CN113817731A - Targeted reduction drug resistance gene blaTEMgRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof - Google Patents
Targeted reduction drug resistance gene blaTEMgRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof Download PDFInfo
- Publication number
- CN113817731A CN113817731A CN202110877460.2A CN202110877460A CN113817731A CN 113817731 A CN113817731 A CN 113817731A CN 202110877460 A CN202110877460 A CN 202110877460A CN 113817731 A CN113817731 A CN 113817731A
- Authority
- CN
- China
- Prior art keywords
- tem
- grna
- pcas9
- mob
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 75
- 239000013598 vector Substances 0.000 title claims abstract description 74
- 239000003814 drug Substances 0.000 title claims abstract description 64
- 229940079593 drug Drugs 0.000 title claims abstract description 64
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 59
- 230000009467 reduction Effects 0.000 title claims abstract description 18
- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 16
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 26
- 238000012546 transfer Methods 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 12
- 230000003115 biocidal effect Effects 0.000 claims abstract description 10
- 241000894006 Bacteria Species 0.000 claims description 18
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 12
- 238000012216 screening Methods 0.000 claims description 12
- 238000000137 annealing Methods 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 8
- 108091033409 CRISPR Proteins 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 6
- 210000000349 chromosome Anatomy 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 108010042407 Endonucleases Proteins 0.000 claims 1
- 102000004533 Endonucleases Human genes 0.000 claims 1
- 102100035460 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000003209 gene knockout Methods 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000008685 targeting Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 14
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 6
- 239000008223 sterile water Substances 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 102100038517 Pyridoxal kinase Human genes 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Targeted reduction drug resistance gene blaTEMThe invention relates to the technical field of biology, in particular to a targeting bla, a gRNA of a drug-resistant plasmid thereof, a transferable knockout vector and application thereofTEMgRNA of drug-resistant gene and drug-resistant plasmid thereof, gene knockout vector capable of horizontal transfer and gene knockout vector in blaTEMApplication in drug-resistant plasmid reduction. The gRNA provided by the inventionTEM‑1、gRNATEM-2 and knockout vector pCas9-Mob-gRNA thereofTEM-1 and pCas9-Mob-gRNATEM-2 can effectively reduce the drug resistance gene blaTEMAnd carry blaTEMThe multi-drug resistant plasmid of (1). Meanwhile, the knockout vector pCas9-Mob-gRNA provided by the invention is utilizedTEM-1 and pCas9-Mob-gRNATEM-2 preparation method is easier to obtain targeted antibiotic drug-resistant gene blaTEMThe knockout vector also provides an effective method and a foundation for knockout and reduction research of other drug-resistant genes.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to targeted blaTEMgRNA of drug-resistant gene and drug-resistant plasmid thereof, gene knockout vector capable of horizontal transfer and gene knockout vector in blaTEMApplication in drug-resistant plasmid reduction.
Background
In recent years, with the heavy use of antibiotics, more and more antibiotics and their metabolites are discharged into sewage treatment systems and even into natural environments, so that the selective pressure of microorganisms in these environments is increasing. These residual antibiotics allow the bacteria to continue to adapt and evolve, and thus acquire antibiotic resistance. Antibiotic resistance is often determined by drug resistance genes, which can be located on chromosomes and plasmids and spread by means of gene vertical transfer and horizontal transfer. At present, the drug resistance gene is regarded as a novel environmental pollutant. Drug-resistant gene blaTEMIt is ubiquitous in the environment, and its coded broad-spectrum beta-lactamase can hydrolyze beta-lactam antibiotics such as ampicillin and endow bacteria with antibiotic resistance. At present, the drug resistance gene blaTEMIt was widely detected on multiple resistant plasmids that can often be transmitted between bacteria of different species by conjugative transfer. When pathogenic bacteria obtain the multi-drug resistant plasmids, the pathogenic bacteria are difficult to kill by corresponding antibiotics, and potential health risks are generated to human beings and animals.
Currently, existing techniques for attenuating antibiotic resistance genes are often accomplished by altering bacterial flora structures or by killing bacteria directly, e.g., by altering loopsEnvironmental conditions change the structure of the bacterial community, thereby influencing the transmission of the multi-drug resistant plasmids, or directly inactivate the bacteria through sterilization means such as disinfection and the like, so that donor bacteria are difficult to transmit the multi-drug resistant plasmids to other bacteria. However, these approaches do not essentially disrupt the multidrug resistance plasmids, which carry the resistance gene bla under the appropriate conditionsTEMThe multiple drug-resistant plasmids can still enter other bacteria through modes such as transformation and the like, and the spread of drug-resistant genes is continued. Therefore, the reduction method of directly knocking out the multi-drug resistant plasmids by the CRISPR/Cas system in a targeted way can fundamentally overcome the problem. Meanwhile, the knockout vector is delivered by utilizing the horizontal transfer of the plasmid, so that the knockout process can be more sustainable, and the migration and the propagation of the multi-drug resistant plasmid can be better controlled.
Disclosure of Invention
To solve the problems of the prior art, the present invention provides targeted bla reductionTEMA gRNA of a drug-resistant gene and a drug-resistant plasmid thereof, a transferable knockout vector and application thereof, and specifically comprises a targeted drug-resistant gene blaTEMThe gRNA, the knockout vector capable of being horizontally transferred, the vector construction method and the application of the gRNA in carrying blaTEMThe application of multiple drug-resistant plasmid reduction can be used for controlling the migration and transmission of drug-resistant genes in the fields of environment and the like.
The realization process of the invention is as follows:
in a first aspect, the present invention provides a targeted knockout of a drug resistance gene blaTEMAnd carry blaTEMThe gRNA sequence of the multiple drug-resistant plasmid is shown as follows:
gRNATEM-1：5’-ATCGAACTGGATCTCAACAG-3’
gRNATEM-2：5’-ACAATTAATAGACTGGATGG-3’
in a second aspect, the invention constructs a gene knockout vector pCas9-Mob capable of horizontally transferring, wherein the vector comprises a knockout protein gene Cas9, a gRNA expression sequence and a horizontally transferring site gene, and a vector map is shown in figure 1.
In a third aspect, the present invention provides a targeted knockout of a drug resistance gene blaTEMAnd carry blaTEMMultiple toleranceTransferable knockout vector pCas9-Mob-gRNA of drug plasmidTEM-1 and pCas9-Mob-gRNATEM-2, said vector map is shown in figures 2 and 3, respectively.
The knockout vector pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 is characterized in that the targeted bla knockout can be achieved either by horizontal transfer using bacterial-to-bacterial conjugation using specific E.coli as donor or by transformation into recipient bacteriaTEMThe corresponding site.
In a fourth aspect, the present invention provides the pCas9-Mob-gRNA described aboveTEM-1 and pCas9-Mob-gRNATEM-2 a method of constructing a knock-out vector comprising the steps of:
(1) separately designing gRNATEM-1 and gRNATEM-2, the sequence of the annealing primer is as follows:
gRNATEM-1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'
gRNATEM-1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
gRNATEM-2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'
gRNATEM-2 reverse primer: 5'-AAAACCCATCCAGTCTATTAATTGT-3'
(2) Phosphorylating the forward primer and the reverse primer, annealing respectively, purifying and recovering to obtain the targeted drug-resistant gene blaTEMAnd carry blaTEMThe annealing system of the gRNA double strand of the multiple drug-resistant plasmid is as follows:
in 50ul reaction system, 5ul T4 PNK buffer, 5ul 10mM ATP, 1ul T4 PNK enzyme, 1ul each of 100uM forward and reverse primers were added, and finally, sterile water was added to make up to 50 ul. After 30min of reaction at 37 ℃, the temperature is increased to 95 ℃ for 10min, and then the temperature is slowly reduced to the room temperature at the speed of 1 ℃/min.
(3) The pCas9-Mob vector described above was linearized by restriction endonuclease cleavage.
(4) The linearized pCas9-Mob vector and gRNA after enzyme digestionTEM-T1、gRNATEMConnecting the-T2 annealed double chains with T4 ligase to obtain the targeted drug-resistant gene blaTEMAnd carry blaTEMTransferable knockout vector pCas9-Mob-gRNA of multiple drug-resistant plasmidsTEM-1 and pCas9-Mob-gRNATEM-2. The linking system is as follows:
linearized pCas9-Mob fragment 0.05pmol, gRNATEM-T1Or gRNATEM-T20.15pmol of annealed duplex, 1ul of T4 DNA ligase, 10ul of ligation buffer, was made up to 20ul with sterile water. The reaction conditions were 25 ℃ incubation for 30min, after completion of the reaction, on ice. And finally, converting the reaction solution into competent cells for screening and amplification.
In a fifth aspect, the method for constructing the pCas9-Mob plasmid comprises the following steps:
(1) and (3) amplifying a replicon OriV, a screening marker gene Chl and a knockout protein gene Cas9 sequence by using the pCas9 plasmid as a template and using a high-fidelity PCR (polymerase chain reaction) technology, and obtaining OriV-Chl-Cas9 after gel purification.
(2) And (3) taking the plasmid carrying the transfer site as a template, amplifying the transfer site of the plasmid by using a high-fidelity PCR technology, and obtaining OriT after gel purification.
(3) The purified oriV-Chl-Cas9 and OriT fragment were ligated using seamless cloning techniques.
(4) The ligation product was transformed into a specific E.coli having integrated on its chromosome the relevant gene required for the horizontal transfer of the pCas9-Mob plasmid.
(5) Screening positive clones by using a screening marker Chl, carrying out amplification culture on bacteria, and extracting a plasmid, wherein the plasmid is the knockout vector pCas 9-Mob.
In a sixth aspect, the pCas9-Mob-gRNA described above is usedTEM-1 and pCas9-Mob-gRNATEMAnd (3) transforming the knockout vector into competent cells of a specific strain to obtain an engineering strain carrying the knockout vector, wherein the strain is escherichia coli.
In the seventh aspect, the pCas9-Mob-gRNA is usedTEM-1 and pCas9-Mob-gRNATEM-2 in the drug resistance gene blaTEMAnd carry blaTEMThe application of the drug-resistant plasmid reduction aspect also belongs to the protection scope of the invention.
Eighth aspect, comprising the knockout vector pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEMMicroorganism of-2 in drug-resistant Gene blaTEMAnd carry blaTEMThe application of the drug-resistant plasmid reduction aspect also belongs to the protection scope of the invention.
The beneficial effects of the invention at least comprise:
(1) the invention provides a targeted drug-resistant gene bla through sequence analysis and experimental verificationTEMAnd carry blaTEMA gRNA of a multi-drug resistant plasmid, the gRNA being capable of directing Cas9 protein to knock out a corresponding site.
(2) The invention provides pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 the transferable knockout vector can enter a recipient bacterium in a transformation mode, can also take specific escherichia coli as a donor, can generate horizontal transfer by utilizing the conjugation between bacteria, and can realize a drug-resistant gene bla in a diversified wayTEMAnd carry blaTEMKnockout of multiple drug-resistant plasmids, a drug-resistant gene bla in the fields of environment and the likeTEMAnd reduction of multiple drug-resistant plasmids provides a technical reference.
(3) The invention also provides pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEMThe construction method of the-2 knockout vector provides effective reference and basis for knockout and reduction research of other drug-resistant genes.
In conclusion, the gRNA provided by the inventionTEM-1、gRNATEM-2 and knockout vector pCas9-Mob-gRNA thereofTEM-1 and pCas9-Mob-gRNATEM-2 can effectively reduce the drug resistance gene blaTEMAnd carry blaTEMThe multi-drug resistant plasmid of (1). Meanwhile, the knockout vector pCas9-Mob-gRNA provided by the invention is utilizedTEM-1 and pCas9-Mob-gRNATEM-2 preparation method is easier to obtain targeted antibiotic drug-resistant gene blaTEMThe knockout vector also provides an effective method and a foundation for knockout and reduction research of other drug-resistant genes.
Drawings
FIG. 1 is a vector map of knock-out vector pCas 9-Mob.
FIG. 2 shows a knock-out vector pCas9-Mob-gRNA according to the present inventionTEM-1 vector map.
FIG. 3 shows a knock-out vector pCas9-Mob-gRNA according to the present inventionTEM-2 vector map.
FIG. 4 shows pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 electrophoretogram of the knock-out vector,
wherein, A: pCas9-Mob-gRNATEM-1 a plasmid vector; b: pCas9-Mob-gRNATEM-2 a plasmid vector; c: the pCas9-Mob vector was linearized.
FIG. 5 shows blaTEMAn electrophoresis chart of a gene in-vitro cutting experiment,
wherein, A: blaTEMA gene; b: sgRNATEM-1 cleavage of blaTEMA gene; c: sgRNATEM-2 cleavage of blaTEMGene injection: the cut fragments are shown in the dotted line frame, and the residual part of the original fragment may be incomplete due to more initial fragments, short cutting time and the like.
FIG. 6 is a fluorescent characterization of the colonies of the adapters under a fluorescent microscope.
Wherein A: NK5449/blaTEM-mcherry colonies; b is a zygospore colony of the control plasmid pCas 9-Mob; c is pCas9-Mob-gRNA TEM1, a zygospore colony; d is pCas9-Mob-gRNA TEM2, a zygospore colony;
FIG. 7 shows pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 transformant colony PCR gel electrophoresis.
Detailed Description
Example 1 construction and validation of a Gene knockout vector pCas9-Mob for horizontal transfer
(1) The plasmid pCas9 is used as a template, a specific primer is designed, a replicon, a screening marker gene and a knockout protein gene Cas9 sequence are amplified by utilizing a high-fidelity PCR technology, and OriV-Chl-Cas9 is obtained after gel purification.
(2) Plasmid carrying transfer sites is used as a template, specific primers are designed, the transfer sites of the plasmid are amplified by utilizing a high-fidelity PCR technology, and OriT is obtained after gel purification.
(3) The purified oriV-Chl-Cas9 and OriT fragments were ligated according to protocol using Novoprotein seamless cloning reagent.
(4) By using CaCl2The method is used for preparing competence, and the ligation product is transformed into the competence of specific Escherichia coli, and related genes required by the horizontal transfer of the pCas9-Mob plasmid are integrated on the chromosome of the Escherichia coli.
(5) Screening out positive clone by using chloramphenicol screening marker, extracting plasmid after bacteria amplification culture, and storing strain.
(6) The pCas9-Mob is sequenced, and the sequence is correct after the sequence is verified, which indicates that the vector is successfully constructed.
The PCR reaction referred to in example 1 employed the PrimeSTAR Max Hi polymerase from Takara, as follows: PrimeSTAR Max Premix 25ul, upstream and downstream primers 1ul, template 1ul, ddH2O22 ul. The PCR reaction procedure was as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s, extension temperature at 72 ℃, speed at 10s/kb, cycle number 30.
The seamless cloning reaction system used in example 1 was as follows: NovoRec plus recombinase 1ul, 5 Xbuffer 4ul, long fragment 0.03pmol, short fragment 0.05pmol, with ddH2O the reaction system was replenished to 20ul and reacted at 50 ℃ for 25 minutes.
Example 2 construction of Targeted drug resistance Gene blaTEMAnd carry blaTEMTransferable knockout vectors for multiple drug-resistant plasmids
(1) Downloading drug-resistant gene bla from NCBI databaseTEMSearching a PAM locus, and screening gRNAs with indexes such as GC ratio, specificity and the like meeting requirements from gRNAs adjacent to the PAM locus, wherein 20-25bp is generally suitable. Preferred grnas for this case are:
gRNATEM-1：5’-ATCGAACTGGATCTCAACAG-3’
gRNATEM-2：5’-ACAATTAATAGACTGGATGG-3’
(2) separately designing gRNATEM-1 and gRNATEMThe sequence of the annealing primer related to the-2 is as follows:
gRNATEM-1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'
gRNATEM-1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
gRNATEM-2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'
gRNATEM-2 reverse primer: 5'-AAAACCCATCCAGTCTATTAATTGT-3'
(3) Phosphorylating the forward primer and the reverse primer, annealing respectively, purifying and recovering to obtain the targeted drug-resistant gene blaTEMAnd a gRNA double strand of a drug-resistant plasmid thereof, each being a gRNATEM-T1 and gRNATEM-T2, annealing system as follows:
in 50ul reaction system, 5ul T4 PNK buffer, 5ul 10mM ATP, 1ul T4 PNK enzyme, 1ul each of 100uM forward and reverse primers were added, and finally, sterile water was added to make up to 50 ul. After 30min of reaction at 37 ℃, the temperature is increased to 95 ℃ for 10min, and then the temperature is slowly reduced to the room temperature at the speed of 1 ℃/min.
(4) The pCas9-Mob vector after restriction endonuclease cleavage referred to in example 1 was recovered from the gel.
(5) The enzyme-cleaved pCas9-Mob vector and gRNA are usedTEM-T1、gRNATEMConnecting T2 with T4 ligase to obtain target drug-resistant gene blaTEMAnd carry blaTEMKnockout vector pCas9-Mob-gRNA of multiple drug-resistant plasmidsTEM-1 and pCas9-Mob-gRNATEM-2。
The linking system is as follows:
linearized pCas9-Mob fragment 0.05pmol, gRNATEM-T1 or gRNATEMT2 annealed double strand 0.15pmol, T4 DNA ligase 1ul, ligation buffer 10ul, made up to 20ul with sterile water. The reaction conditions were 25 ℃ incubation for 30min, after completion of the reaction, on ice. Finally, the reaction solution is transformed into competent cells.
(6) Agarose electrophoresis was used to knock out vector pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNA TEM2, the glue pattern is shown in figure 4, and the result shows that the size is in accordance with the expectation. And (3) sequencing the knockout vector respectively, and determining that the sequence is correct after the sequence is verified, thereby indicating that the vector is successfully constructed.
Example 3 validation of gRNA Using in vitro Targeted cleavage experimentsTEM-1 and gRNATEM-2 pairs of blasTEMEffectiveness of
(1) Design and Synthesis of Forward primer gRNATEM-1-F and gRNATEM-2-F, the sequence is as follows:
gRNATEM-1-F：
5’-TTAATACGACTCACTATAGGATCGAACTGGATCTCAACAGGTTTTAGAGCTAGAAATAG-3’
gRNATEM-2-F：
5’-TTAATACGACTCACTATAGGACAATTAATAGACTGGATGGGTTTTAGAGCTAGAAATAG-3’
(2) adding 10ul of 2 XsgRNA reaction buffer solution into a 20ul system, 2ul of the forward primer, 2ul of enzyme mixed solution and 6ul of sterile water, and carrying out in-vitro transcription for 1h to obtain sgRNATEM-1 and sgRNATEM-2。
(3) Taking appropriate amount of purified blaTEMAdding 2ul reaction buffer solution, 1ul Cas9 enzyme mixture and 1ul sgRNATEMSupplementing the solution to 20ul with sterile water, reacting at 37 ℃ for 1h, and reacting at 70 ℃ for 10 min.
(4) Mixing the above blaTEMGene fragment, sgRNATEM-1 and sgRNATEM-2 post-cleavage blaTEMThe gene fragment was subjected to electrophoresis using 1% agarose gel, and the results are shown in FIG. 5. The results show that gRNATEM-1 and gRNATEM-2 pairs of blasTEMHas targeting cutting effect.
Example 4 verification of the transferable knock-out vector pCas9-Mob-gRNA by means of fluorescence signalsTEM-1 and pCas9-Mob-gRNATEMKnockout Effect of-2
(1) Will carry blaTEMPlasmid bla of Gene and Red fluorescent protein GeneTEMColi NK5449, which encodes Rif and Nal resistance on the chromosome, and positive clones (NK 5449/bla)TEM-mcherry)。
(2) Carrying a control vector pCas9-Mob and a knockout vector pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEME.coli carrying bla of-2TEMAnd (3) inoculating the activated recipient strains of the-mcherry plasmid into LB liquid culture media with corresponding resistance respectively after streak activation, and placing the activated recipient strains in a constant-temperature shaking incubator for shaking culture at 37 ℃ for 16 hours at the speed of 200 rmp.
(3) And (3) respectively centrifuging the bacteria liquid after the amplification culture in the step (2), discarding the supernatant, adding a proper amount of PBS (phosphate buffer solution) into a bacteria shaking tube to resuspend the bacteria liquid at the bottom, centrifuging again, discarding the supernatant again, and repeating for 3 times. Finally, the OD600 values of the bacterial liquid were all adjusted to 0.7 (+ -0.05) with PBS buffer.
(4) Experimental groups: 2.5ml of the resuspended carried bla in PBS were added to a 10ml centrifuge tubeTEMRecipient bacteria of the mcerry plasmid with 2.5ml PBS resuspended containing pCas9-Mob-gRNATEME.coli containing a-1 knock-out vector or a vector containing pCas9-Mob-gRNATEM-2 vector knockout E.coli. Control group: 2.5ml of the resuspended carried bla in PBS were added to a 10ml centrifuge tubeTEMRecipient bacteria of the mcerry plasmid were treated with 2.5ml of E.coli containing pCas9-Mob vector resuspended in PBS.
(5) After culturing for 16h at 37 ℃, 100ul of each bacterial liquid of the experimental group and the control group in the step (4) is taken and coated on three resistant LB solid culture media of Nal, Rif and Chl, and then the zygospores are screened.
(6) Observing the colonies on LB plate using a fluorescent microscope, if blaTEMWhen the gene is knocked out, the red fluorescent protein gene on the same plasmid disappears, and the bacterial colony does not fluoresce; otherwise, fluorescence is emitted.
(7) The results are shown in FIG. 6, in which the Zygomycetes of pCas9-Mob fluoresced red, and pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEMNo significant fluorescence was observed in the Zygosaccharomyces-2 colonies, indicating that the horizontally transferable vector pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 efficient knock-out of a carrier blaTEMA drug-resistant plasmid of the gene.
Example 5 confirmation of pCas9-Mob-gRNA by colony PCRTEM-1 and pCas9-Mob-gRNATEM-2 knock-out Effect on multiple drug-resistant plasmids
(1) Will carry blaTEMThe multiple drug-resistant plasmid RP4 is transferred into Escherichia coli NK5449, the chromosome of the strain codes Rif and Nal resistance, positive clone (NK5449/RP4) is selected, and competent cells are prepared after LB amplification.
(2) Equal amounts of pCas9-Mob, pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 carrierThe cells were transformed into 100ul of the above competent cells, respectively, left to stand for 30min, then subjected to water bath at 42 ℃ for 60 sec, and then transferred to ice to be cooled for 5 min. Adding 1ml LB liquid culture medium into the cooled competent cells, mixing uniformly, and then putting the mixture in a shaker at 37 ℃ and 200rpm for recovery for 60 minutes. Absorbing recovered competent cells 100ul, coating the recovered competent cells on LB culture medium added with Nal, Rif and Chl for three resistances, screening transformants, and placing the transformants in a constant temperature incubator for 15 hours.
(3) Designing and identifying primers according to the sequence of RP4 plasmid, and respectively selecting pCas9-Mob, pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEMTransformants of-2 5 strains each, colonies were diluted and subjected to PCR experiments. If the RP4 plasmid is knocked out, no obvious bright band appears in the PCR result, and otherwise, an obvious bright band exists.
(4) The experimental results are shown in FIG. 7, the pCas9-Mob transformant has obvious bands, and pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEMThe transformant of-2 had no clear bright band. Thus, pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEMThe-2 knockout vector can effectively reduce carrying blaTEMThe multi-drug resistant plasmid of (1).
In conclusion, the gRNA provided by the inventionTEM-1、gRNATEM-2 and knockout vector pCas9-Mob-gRNA thereofTEM-1 and pCas9-Mob-gRNATEM-2 can effectively reduce the drug resistance gene blaTEMAnd carry blaTEMThe multi-drug resistant plasmid of (1). Meanwhile, the knockout vector pCas9-Mob-gRNA provided by the invention is utilizedTEM-1 and pCas9-Mob-gRNATEM-2 preparation method is easier to obtain targeted antibiotic drug-resistant gene blaTEMThe knockout vector also provides an effective method and a foundation for knockout and reduction research of other drug-resistant genes.
Sequence listing
<110> Zhejiang university
<120>Targeted reduction drug resistance gene blaTEMgRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atcgaactgg atctcaacag 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acaattaata gactggatgg 20
Claims (10)
1. Specific targeted antibiotic drug resistance gene blaTEMAnd carry blaTEMA gRNA of a drug-resistant plasmid, characterized in that the encoded nucleotide sequence thereof is a gRNATEM-1：5’-ATCGAACTGGATCTCAACAG-3’，gRNATEM-2：5’-ACAATTAATAGACTGGATGG-3’。
2. A knockout vector pCas9-Mob capable of horizontal transfer, which is characterized in that the vector comprises a knockout protein gene Cas9, a gRNA expression sequence and a horizontal transfer site OriT.
3. Contains the specific targeted drug-resistant gene bla of claim 1TEMAnd carry blaTEMgRNA of multiple drug-resistant plasmidsTEM-1、gRNATEM-2 a transferable knock-out vector pCas9-Mob-gRNATEM-1 or pCas9-Mob-gRNATEM-2。
4. Construction of the knockout vector pCas9-Mob-gRNA of claim 3TEM-1 or pCas9-Mob-gRNATEM-2, characterized in that it comprises the following steps:
(1) the gRNA of claim 1, wherein the gRNA is a recombinant gRNA that is a recombinant for a recombinant gRNA that is a recombinantTEM-1 and gRNATEM-2, designing annealing primers by the sequence:
gRNATEM-1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'
gRNATEM-1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
gRNATEM-2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'
gRNATEM-2 reverse primer: 5'-AAAACCCATCCAGTCTATTAATTGT-3'
(2) Mixing gRNATEM-1 and gRNATEMThe forward primer and the reverse primer of-2 are annealed separately to form double strands and phosphorylated with the T4 PNK enzyme;
(3) inserting the plasmid horizontal transfer site OriT into the pCas9 plasmid by using a seamless cloning technology to construct a transferable pCas9-Mob plasmid, linearizing the plasmid by using endonuclease, and carrying out gel recovery;
(4) phosphorylated gRNAs using T4 ligaseTEM-1 and gRNATEM-2 the annealed double strands are respectively connected with linearized pCas9-Mob vectors, and the obtained vector is the transferable knockout vector pCas9-Mob-gRNA of claim 3TEM-1 and pCas9-Mob-gRNATEM-2。
5. The method of claim 4, wherein: the construction method of the pCas9-Mob plasmid in the step (3) comprises the following steps:
(1) using pCas9 plasmid as a template, amplifying replicon OriV, screening marker gene Chl and knockout protein gene Cas9 sequences by using a high-fidelity PCR technology, and obtaining OriV-Chl-Cas9 after gel purification;
(2) taking a plasmid carrying a transfer site as a template, amplifying the transfer site of the plasmid by using a high-fidelity PCR technology, and obtaining OriT after gel purification;
(3) connecting the purified OriV-Chl-Cas9 and the OriT fragment by using a seamless cloning technology;
(4) transforming the ligation product into specific Escherichia coli, wherein relevant genes required by horizontal transfer of pCas9-Mob plasmids are integrated on a chromosome of the Escherichia coli;
(5) screening positive clones by using a screening marker Chl, carrying out amplification culture on bacteria, and extracting a plasmid, wherein the plasmid is the transfer knockout vector pCas 9-Mob.
6. The gRNA of claim 1TEM-1 or gRNATEM-2 in antibiotic resistance gene blaTEMAnd carry blaTEMThe knockout and reduction of the multi-drug resistant plasmid.
7. The knock-out vector pCas9-Mob-gRNA of claim 3 or 4TEM-1 or pCas9-Mob-gRNATEM-2 in antibiotic resistance gene blaTEMAnd carry blaTEMThe knockout and reduction of the multi-drug resistant plasmid.
8. The knock-out vector pCas9-Mob-gRNA of claim 3 or 4TEM-1 or pCas9-Mob-gRNATEM-2, characterized in that it can enter the recipient microorganism both by transformation and by horizontal transfer.
9. Comprising the knock-out vector pCas9-Mob-gRNA of claim 3 or 4TEM-1 and pCas9-Mob-gRNATEMThe microorganism of-2, which is Escherichia coli.
10. The microorganism according to claim 9, wherein said microorganism has a drug resistance gene blaTEMAnd carry blaTEMThe knockout and reduction of the multi-drug resistant plasmid.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110877460.2A CN113817731B (en) | 2021-07-31 | 2021-07-31 | Targeting drug resistance gene bla reductionTEMGRNA of its drug-resistant plasmid, transferable knockout carrier and application thereof |
| PCT/CN2021/142756 WO2023010789A1 (en) | 2021-07-31 | 2021-12-29 | Grna for targeted reduction of drug-resistant gene blatem and drug-resistant plasmid thereof, transferable knockout vector, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110877460.2A CN113817731B (en) | 2021-07-31 | 2021-07-31 | Targeting drug resistance gene bla reductionTEMGRNA of its drug-resistant plasmid, transferable knockout carrier and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113817731A true CN113817731A (en) | 2021-12-21 |
| CN113817731B CN113817731B (en) | 2024-08-30 |
Family
ID=78924129
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110877460.2A Active CN113817731B (en) | 2021-07-31 | 2021-07-31 | Targeting drug resistance gene bla reductionTEMGRNA of its drug-resistant plasmid, transferable knockout carrier and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113817731B (en) |
| WO (1) | WO2023010789A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114561390A (en) * | 2022-03-15 | 2022-05-31 | 浙江大学 | Targeted reduction tigecycline drug resistance gene tetX, gRNA of drug resistance plasmid of tigecycline, transferable knockout vector and application of vector |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108135949A (en) * | 2015-08-14 | 2018-06-08 | 内梅西斯生物有限公司 | Delivery vehicle |
| CN109371048A (en) * | 2018-11-12 | 2019-02-22 | 四川大学 | A method of polymyxins drug resistant gene mcr-1 in Escherichia coli is knocked out using CRISPRCas9 technology |
| CN110423772A (en) * | 2019-07-17 | 2019-11-08 | 上海科技大学 | One kind being used for Acinetobacter bauamnnii cytosine base editor plasmid and its application |
| CN111378660A (en) * | 2020-02-29 | 2020-07-07 | 浙江大学 | sgRNA targeting tetracycline resistance gene tetA, knockout vector thereof, vector construction method and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463003A (en) * | 2015-12-11 | 2016-04-06 | 扬州大学 | Recombinant vector for eliminating activity of kanamycin drug resistance gene and building method of recombinant vector |
| CN107384926B (en) * | 2017-08-13 | 2020-06-26 | 中国人民解放军疾病预防控制所 | CRISPR-Cas9 system for targeted removal of bacterial drug-resistant plasmids and application |
| US20230046246A1 (en) * | 2019-12-13 | 2023-02-16 | The Regents Of The University Of California | Reducing Antibiotic Resistance in Bacteria Using Pro-Active Genetics |
-
2021
- 2021-07-31 CN CN202110877460.2A patent/CN113817731B/en active Active
- 2021-12-29 WO PCT/CN2021/142756 patent/WO2023010789A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108135949A (en) * | 2015-08-14 | 2018-06-08 | 内梅西斯生物有限公司 | Delivery vehicle |
| CN109371048A (en) * | 2018-11-12 | 2019-02-22 | 四川大学 | A method of polymyxins drug resistant gene mcr-1 in Escherichia coli is knocked out using CRISPRCas9 technology |
| CN110423772A (en) * | 2019-07-17 | 2019-11-08 | 上海科技大学 | One kind being used for Acinetobacter bauamnnii cytosine base editor plasmid and its application |
| CN111378660A (en) * | 2020-02-29 | 2020-07-07 | 浙江大学 | sgRNA targeting tetracycline resistance gene tetA, knockout vector thereof, vector construction method and application |
Non-Patent Citations (3)
| Title |
|---|
| XIE ET, AL.: "sgRNAcas9:a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites", PLOS ONE, vol. 9, no. 6, 30 December 2014 (2014-12-30) * |
| 张冰;王鹏飞;段广才;梁文娟;洪丽娟;王琳琳;郭向娇;杨海燕;郗园林;: "CRISPR/Cas系统间隔序列同源质粒耐药信息与志贺菌耐药的关系", 中国病原生物学杂志, no. 10, 26 October 2016 (2016-10-26) * |
| 徐兰等: "饮用水活性炭净水器对抗生素抗性基因的去除效果", 环境工程, vol. 39, no. 6, 30 June 2021 (2021-06-30) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114561390A (en) * | 2022-03-15 | 2022-05-31 | 浙江大学 | Targeted reduction tigecycline drug resistance gene tetX, gRNA of drug resistance plasmid of tigecycline, transferable knockout vector and application of vector |
| CN114561390B (en) * | 2022-03-15 | 2024-02-23 | 浙江大学 | Targeting cut-down tigecycline drug-resistant gene tetX and gRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023010789A1 (en) | 2023-02-09 |
| CN113817731B (en) | 2024-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4303597B2 (en) | Construction of novel strains containing minimized genomes by Tn5-binding Cre / loxP excision system | |
| CN106119269B (en) | Method for preparing linear single-stranded DNA in escherichia coli | |
| CN101633901B (en) | Escherichia coli strain for recombined engineering | |
| CN110699407B (en) | Preparation method of long single-stranded DNA | |
| JP2021512617A (en) | Genome editing using CRISPR in Corynebacterium | |
| CN103555644A (en) | Host-vector system for antibiotic-free cole1 plasmid propagation | |
| CN106755037A (en) | Streptomyces virginiae IBL14 type I-B-sv14 CAS gene editing system | |
| Kwong et al. | Staphylococcus aureus multiresistance plasmid pSK41: analysis of the replication region, initiator protein binding and antisense RNA regulation | |
| KR20150140663A (en) | Methods for the production of libraries for directed evolution | |
| CA3123103A1 (en) | Cis conjugative plasmid system | |
| JPH04346787A (en) | Method for obtaining dnarecombinant vector, microorganism and dna, method for obtaining recombinant protein and recombinant protein | |
| CN103160495A (en) | Disconnected dependent rapid cloning method | |
| CN111378660B (en) | sgRNA targeting tetracycline resistance gene tetA, knockout vector thereof, vector construction method and application | |
| Pesce et al. | Stable transformation of the actinobacteria Frankia spp | |
| CN113817731A (en) | Targeted reduction drug resistance gene blaTEMgRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof | |
| CN112011539A (en) | Cell line for targeted knockout of porcine GDPD2 gene based on CRISPR-Cas9 technology and construction method thereof | |
| CN114561390B (en) | Targeting cut-down tigecycline drug-resistant gene tetX and gRNA of drug-resistant plasmid thereof, transferable knockout vector and application thereof | |
| CN118139979A (en) | Enzymes with HEPN domains | |
| US20230183678A1 (en) | In-cell continuous target-gene evolution, screening and selection | |
| KR20240049306A (en) | Enzymes with RUVC domains | |
| CN111748539B (en) | CRISPR/LpCas9 gene editing system and application thereof | |
| EP1156114A1 (en) | Vectors for use in transponson-based DNA sequencing methods | |
| CN112921040A (en) | Construction method of pig TFF1 gene knockout cell line based on CRISPR-Cas9 gene editing technology | |
| CA2492966C (en) | Method for the expression of unknown environmental dna into adapted host cells | |
| Cohen et al. | Biotechnology: principles and processes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |