CN116334108B - Novel anti-phage element and application thereof - Google Patents

Novel anti-phage element and application thereof Download PDF

Info

Publication number
CN116334108B
CN116334108B CN202210879453.0A CN202210879453A CN116334108B CN 116334108 B CN116334108 B CN 116334108B CN 202210879453 A CN202210879453 A CN 202210879453A CN 116334108 B CN116334108 B CN 116334108B
Authority
CN
China
Prior art keywords
phage
seq
novel anti
resistance
chassis
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.)
Active
Application number
CN202210879453.0A
Other languages
Chinese (zh)
Other versions
CN116334108A (en
Inventor
郭云学
王晓雪
汤开浩
古嘉瑜
林世团
林兼仲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Sea Institute of Oceanology of CAS
Original Assignee
South China Sea Institute of Oceanology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Sea Institute of Oceanology of CAS filed Critical South China Sea Institute of Oceanology of CAS
Priority to CN202210879453.0A priority Critical patent/CN116334108B/en
Publication of CN116334108A publication Critical patent/CN116334108A/en
Application granted granted Critical
Publication of CN116334108B publication Critical patent/CN116334108B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01001—Phosphorylase (2.4.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01011—6-Phosphofructokinase (2.7.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/385—Pseudomonas aeruginosa
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a novel anti-phage element and application thereof. The nucleotide sequence of the novel anti-phage element is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6. The novel anti-phage element discovered in the invention can not only expand phage resistance element library and enhance the recognition of anti-phage element members, but also be used for modifying escherichia coli to enhance the resistance to different phages, and has extremely strong fermentation industrial application value.

Description

Novel anti-phage element and application thereof
Technical Field
The invention belongs to the field of biology, and particularly relates to a novel anti-phage element and application thereof.
Background
Phages are viruses that infect bacteria, and are also the most abundant form of life in the biosphere. Host bacteria, particularly pathogenic and environmental bacteria, evolved a wide variety and well-designed anti-phage elements during game play with phage. The more classical anti-phage elements include modified restriction systems and CRISPR-Cas systems. Modification restriction systems are widely present in 75% of bacterial genomes and are generally composed of DNA methyltransferases, restriction endonucleases and target recognition modules that can rapidly recognize and degrade nucleotides of specific phage origin. When the host bacteria are infected by phage, the CRISPR-Cas system integrates the CRISPR sequence of the host bacteria into a phage genome, and in the subsequent infection process, apoptosis protease of the host bacteria can rapidly detect and cut the specific sequence of the host bacteria so as to achieve the anti-phage effect. In addition, the anti-phage elements of interest in recent years include reverse transcription subsystems, toxin-antitoxin systems, etc., which can exert an inhibitory effect at various stages of phage infection of host bacteria, involving adsorption, injection infection, replication, release, etc. of phage. Rapid developments in pan genome sequencing technology have led to the discovery of more new anti-phage elements, which are typically clustered by two or more adjacent genes. Furthermore, related studies have found that genes encoding the defense system frequently occur in variable genomic islands or are associated with mobile genetic elements. Recent studies have found that endogenous mobile genetic elements in vibrio vulnificus mediate anti-phage evolution of bacteria, and that the number of defenses in a single bacterial genome can be as high as 6 to 12, indicating that protection of phage defenses is cumulative, with defenses accounting for over 90% of the variable non-core genome, which is why many similar bacterial genomes differ most by the composition and number of mobile genetic elements. It is of interest that these mobile genetic elements also travel quite rapidly between bacteria. Based on the characteristics of wide existence and quick propagation of the anti-phage element, the technology can be popularized and applied. CRISPR-Cas9 gene editing technology has a tremendous impact on the healthcare industry, however, while different types of anti-phage elements are increasingly being discovered, reports on their use in the fermentation industry as well as phage therapy remain lacking.
The fermentation industry usually uses conventional methods for preventing virulent phage contamination, such as rotation of the species, ventilation quality, screening of resistant strains for phage receptor mutations, etc., but these methods cure the symptoms but not the root cause, and still result in impaired production in the presence of new phage. In addition, there are a wide variety of temperate phages in the environment that, after infection of the host, integrate its genome into the host genome, silence, and enter the lytic cycle in a specific environment, and contamination with such temperate phages is hardly detected, but reduces the production efficiency, and may in part result in production failure. The discovery and transformation of the novel element of the high-efficiency anti-phage not only can provide theoretical basis and resources for developing anti-phage products based on phage-host interaction in fermentation industry, but also can develop related medical agents aiming at the anti-phage element, and has important practical significance in the aspect of assisting phage in treating antibiotic resistant bacteria.
Phage are now looking for host bacteria and trying to complete the infection process, which may be called a pore-free entry. In industrial fermentation production, engineering strains, particularly escherichia coli, are easy to be polluted by phage, and if the virulence of the polluted phage is not strong, the polluted phage can even be integrated into a host genome, and the polluted phage coexists with the host in a mild phage form and is difficult to detect. They exist like a timed bomb and can be activated at any time to enter a cracking cycle, killing the host, and causing huge economic loss. In addition, phage are important transmitters of antibiotic resistance genes and host normal metabolism interference genes, and are one of main factors restricting the production process. Host bacteria evolved a wide variety of genetic elements against phage during their struggle with phage, but most were identified by high throughput screening methods, lacking systematic knowledge. If one were to develop the use of these anti-phage elements to aid in the fermentation industry, one would need to investigate in detail their function and mechanism of combating phage infection.
Disclosure of Invention
It is an object of the present invention to provide a new class of anti-phage elements which can enhance the effect of chassis cells against phage infection.
The invention discloses a new type of anti-phage element, which is used for detecting the anti-phage effect of the element, analyzing the distribution breadth of the element, transferring the element from different sources into escherichia coli, and detecting the resistance condition of different escherichia coli phages so as to realize the effect of enhancing the anti-phage infection effect of chassis cells through modifying the chassis cells, thereby realizing the purpose of the invention.
The nucleotide sequence of the novel anti-phage element is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.
It is a second object of the present invention to provide a chassis cell comprising the novel anti-phage element described above.
It is a third object of the present invention to provide the use of the novel anti-phage elements described above for increasing the resistance of chassis cells to phage infection.
A fourth object of the present invention is to provide a method for increasing the resistance of chassis cells to phage infection by transferring the novel anti-phage element described above into E.coli to increase the resistance of the host to phage.
The chassis cells can be escherichia coli, pathogenic bacteria pseudomonas aeruginosa, salmonella and vibrio.
The phage may be phage PAP8, PAO-L5, QDWS, PAP-L5, E.coli phage T1, T4, T5, T7, EEP, lambda or M13.
The invention focuses on finding a brand-new anti-phage element with wide distribution, evaluates the anti-phage effect, realizes transformation of chassis cells by transferring the element into escherichia coli, and enhances the resistance of a host to phage.
Phage contamination is common in the laboratory and fermentation industries, and some temperate phages integrate their genome into the host genome and are difficult to find and detect. The prevention and improvement of the phage pollution problem in the laboratory mainly uses methods of cleaning related tools, replacing strains and the like. Phage contamination in the fermentation industry is usually prevented by means of purification of the production environment, strain rotation, etc., but there is no efficient and durable solution. The invention creates and discovers a novel anti-phage genetic element with good anti-phage effect. The novel anti-phage elements are widely distributed in different bacteria, such as pathogenic bacteria of pseudomonas aeruginosa, salmonella, vibrio, escherichia coli and the like, and are shown to have anti-phage effect on phage of different sources. In industrial fermentation, the escherichia coli is the engineering bacterium which is most easily polluted by phage, and KKP anti-phage elements from different sources are introduced into the escherichia coli, so that the effect of the escherichia coli on phage infection resistance can be enhanced. And KKP elements from different sources have different resistances to different phages, if the KKP elements are combined and designed, the super-strong anti-phage engineering bacteria with resistances to different coliphages are very likely to be developed, and a feasible scheme is provided for solving industrial fermentation phage pollution from the source.
In conclusion, the novel anti-phage elements found in the invention can expand phage resistance element libraries, enhance the knowledge of members of the anti-phage elements, and can be used for modifying escherichia coli to enhance the resistance to different phages, thereby having extremely strong fermentation industrial application value.
Description of the drawings:
FIG. 1 is the KKP genetic element consisting of three genes, pfkA, pfkB and PfpC.
FIG. 2 is a process for inserting KKP integration into the P.aeruginosa PAO1 genome.
FIG. 3 is a three-component gene cluster anti-phage effect in P.aeruginosa, wherein-KKP is PAO1 without Pf6 and +KKP is PAO1 incorporating the KKP three-component gene cluster.
FIG. 4 is a prophage of the KKP three-component gene cluster widely distributed across a number of different bacteria.
FIG. 5 shows that E.coli harboring KKP from different sources has a strong resistance to infection by E.coli phage.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1:
1. comparative analysis of the genomes of two filamentous phage Pf4 and Pf6 coexisting with Pseudomonas aeruginosa MPAO1 revealed that Pf6 carries a three-component genetic element consisting of two kinases (PfkA and PfkB) and a phosphorylase (PfpC) (FIG. 1), abbreviated as KKP (kinase-kinase-phosphotase), the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention is derived from the same methodShewanella sp.W3-18-1、Escherichia coli15EC039、Escherichia coli strainThe KKP three-component gene cluster in Salmonella enterica subsp.enterica serovar Typhi str.CT18, vibrio tasmaniensis 10.10N.222.48.A2 was also tested, and the sequence of the KKP three-component gene cluster is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
2. Since KKP cannot be knocked out from MPAO1 genome, we integrated it into PAO1 genome which does not contain Pf6 and is highly similar to MPAO1 genome as follows (FIG. 2).
The specific operation is as follows:
A. inoculating Pseudomonas aeruginosa MPAO1 on LB plate from-80deg.C refrigerator, culturing at 37deg.C overnight, selecting MPAO1, inoculating to LB liquid culture medium, and shake culturing at 37deg.C to OD 600 About 1.
B. 1ml of the bacterial liquid was centrifuged at 12000rpm for 1min, and the bacterial cells were collected. The genomic DNA of the bacteria was extracted using the Tiangen bacterial genome extraction kit (cat# DP 302-02).
C. Primer pairs KKP-F1/R1, KKP-F3/R3 and KKP-F4/R4 are adopted, and MPAO1 genomic DNA is used as a template. The primer pair KKP-F2/R2 is adopted, and plasmid pEX18Gm is used as a template. PCR amplification (conditions: 95 ℃ C., 10min;95 ℃ C., 30s,60 ℃ C., 30s,72 ℃ C., 90s,35 cycles) was performed using Takara Primer Star reagent (cat# AL 52850A), and the PCR product was subjected to agarose gel electrophoresis and then recovered using Omega gel recovery kit (cat# D2500-02).
D. Vector pEx18Ap was double digested with EcoRI and HindIII, followed by agarose gel electrophoresis and gel recovery (recovery of PCR fragments as above).
E. The recovered vector and the PCR fragment were ligated according to the instructions using the One Step multiset cloning kit (cat# C113-01) from Norwegian Co.
F. The ligated ligation product was transformed into auxotrophic E.coli WM3064 competent cells (0.3 mM diaminoacrylic acid was added to the medium during cultivation), PCR was performed on the correct transformants using the pEX18Ap-F/R primer pair and sent to the company for sequencing. Thus, a WM3064 strain containing a recombinant plasmid was obtained.
G. The WM3064 strain (donor strain) containing the recombinant plasmid and Pseudomonas aeruginosa PAO1 (acceptor strain) carrying no Pf6 were cultured to an absorbance of 600nm to 1.0. 4mL of donor bacteria were mixed with 1mL of recipient bacteria, washed 3 times with LB medium, and centrifuged at 3000rpm for 5min. The final suspension was performed with 100. Mu.L LB, dropped onto LB solid plates containing 0.3mM diaminoacrylic acid and 1.5% agar, and the plates were left to stand at 25℃for 8 hours. Bacteria were serially diluted and plated onto LB solid plates containing 30. Mu.g/ml gentamicin and 1.5% agar, and recipient strains that successfully bound the metastasis were selected. Single-crossover strains which were successfully transferred in conjugal and contained suicide plasmids were cultured at 37℃with salt-free LB medium (formulation: 1% peptone, 0.5% yeast extract, dissolved in distilled water) and then PCR-verified with primers PfkC-F and PfkA-R. Next, for the strains that were confirmed to be correct, double-crossover screening was performed, using a salt-free LB solid medium containing 10% sucrose and 1.5% agar. The obtained strain is subjected to gentamicin resistance screening, PCR verification is carried out on the strain losing gentamicin resistance by adopting a primer pair conf-F/R, and DNA sequencing is carried out. Thus, a engineered PAO1 with integrated three-component gene clusters was obtained.
3. Performing infection experiments on the phage of pseudomonas aeruginosa stored in a laboratory, performing 1% dilution on PAO1 cultured at 37 ℃ overnight and modified PAO1 integrated with three-component gene clusters by adopting a double-layer agar plate method, and then culturing until the OD600 is about 1; at this time, 3ml of the bacterial liquid was mixed with 10ml of R-top medium (temperature about 55 ℃ C.) (formulation: 1% tryptone, 0.1% yeast powder, 1% NaCl,0.8% agar) and plated on LB plates, and left standing on an ultra clean bench for 10min for 4 P.aeruginosa phages PAP8, PAO-L5, QDWS and PAP-L5 spot plate operations. Phage were pressed to 10 1 ～10 8 The phage resistance was measured by the number and intensity of plaques by performing gradient dilution, plating by pipetting 5. Mu.l each, and incubating overnight at 37 ℃. As a result, KKP exhibited resistance to phages PAP8, PAO-L5, QDWS and PAP-L5 (FIG. 3).
4. The three-component gene cluster was predicted by bioinformatics and found to be widely distributed in different prophages of various bacteria including Shewanella marine W3-18-1 (prophage P4), salmonella enterica serovar Typhi str.CT18 (prophage P4), E.coli MPEC4969 (prophage P2), E.coli 15EC039 (prophage P2), vibrio 10N.222.48.A2 (prophage P2) (FIG. 4).
5. And (3) synthesizing the four three-component gene clusters in the step (4) by a gene synthesis method, and cloning the four three-component gene clusters into pHERD20T vectors respectively. The vector adopts NcoI and HindIII as cleavage sites, and the Escherichia coli K12 strain MG1655 is used for transformation. Laboratory-preserved E.coli phages T1, T4, T5, T7, EEP, lambda and M13 were tested against phages using the same double-layer agar plate method as in step 3. Only 0.3% of arabinose is used for inducing the expression of the three-component gene cluster, the induction time is 3h (figure 5), and as can be seen from figure 5, the three-component gene cluster can improve the infection of the bacterial strain to phage.
TABLE 1 primers used in the present invention
ATGTTTCAAAGGCTATTGCAAAAACACCTTGCCAGAGGAATTCTTGGCAGAAAAATGTTATCTATCGACAAAGGTTCTATTGCCTTAGCTTCAGATCTAGGTCTGAAGAGAACTGAGAATCAAGACAGAACCGCTTTAATGAAATTTAGATCTTCAACAGCTTCGTATACTGTCATAGCCGTAGTTGATGGAATGGGTGGTATGAGAGATGGGGAGAAGTCTGCTGAAATAGCTATTTCAACTTTCTTGTGCTCTATTATGGAAAATGTTCATTTGGGTTCTGAACATGCAATAATGCAAGCCACGATGACTGCCAATAACGCTGTATTCGAATTTACAAATGGTAAAGGTGGAAGTACCTTATCCGCAATTTTATTAGCTAGCGACGGCACTCATATGACCGTTAATGTCGGGGACAGTCGAATTTATGCAAAGGAGTCTATCTTTGGCAAAGTAATTAGACTTACCGTTGATGATTCGTTAGCGGAAACCGTTGGAGGAAGCGGTACAGAATTATTGCAGTTCATAGGTATGGGGGAAGGAATTAGACCACACGTAGTTCCGCTGCCACTTGAAGCTAAGCAAGTATATCTGACAACTGATGGTGTCCATTACATTGAACCAAACACATTGTCTGATATTATAAAACATGCAGAAAAAATCACTCAGGTTGTAGAGCGGTTGATAGCAACTGCACGTTGGTGTGGAGGCCCAGATAACGCTACAGTTAGTGCTCTTGATTTAGAGCTATTAAACTTTGAGGAGTCCCTGGATGATGCCTCGATAGTGCAAATATCTGATCCACATAGCTCTACACAATTCATATTCCCCCAATTTCAGTTGGAAAGTGAAGTATCTCTCCCAGAGACAAGTGTTCAAACTCAAAATAATTCTATTAATACAGCTCAATCTGAGAAATCGGCTGCTCCTAGCAGAACCTCAACTTTGGTCGAAGAAAAAGAATCGATTACTGAAACAGTCAAAGTTGAAGCTAAAACCCCGCCTAAGAGAAAGAAACGTCAATCAAAGAAGGCTGTTGATCATCTAGATTCCGCAGATGAAGTACAAATTAAGATGACCATTTTTGATGAAGCAGGTTGCGAAAGTCATGAGGTTGAAGATGATGATTCCAAGTAGATATGAACTC TGTGGTAACAACGATACTGGTGGTATGGGTGACATTCTCTATTGCAAAGACAAACATTTACAACGCGATGTCATAAT TAAACTCTTAAAAGGTGATGCTGAACAACGAAGGTTAATTGACGAGCAAAAATCTCTTATTCAGCTCCGTTCTAAAC ATGTAGTTCAATTGTACGACGTAGTTAATATTGATAATCAATCCGGTTTAGTATTAGAGTTTATTAAAGGCGAGGAC CTCAAAAATGGTCTTTATGAGTCGAATATGAAAGGACTCATAGAAGTATTGTGGCAAGTTGCGTGCGGTTTATCTGA CATACATAAGGCAGGAATTATTCATCGTGATATCAAACCAAACAATATTCGTCGAGATAACATCGGAGTTATCAAGG TCTTTGATTTTGGGCTTTCGAGAAAACTTGATAGCGCTAAAACACATAGTGTCATTGGTACTGTGGGGTATATGGCA CCTGAATTATGGAAATGTGGTGAAGTTGAGTTTACCACAGCTGTAGATGTTTATGCCTTCGGTATTACGGCAATGGC TCTTTCAAATGCTATTGTCCCAAGAGAACTTTTAGAGTTCCCTCCTCGTTGCGCTAACAATGGTTGGGTTAAAAATA GTTTACCAAGCTTAGATTCGGATATTGCAGAGATTCTAGAGCGATGCCTAGAATACAAACCAGAGGCTCGCCCCACA ATCGATCAGGTTGAAAAAATACTAAGAAAGCATCTCCTGAAAGACAAACACAAAGGTCTTATTGTGATGGGGAATGA AGTTAGAGAGCTTAATAATCAAAATAGACGTGCTAAGATTTCATCTATTCATGGTAATAATTTAAATGGTGAAATTA CCATTGAATATGATAGTTATGAATTTAAAGTTGCTGCTATCGGTGGTTCGGTAACAGTAAATAATGAAGTGATCAAA GTCGGTTATATCCTGCCAGGTGCTTCCGTTATTACTCTAGGTTCCGACTCTAATCGGCGTTTTGTTACTTTCGATAT ATCTAACCCAGAGGTGGTTTCATGATCACATCGAATACTCTGATCGGTGGCCGTTATATGGTTCACCAACACATTGGTGCTGGCGGTATGCAAGATGTTTATCTGGCGCTAGATCAATTCCTCGGTAATTATGTCGCACTTAAGACGCCTCAGCCTGGCCAGAAAACCAGACGATTCCAAGCTAGTGCTGTAATTGCAGGTAGAGTAAATCACCACAATGTAGCCAAGACATTAGATTACTTTGAAGAAAATGGAAATGTTTACCTAATTGAGGAATTTGTTAAAGGTGAAACTCTTGAAGATAAGATTAAGCAGAGAAAATTCCTTGATCCTCATCTAGCCGCTAGAACTATACACCTATTAGCAAAGGGGGTCAGAGCCTCACACATTCAAGGAGTAATACACAGAGATTTGAAGCCAAGTAATATTATGGTTGACTCTAGTACTGGTATCGAAGAGCTAAAGATTACCGATTTTGGTATTGCAACTTTTACCGACGAAGTATTTCAAGAAGAGGCCGACTCCGGCGATATTACTCGTTCCACTTCTGGGACAGTGAAAGGTGCTCTTCCATTTATGGCACCTGAAATGATGTTTCGTAAAAAAGGGGATAGTATTACTCCGGCCTTAGATATTTGGTCAATTGGGGCGATGATGTTCAAAATACTAACAGGTGAGTACCCATTTGGTGTTTTTCTCGATGCTGCTGTGAATGTCAAAACAAGAAATAGGCTAGATTGGCCTGCTTTTATGACTTCTAACGCACAGTTTTCCCCATTATGCAGGGAACTTCAGAAGATAATAGATAGCTGTTTGGAGTATGAACCAACCAAACGCCCTACGGCTGATGCTCTTGTGAAAATGTGCCAAAATTTGTGCTATCAAACTTCTGAACGTTTTGAAGCGACGGTTACGAGGATGATTCAAAACGGATATAGTGGCTTTGCTTCAAACCCTCAACATAGCGTATTTTTTAGTATCCATAGCATCTACGGAGCTTCTAGGGTTAATAGTGGAAGTAAAATTACGTACTCAAAGTTTCCGGGCACCCCTAATTTTAGAGCTCACCCAGTCATTATCTTAAATTAA
GTGAGCCGTGATTCTTACGAAATCCTTCATGAGCATATTCACGGATGGCTACATCGAAAAAATATAGCATCCTCAGTGCGTCGTGTCTCAACCTTACCAGTGGCTATAGCTACTGATATTGGGTTGGTACGGAAGGAAAACCAAGATAGGGTTGCTATATTGAAATTCCGCCCAAGTAGCAAAGCTAAAGATATCGTTGTTGTTGCGTTAGCCGATGGTATGGGGGGGATGGAGGGGGGTGCCAATGCAGCATCTTTAACTTTATCTACATTTTTTACTGAAATAATAAGAAATTCTCATTTACCAATACGTTCTTGTCTTGAGAAAGCTGTGCTACAAGCGAATGATTCTGTTTTGAATGTATATAAGGGTAATGGAGGGGCGACATTATCAGCGATAGCTTTAGAAGATGATGATAATATTACCGCAGTAAATGTAGGTGATAGCCGTATTTATTATGTCTCGCATGAAGAAACTACTCAGTTGAGCGAGGACGATACTTTAGTTGCCTTAGCCAAAAAGTATAATAATCATCTAAATATGGATCCACAGGATATTGATTTACGCTTTGGTGGCGAATTAGTACAATTTATAGGCATAGATGGCACATTGGAAATACACTTTCATCATATCCAAGCCTTGGAGAGTGGCGTAATTATTTTAAGTTCTGATGGTGCGCATTCTATCGGTAAGGATAATTTAAGAAAGTTATATGTGCACTCGGCGAATCTAGGTGTTTATTCTAGGCGGGTTATTGATCTTGCTAGTTGGTTTGGTGGTTTCGACAATGCAAGTATAGCAGTTATAGACCTTTTTAATACTCTAAAAGAGTTAGATGTTTCATCTGGAGATGTAATAAATCTCTGGGATCCATTTGGTGAATTAAAAGTCATTAGCGTGCCGAATAAGTCATCTTCATTAGAGCCTTTAAATACAGTTGAGCTTGAAAAAAATAATAAAGGCACTTCTGGTGTTAGAAAAGTGTCAAACAAAAAAAATACTGATTCAGTCGTTAGCGATATAAACAAAGCAACAACAAAAAGAAAGGCAAGAACAAAAAATAAGAATAAATCTCTTCAGGAATTAGATGAGAAAAAAAATGGGTTAAATAAAAATAATAGACTTGATTGTATTTCACAACTTGACATGTCATTCCTTGAAAGGAATTCAATAAAAGGAGATGGTGATGATGAGTGATTTTCTCCCAGAAAGATATCAAGTGGT TGGGGATCCTGATTTAGGGGGATTCGGTAGTGTAATCAAATGCCGCGATTCTCATCTTGAGAGATTTGTTGCAATAA AGACTATAAATGATCCATCAGATACAGAGCGAATGAAAGACGAGTTGGCTGCTCTAATGACACTACGTTCAAAACAT GTTGTTGAACTGTTTGATGTGATTAATTATGCTGAAGGCAATCTTGCAATTGTTGAAGAGTTTATCGATGGTCCATC GTTGAATGAAGTTAATAATAAAATTACTACAGTAGGGGAGCTTATTAAGATTTTGTGGCAAATAGCATCAGGTATTT GCGAGATACATGAACATGATATCATTCATCGTGATATAAAGCCTGGGAATATGAAGATTGATAAAGAAGGGCTTGTA AAAATATATGATTTTGGCTTGTCAAGAAAAATAGATAATGCAAAAACAATTGGGTTTAAAGGTACCCCAATTTTTGC AGCTCCTGAGTTGTATTTGCAGAACGTAGACTTTACTAAAGCAATTGATACATATGCCTTTGCTGTTACAGCAATGT GCTTAGCTAAAACCCCTGTCCCAGATGAATTGACCCGTTACCCTAAGATTCTGACATCTAATCCATTTGATTTGTCG GTAATAAAATTACCAAGTATTGTAAAGGAATTGTTTTTCAAATGTCTTGATGCAAATCCTCAAGCTAGGCCCCCTAT GAAAGATGTTTGCGATGTTTTGAAAAAAATATTATTGCACAACTCTCATCGAGCATTGCTTATATCTGATAATAAAA AACCAGTAGTGCTCTCAGCTACACACAAGACGGAGTCTTATAACAATCCAGGGGTGGGTAGTGTGGAAATTACTTAC TCTGGTTCCGAGTTTTATATTTCAGATATATCAGGGGATGTCTATGTTAATAACATTAGGGCTAAAAAACGAAATTT ATTGCCTAGCTCATGCGTGATAATACTTGGCCCTGCCGGAAGAACAACTACAAAACGTATATTTATCACATTTGATC TTTCTCATCCGGAGGTTGTGTTATGATTGAGTTGGTTCCTGGGACTAATATAAATCGTTATACTATTATCAGCGAAATTGGTGAGGGGGGGATGCAAAAGGTTTACCTTGCGAATGATAAGATATTAAATAGGCAAGTTGCTCTTAAGACCCCTAAAAATAAGTCTGCTGAAAAACGATTCCATAGAAGTGCTATTTTAGCATCTAGGGTCAACCATCCTAACGTCGCTAAAACATTAGATTATTTTGCCGAAGATGGACGTGAATTTTTAACGGAGGAATTTATCGATGGAGTAGATCTGGATAAAGCATTGTTGAGTAGCTATACAAGTGTTGATCCTTACTTGACTGCAAAGATATTTCATAACTTAGCGAAGGCTCTTTCGGCTTCCCATCATGTGGATGTAATACATCGAGACCTCAAACCTTCTAACATAATGGTTATTGGAGGAGTTAGTGCTACAGGTGTTAAAATCACCGATTTTGGAATTTCAAAAATGGCCGGTGATGAAATTGATGAGGCCGCAAAGAATGGGCAAGGATCGATTACTTCATCTCAAACAGCTATGGGGGCATTGCCATATATGGCCCCGGAAATTATACAAAGTCAGGGGCAAGTTTCAAAACCATCTGATGTCTGGGCATTAGGTGCGATGATGTTCAGAATCCTCACGGGAGAGTATCCTTTTGGATTAGGGTATATGGCTATTCCGAACATCTTATCTGGAAAGCATACTCAATATCCTGATTTTATTAAGTCAAATAAGCAGTTTGCTCCGCTGGCAAATGAAATTATAGATATAATTGAAAAATGTTTAAATCTAGACCCTTCTAAACGCCCCACTGCAGATGAGCTCGTGTCATTATGTGGTCAATTATGCTATCCGGTTTGTAATAGAGAAGAAGGAGTAATAGGTGATACTAGACTAGCTTATGGTTTCATTCGTATACCAAACCAACCACAAGTATTTTTTCATTACGATAGTGTGTATGGTAGTAAACCAGTGAGTAATGATAAGGTGATTTTTTCAAAGTTCTTGGGAGGGGGCCATGACCGGGCTCATCCAGTTATCAAGGCTAAGTAG
GTGAGCCGTGATTCTTACGAAATCCTTCATGAGCATATTCACGGATGGCTACATCGAAAAAATATAGCATCCTCAGTGCGTCGTGTCTCAACCTTACCAGTGGCTATAGCTACTGACATTGGGTTGGTACGGAAGGAAAACCAAGATAGGGTTGCTATATTGAAATTCCGCCCAAGTAGCAAAGCTAAAGATATCGTTGTTGTTGCGTTAGCCGATGGTATGGGGGGGATGGAGGGGGGTGCCAATGCAGCATCTTTAACTTTATCTACATTTTTTACTGAAATAATAAGAAATTCTCATTTACCAATACGTTCTTGTCTTGAGAAAGCTGTGCTACAAGCGAATGATTCTGTTTTGAATGTATATAAGGGTAATGGAGGGGCGACATTATCAGCGATAGCTTTAGAAGATGATGATAATATTACCGCAGTAAATGTAGGTGATAGCCGTATTTATTATGTCTCGCATGAAGAAACTACTCAGTTGAGCGAGGACGATACTTTAGTTGCCTTAGCCAAAAAGTATAATAATCATCTAAATATGGATCCACAGGATATTGATTTACGCTTTGGTGGTGAATTAGTACAATTTATAGGCATAGATGGCACATTGGAAATACACTTTCATCATATCCAAGCCTTGGAGAGTGGCGTAATTATTTTAAGTTCTGATGGTGCGCATTCTATCGGTAAGGATAATTTAAGAAAGTTATATGTGCACTCGGCGAATCTAGGTGTTTATTCTAGGCGGGTTATTGATCTTGCTAGTTGGTTTGGTGGTTTCGACAATGCAAGTATAGCAGTTATAGACCTTTTTAATACTCTAAAAGAGTTAGATGTTTCATCTGGAGATGTAATAAATCTCTGGGATCCATTTGGTGAATTAAAAGTCATTAGCGTGCCGAATAAGTCATCTTCATTAGAGCCTTTAAATACAGTTGAGCTTGAAAAAAATAATAAAGGCACTTCTGGTGTTAGAAAAGTGTCAAACAAAAAAAATACTGATTCAGTCGTTAGCGATATAAACAAAGCAACAACAAAAAGAAAGGCAAGAACAAAAAATAAGAATAAATCTCTTCAGGAATTAGATGAGAAAAAAAATGGGTTAAATAAAAATAATAGACTTGATTGTATTTCACAGCTTGACATGTCATTCCTTGAAAGGAATTCAATAAAAGGAGATGGTGATGATGAGTGATTTTCTCCCAGAAAGATATCAAGTGGTTGGGGATCCTGATTTAGGGGGATTTGGTAGTGTAATCAAATGCCGCGATTCTCATCTTGAGAGATTTGTTGCAATAAAGACTATAAATGATCCATCAGATACAGAGCGAATGAAAGACGAGTTGGCTGCTCTAATGACACTACGTTCAAAACATGTTGTTGAACTGTTTGATGTGATTAATTATGCTGAAGGCAATCTTGCAATTGTTGAAGAGTTTATCGATGGTCCATCGTTGAATGAAGTTAATAATAAAATTACTACAGTAGGGGAGCTTATTAAGATTTTGTGGCAAATAGCATCAGGTATTTGCGAGATACATGAACATGATATCATTCATCGTGATATAAAGCCTGGGAATATGAAGATTGATAAAGAAGGGCTTGTAAAAATATATGATTTTGGCTTGTCAAGAAAAATAGATAATGCAAAAACAATTGGGTTTAAAGGTACCCCAATTTTTGCAGCTCCTGAGTTGTATTTGCAGAACGTAGACTTTACTAAAGCAATTGATACATATGCCTTTGCTGTTACAGCAATGTGCTTAGCTAAAACCCCTGTCCCAGATGAATTGACCCGTTACCCTAAGATTCTGACATCTAATCCATTTGATTTGTCGGTAATAAAATTACCAAGTATTGTAAAGGAATTGTTTTTCAAATGTCTTGATGCAAATCCTCAAGCTAGGCCCCCTATGAAAGATGTTTGCGATGTTTTGAAAAAAATATTATTGCACAACTCTCATCGAGCATTGCTTATATCTGATAATAAAAAACCAGTAGTGCTCTCAGCTACACACAAGACGGAGTCTTATAACAATCCAGGGGTGGGTAGTGTGGAAATTACTTACTCTGGTTCCGAGTTTTATATTTCAGATATATCAGGGGATGTCTATGTTAATAATATTAGGGCTAAAAAACGAAATTTATTGCCTAGCTCATGCGTGATAATACTTGGCCCTGCCGGAAGAACAACTACAAAACGTATATTTATCACATTTGATCTTTCTCATCCGGAGGTTGTGTTATGATTGAGTTGGTTCCTGGGACTAATATAAATCGTTATACTATTATCAGCGAAATTGGTGAGGGGGGGATGCAAAAGGTTTACCTTGCGAATGATAAGATATTAAATAGGCAAGTTGCTCTTAAGACCCCTAAAAATAAGTCTGCTGAAAAACGATTCCATAGAAGTGCTATTTTAGCATCTAGGGTCAACCATCCTAACGTCGCTAAAACATTAGATTATTTTGCCGAAGATGGACGTGAATTTTTAACGGAGGAATTTATCGATGGAGTAGATCTGGATAAAGCATTGTTGAGTAGCTATACAAGTGTTGATCCTTACTTGACTGCAAAGATATTTCATAACTTAGCGAAGGCTCTTTCGGCTTCCCATCATGTGGATGTAATACATCGAGACCTCAAACCTTCTAACATAATGGTTATTGGAGGAGTTAGTGCTACAGGTGTTAAAATCACCGATTTTGGAATTTCAAAAATGGCCGGTGATGAAATTGATGAGGCCGCAAAGAATGGGCAAGGATCGATTACTTCATCTCAAACAGCTATGGGGGCATTGCCATATATGGCCCCGGAAATTATACAAAGTCAGGGGCAAGTTTCAAAACCATCTGATGTCTGGGCATTAGGTGCGATGATGTTCAGAATCCTCACGGGAGAGTATCCTTTTGGATTAGGGTATATGGCTATTCCGAACATCTTATCTGGAAAGCATACTCAATATCCTGATTTTATTAAGTCAAATAAGCAGTTTGCTCCGCTGGCAAATGAAATTATAGATATAATTGAAAAATGTTTAAATCTAGACCCTTCTAAACGCCCCACTGCAGATGAGCTCGTGTCATTATGTGGTCAATTATGCTATCCGGTTTGTAATAGAGAAGAAGGAGTAATAGGTGATACTAGACTAGCTTATGGTTTCATTCGTATACCAAACCAACCACAAGTATTTTTTCATTACGATAGTGTGTATGGTAGTAAACCAGTGAGTAATGATAAGGTGATTTTTTCAAAGTTCTTGGGAGGGGGCCATGACCGGGCTCATCCAGTTATCAAGGCTAAGTAG
ATGTTTACAGAACGACTTGCTCGCTGGTTAGCTCGTTCTTCGGCCAAAAGCGGCATTAACCGGCCAGAAGACCTCAACGCTGTCCTTAGCACGGATATAGGACTGGTTAGAGCTGAGAATCAGGATCTAATAGCCGCGATTAGAGTTAACACTCCGTCAAACGTTGGCAATCCTTTTTTTGCAATGGCGTTATTAGATGGCATGGGTGGAATGCAAGATGGAAAGCAATGCGCAACAATTGCTTTATCAACTTTATTCTATTCTTTGATTAAGTTTAGAAGTGATCCTCCCGAGTCTCGGTTATTAAAAGCGACTTTAGAAGCAAACTCCGTTGTATATGACTATGCAAAAGGACATGGCGGTTCAACATTATCCGCTGTAATTATTGAAAATGGGTCTGCTCCTGTAATTGTCAACGTTGGCGACAGTCGAATATATAGCTTTTCTCTGGACTGTGGACTTACAGCAATTAGCAGTGATGACTCTCTAGAAGCATTGGGTGGCAGAGGGCGCGGATTACTCCAGTTTATAGGAATGGGGGAATCTATAAAGCCTCATATCAATATCTTAGATAAAAATCATAAAAATATAATATTGACATCCGATGGAACTCATTTCATTTCTCATTCAGCATTCGAAGAGTTATTAAGTCATTCATCTGATTTTTCTACATCAGCGCAGAGAATAGCTCAATATGTTCAATGGTGCGGTGCGAAAGATAACGCTTCATTTGGAATTATTAATTGCAATGATATAGAAAACAGCCTCAACTCCCATAAAGATATTGGTGTAGAGTTATGGGACCCTCATGGGAATCTACATATCATGTGGATGAAAAATTATCCTGCAGCGCAGAATTACTTCTCTCAAAATATAGTGGATGATCAAGATAAAGAACCTTCACCTATTATAGACGATGATGGTTTTGAAAATAAAAAAACACTAAACAATCCTTCAACAAAAAATCTAGAATTAGATTCTGAAACACCACAAAGAGAGCTATTCTCAAACGAATCCCCAGAAAAATCTCAAGATCCATCCATTACAAGCAAAGCAATCAAACAAAGAAAAAACAAAGATAAAAAGAAAGCTATTGAAAAGATCAAAAAAGACCAATCTGTAATGATAAATATCAAGGATGAGGAAA ACAAAAATGAAGATTAATCACGTCCTACCAGAAAGATATTCATTAAAGAGCACTGAACTCGGTGGTGGCATGGGGGACATTTTAATATGTAAGGATAATCATTTAGATAGAGATGTAATTGTAAAGTTGTTAAAGGATGGAGAAGAAGAGAGACGTTTACTAGATGAACAGAAAGCGCTACTCAAACTTCGTTCTAAACATGTAGTACAACTTTATGATTTAATTGACATAACAGTCTCCGAAAAAACTAAAAAAGGATTAGTTCTGGAGTATATTAACGGAGTGGATTTAAATTATAACCCGTCAGAAAGTCACCCCGAAAAACTAAAGAAATTATGGCAGATAGCATGCGGGTTAAGTGACATCCACTCTGCTAAAGTAATTC ACAGAGATATAAAGCCCAATAATATTAGAGTAGACGAGAATAAAATTGTTAAAATATTAGATTTTGGTTTAGCAAGGACCTCAGGCACAGAAGCATTCACTCATTCTGTTATTGGAACCTTAGGATATATGGCTCCTGAACTATGGAAGAGAAAAAACATTAGTTTCGATCAAAAAATTGATGTTTATGCATATGGTGTCCTCGTTTTAGATTTATTCGGCATAGAAAAACCAGATGAATTATACGAACATCCCCCGGCCGCGATAACCAATATACCTGAATTAGGAAAGATACTTCCAAAGGACTTAGCCAGAACTTTCATTAGTTGCTTAAGCCATGACAAATATGCTCGACCGGCAATGTCTTCGGTTAGAGATCAAATAGCTAAATACCTATTAAAAGATAGACACCGTGCCCTCTTCGTCCTGAATGGAAAGAAATATGAAATAAATGCTAAAAATAAAAGTGTTACGATCACTTGGGGTACTAGTGGAAGTATGGAAATAGTATATGATGGTTTCGACTTTAAAGTAGGTAATTTTTCAGGAAGTGCGACAATTAATAACCAACAAGTGATAACAAATAAAGTATTTCCTAGCTGCAGTGTAATTACTCTTATAAATGAAAAATCTAGAAGCTTTGTCACCTTTGATATATCTAGACCGGAGGTAATATCATGATTGAAGTCGGAAGAATCATTGCAGAACGCTATAAGATTTTATCTTATGTTGGTAAAGGTGGGATGCAAGACGTATATAAAGTTTTAGATCTAAAGCTGGATTTAGATTTAGCTTTAAAGACTCCACTTCCTGGTTTGGAAAGCAAAAGATTTCTTAAAAGTGCCAAAATCGCTGCAAAAATAAACCATCACAATATAGCAAAAACCTTTGATTATGTTGAAGATAATGGCAATATATTTTTAGCGGAAGAATTTGTTGAAGGTGAAAACCTTGAGGAAAAATTGCGCCACTTTGATTTTTTAGACCCACATTATGGCGCTTGTATACTACATAACCTTGCTAAAGGAATAATGGCATCTCATAAAGCAGATGTTATTCATAGAGATTTAAAACCGAGTAATGTAATGGTGTCTGGCGGAGTACAAATTTCGAATCTAAAAATTACAGATTTTGGAATAGCTACGCTAACACAAGAACTTTTTGATGAAGCTGCTGCCAGTGGTGACCTAACAAGATCGACCTCTGGTACGATAAAAGGTGCTTTACCCTTCATGTCTCCTGAATTAATGTTTGGTAAAAAGGGTAAACCTATAGAAGCATCAACTGATATTTGGCCATTAGGTGCAATGATGTTTAAATTATTAACAGGAGACTATCCATTTGGCGTTTATTTAGATGCTGCTGTTAATGTTAAAACAAAAAATAGAATGGAATGGCCAACCTTTATGACTGCCAATCCACAATATCAAAGTTTATCACAAGATCTACAAAAAATTGTAGATAAGTGCTTAAGTTATGACTCGGACAAGAGGCCTACAGCTGAAACGCTTGTTAAGGCATGTGAAACCCTTTGTTATTTATCCGAAGAACGTCATGTCGGTCGCGTCAATAATCTTATTCAAGGAGGGATAAGTGGATTTATTGATGGGACACCTTCTAACTCCTTTTTTAGTATGGAAAGTGTATACGGTTCGAGGTACCCAAACACCTCAACAAGAAATACTGTTTGTTATTCCACTTTTGATGGACACCCATGGCCTAGAGCACACCCTGTAATTCTTTGGAAAGATTGA
ATGCAAGATATATTAACAAAAAGGCTAAATAGACCTGTAAATGGGAACCGGTCATCCGTTGTACATGAAGTCGGAGCGACGTTAGCGACAACTGTCGGGCTTATCAGAACTGAAAATGAAGATCGGGCAATTTCTGCTCGTTTCTATAGTTCAAAAATGGAGCGCTACATATACTTTTATATTCTTAGTGATGGTATGGGAGGTATGGTTAATGGGGGGCTTGCCGCAACTCACACTGTCTCAATGTTTTTAAGCTCTATAATACCTCTTATCGAGTCAGGCATTGAAATAGATCAAGCGATACAACAGTCTGTGTTTTCTGCTCATCAGATCGTTTCCGAATCAACAAACGGTAAAGGTGGAGCAACTTTATCAGCTATAGTCAGCCATGAACCAGGTGAATTCTTTACCGTCAATGTTGGTGATAGTCGAATTTATCAATGCACAACAAGCAATTCTATTTTTCAAATCACTGAAGATGACGATGTAAAAAGCTTCCTCGAAAAACTCAACGGAATCGAACTAAACAGCCTAATAACGAAGAGAAATGGTCTAACAAAGTATATAGGCATGGAGGGAGAATTAGAGGTCACTGTTGAGTCATGCTCTGCACTCTGCGATTTGTTAGTTATTTCTGATGGTATAAGCCAAATTGGAGAAGCTAACTTAGTTGGCCTATATGAAAATAAGACAACCGATAGTGAATTCGTTCAACGTTGTATACATTTGTCCAATTGGCTTGGCGGTCATGATAACGCAACAGCAATCTATGCTTCTTTGGCAAACCTAACATATCACCCAGAAACTACCGAAGTCACCCATAACTGCCTTGAAGTTTGGGATTGCCATGGTTACATCATTATTCCCTTAGCACAACTACCTTCGAAAGGTAGACACCCAAAAGAAAAAACGAAAAAAGTACGAAAAAAAACAGCTGCGAAAAAAATAATAAAAAGTGATAAAGAAGACTCTCGCAACGATTATAACCTAGAGATAAATCAAAAATCATTATTCTCATCTGACGATGTCGGAGAGATTTCGCCTGATAATAACAGTGTATTTGATGAAGGTGCAGGACTTGACCTAGCCAAAATCGAAGATAATAAAAATAATCTAGATAAATAGAATTAAACGTTGGTGACAATTATGGCTAGTACAAGATATGAATACCTAAAACACATAGACGATGGCGGTTATGGTAGTGTCTCTCTTTATAGAGATATATTCCTTGATCGTGAAGTCGCAATAAAAACAATCCCCATTAGTAAGAAGAACAATACGATTGAGGAAGTAAACCTACTCAAATCAATTGCGTCTAAGCATGTTGTTGGATTGTATGACGTTATTCAAACCCAAACTAACATTGAAATATATCAAGAATACTTAGATGGGGAAGATCTAGCTAGCAAAGTAGGTCAGTGTCATGGCCCAGAATTTTTGAGCCTTGCCTATCAGCTAGCATCTGGACTACGTGATATACATTCATCAGGAATATGTCACCGAGATATAAAGTTGGATAATGCGAAATTTGACAAACAAGGTGTACTTAAAATTTTTGATTTTGGTGTATCTCGTATCGGTGATCCGCACCAAACCGTAAATGGTCATGGTACATTAGAATACCTAGCTCCTGAAGCCTTTGGATTATACACGCAAGACTCCGTTGTACTTTCATTTGCGGTCGATATCTATGCACTTGGAGTGACCTTACACAAACTTGCATTTTCGGGTATATGTAAATTCAATAAAACGCTTAATCCGCAACCAGAAAGCCCTCGATTTGCTGAGCTTGGCTTTTGTTCAAAGCTAACATCACTCTTAAATAAATGTGTTTCAGAAAATAAAACGGAGAGACCCTCAGCAAACAGTTTAGTTGCTGAATTACAAAGAGAGCTCCTACGAAATAAACATACTGGTTTGTTTGTTGCACCGAAAGGCTCCCATAATATTGACCAAGCTCATCCAAAAACAAGAATTAAAATTTCTGATGACTTGTCAATTATTATCAATTACAACGGATACGACTTTTATGTTACTAAAGTTGAAGGACATGTTTATATAAATAATGAAATTGTAGATGTGGGTAAGGTGCTTTATGGAGCATGCGTCCTGACATTTGTTCGAGATGCCAATAACCGTTTCTTTGTTTCGTTTTCTTCATCTCATCCGGAGATAGTATTATGAGTCATATTCACAAACCTAACGACATCATTGCAGATCGATACGTGATCGAAAATTATATCGACGAAGGTGGTATGCAACAAGTCTACTCAGCGATAGACAAAAACATTGGACGAAAAGTCGCTTTAAAGACACCAAAAAATGATTCGGCTAGCCTGAGATTTAAAAGTACCGCAATTTGTAGTGCTCGTGTTATTCACCCTAACGTGGCGAAAACTCTAGATTACTTTGGCTTTGATAAACGCGAATACTTAATTGAAGAACTTATTGACGGAAAAGATTTAAACACTGTATTCAGAAATAATTTCTCATATTTAGATCCATGTCTCGTTGCCTTCATAGGACATCATCTATCAAAAGCAGTAGCTGCATCTCATAGCGCAGACGTCGTCCAT AGGGATCTCAAACCAAGTAACATTATGATTGTAGGGGGCGAGAAGTTTAGAGATATTAAAGTCACTGACTTTGGTATTGCAAAACTTGTTGATGATGAAATCAACGAGGTTTTTTCTGATACCGAAAATGTTGAAAGCTCCATTGCTGGCTCAAAAACACTCGTTGGTGCTCTACCTTATATGGCCCCAGAAATTGTTTTAAACAAAACAAAAGCTGGAAAACATATTGATATTTGGTCAATAGGTGCAATTATGTATTTCCTACTAACAGGAAAAACACCTTTCACATCGCAATTTGCCCAAATCGTCATTAACTATCATACTCAGAAATCTATCGATCCAATCCTTCATATGGATACATCTCGCCATTTAAACCCGTTAGGCAATCAATTACTAGGCATTATCAAAAGTTGCTTGGATTACGACTATTCAAATCGTCCAAATTCTGAGCAATTAGTTCAAATGTTTTCTTCCCTTTGTTACCCTATTCAAGAAAGAAAATATGGACATATCAAGTATCGACGAGGCACACATGGTTGGGGCTTCATTAAAAACATAGGCCCTAACGATACATTTTATCATACTGAAGAAGTTTTTGGACTTCAGGCTTCCCATAGTGAGCGAGTGTGCTTCTCTGAGTACCCTGGATTACCTCAAGCAAGGGCATTTCCAATTATTCGCTGTAAATAA。

Claims (4)

1. The novel anti-phage element is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. A chassis cell comprising the novel anti-phage element of claim 1, said chassis cell being the pathogenic pseudomonas aeruginosa PAO1.
3. Use of the novel anti-phage element of claim 1 for increasing the resistance of a chassis cell, said chassis cell being the pathogenic bacterium pseudomonas aeruginosa PAO1, said phage being the phages PAP8, PAO-L5, QDWS, PAP-L5.
4. A method for improving the phage infection resistance of chassis cells, which is characterized in that the novel phage-resistant element of claim 1 is transferred into chassis cells to enhance the resistance of a host to phage, wherein the chassis cells are pathogenic bacteria pseudomonas aeruginosa PAO1, and the phage are phages PAP8, PAO-L5, QDWS and PAP-L5.
CN202210879453.0A 2022-07-25 2022-07-25 Novel anti-phage element and application thereof Active CN116334108B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210879453.0A CN116334108B (en) 2022-07-25 2022-07-25 Novel anti-phage element and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210879453.0A CN116334108B (en) 2022-07-25 2022-07-25 Novel anti-phage element and application thereof

Publications (2)

Publication Number Publication Date
CN116334108A CN116334108A (en) 2023-06-27
CN116334108B true CN116334108B (en) 2024-03-26

Family

ID=86882757

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210879453.0A Active CN116334108B (en) 2022-07-25 2022-07-25 Novel anti-phage element and application thereof

Country Status (1)

Country Link
CN (1) CN116334108B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899450A (en) * 2010-04-27 2010-12-01 中国科学院水生生物研究所 Virulent cyanophage PaV-LD genome and preparation method and application thereof
CN108410840A (en) * 2018-04-03 2018-08-17 大连理工大学 A kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and application
CN111849848A (en) * 2020-07-23 2020-10-30 江南大学 Construction and application of bacteriophage-resistant escherichia coli chassis cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899450A (en) * 2010-04-27 2010-12-01 中国科学院水生生物研究所 Virulent cyanophage PaV-LD genome and preparation method and application thereof
CN108410840A (en) * 2018-04-03 2018-08-17 大连理工大学 A kind of Pseudomonas aeruginosa phage endolysin and its encoding gene and application
CN111849848A (en) * 2020-07-23 2020-10-30 江南大学 Construction and application of bacteriophage-resistant escherichia coli chassis cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Excisionase in Pf filamentous prophage controls lysis-lysogeny decision-making in Pseudomonas aeruginosa;Yangmei Li等;《Mol Microbiol .》;第111卷(第2期);第495-513页 *
Fine structure analysis of lipopolysaccharides in bacteriophage-resistant Pseudomonas aeruginosa PAO1 mutants;Libera Latino等;《Microbiology (Reading) . 》;第163卷(第6期);第848-855页 *
细菌抗噬菌体防御***研究进展;贡嘉澳等;《生命科学仪器》;第20卷(第02期);第17-26页 *

Also Published As

Publication number Publication date
CN116334108A (en) 2023-06-27

Similar Documents

Publication Publication Date Title
Smith et al. Comparative genomics of Shiga toxin encoding bacteriophages
Shariat et al. CRISPRs: molecular signatures used for pathogen subtyping
Asadulghani et al. The defective prophage pool of Escherichia coli O157: prophage–prophage interactions potentiate horizontal transfer of virulence determinants
Forsberg et al. Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome
Weigele et al. Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus
Pope et al. Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4
Tao et al. Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9
Summer et al. Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex
Chénard et al. Viruses infecting a freshwater filamentous cyanobacterium (Nostoc sp.) encode a functional CRISPR array and a proteobacterial DNA polymerase B
Kot et al. DPS–a rapid method for genome sequencing of DNA-containing bacteriophages directly from a single plaque
Willner et al. From deep sequencing to viral tagging: recent advances in viral metagenomics
Park et al. Evolution of the Stx2-encoding prophage in persistent bovine Escherichia coli O157: H7 strains
Amy et al. Identification of large cryptic plasmids in Clostridioides (Clostridium) difficile
Kvitko et al. φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity
Yoshida-Takashima et al. Genome sequence of a novel deep-sea vent epsilonproteobacterial phage provides new insight into the co-evolution of Epsilonproteobacteria and their phages
Garin-Fernandez et al. Looking for the hidden: Characterization of lysogenic phages in potential pathogenic Vibrio species from the North Sea
Demina et al. Virus-host interactions and genetic diversity of Antarctic sea ice bacteriophages
CN116334108B (en) Novel anti-phage element and application thereof
Merabishvili et al. Digitized fluorescent RFLP analysis (fRFLP) as a universal method for comparing genomes of culturable dsDNA viruses: application to bacteriophages
Owen et al. Detection of bacteriophages: sequence-based systems
US20210403995A1 (en) High-throughput methods to characterize phage receptors and rational formulation of phage cocktails
Hendrix Phage evolution
Zhang et al. Unexplored diversity and ecological functions of transposable phages
Muntyan et al. BACTERIOPHAGES OF SINORHIZOBIUM MELILOTI NATIVE TO ALFALFA ORIGIN OF DIVERSITY AT THE CAUCASUS
Koert et al. Evidence for shared ancestry between Actinobacteria and Firmicutes bacteriophages

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