaacc gtacg 45 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 ccatgatcat gctaacctta gcag 24 <210> 37 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 gctgaagtgc attcatagat aaatctttta ttaatcctat ttctttaatt ggtcg 55 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 gatttatcta tgaatgcact tcagcc 26 <210> 39 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 gcaacacctt cttcacgagg cagacccaag agctttaatt cctatcgg 48 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 gggtacaact ccacatttac cag 23 <210> 41 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 tgtaattgtc ccatttttat taatcatcct ctgtttagct tattcaatat gc 52 <210> 42 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 gattaataaa aatgggacaa ttacatcc 28 <210> 43 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 gcaacacctt cttcacgagg cagacgcaac cagtctgtaa taattggc 48 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 gatattggtt tacgtggatg gc 22 <210> 45 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 tccatacaca tatcctctgt ttagctcaag cataactcac atccttctgt 50 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 gctaaacaga ggatatgtgt atggaac 27 <210> 47 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 gcaacacctt cttcacgagg cagactcatt agcgcaaaac cagtcc 46 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 attgtttttt gttcggtgtt cg 22 <210> 49 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 gtccaattcc aataaaatca agcatctgac tacatcctat tcagaatctt aagc 54 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 atgcttgatt ttattggaat tgga 24 <210> 51 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 gcaacacctt cttcacgagg cagactgctg caataacaca atcagcc 47 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 gactgaccca aacacctcaa 20 <210> 53 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 ggaaatgctt cttgggcata atc 23 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 cacgtggtgt taacgtctct ac 22 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 cacgaatcat tgcgccttta tc 22 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 gctactcatt acgtcaggtt cag 23 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 57 ccactgagga ataacgtgtg g 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 aaagatggcg agtcacagta g 21 <210> 59 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 cccaatagct ctccggtatt ag 22 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 gtagtaactg cgactcgtac ac 22 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 gccgtagctt gctggataa 19 <210> 62 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 cgttgtggtc ttaggtgcta tt 22 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 cccttcattg cgagtggtta 20 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 gtgcgtttat ggaatggatg g 21 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 agggacaatt acggcataag g 21 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 caaagcttgg cacatagatt cc 22 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 cttgctgact gagaccttct t 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 ggcaaagtca ggatcgctat t 21 <210> 69 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 acgtgagctt gatgtgttag tt 22 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 catggcttag gaatgggact t 21 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 ggccgcttga gtaactagat g 21 <210> 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 tgatgtggag gtagtgtgta atg 23 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 gctttctggt aaatgtggag ttg 23 <210> 74 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 gagcagccaa tttcagatca ag 22 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 gagcttgcca aggatgtagt 20

Claims (10)

A protein encoding a protein exhibiting resistance to an antibiotic which comprises a polyclonal region including R6K ori, a mob RP4 gene, a cat gene, a sacB gene, and a restriction enzyme cleavage site cleaved at a blunt end, A suicide vector for producing a mutant strain of a multidrug-resistant strain.
The method according to claim 1,
Wherein the suicide vector exhibits a property of not replicating in a multidrug-resistant strain.
The method according to claim 1,
Wherein the suicide vector has the cleavage map shown in FIG. 1B.
The method according to claim 1,
1. A suicide vector comprising the polynucleotide of SEQ ID NO: 1.
The method according to claim 1,
Wherein a DNA insert fragment capable of mutating the target gene contained in the genomic DNA of the multidrug resistant strain is introduced into the polyclonal region.
6. The method of claim 5,
Wherein the DNA-inserted fragment comprises a base sequence adjacent to an upstream of a target gene located in the genomic DNA of the multidrug-resistant strain, a base sequence adjacent to the downstream of the target gene, and a DNA encoding a protein exhibiting resistance to an antibiotic A suicide vector that sequentially contains the nucleotide sequence of the gene.
The method according to claim 1,
Wherein said multidrug-resistant strain is an Acinetobacter baumannii strain.
A kit for producing a mutant strain of a multidrug-resistant strain comprising the suicide vector according to any one of claims 1 to 7.
9. The method of claim 8,
A DNA insert fragment capable of mutating a target gene contained in the genomic DNA of the multidrug-resistant strain, and a restriction enzyme for cleaving the polyclonal region of the suicide vector into a blunt end.
10. The method of claim 9,
Wherein the restriction enzyme is an enzyme selected from the group consisting of PstI, FspI, NotI, BglII, and combinations thereof.

Images