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WO2018030578A1 - Composition de régulation de la formation de biofilms, et procédé de régulation de la formation de biofilms au moyen de la composition - Google Patents

Composition de régulation de la formation de biofilms, et procédé de régulation de la formation de biofilms au moyen de la composition Download PDF

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WO2018030578A1
WO2018030578A1 PCT/KR2016/011603 KR2016011603W WO2018030578A1 WO 2018030578 A1 WO2018030578 A1 WO 2018030578A1 KR 2016011603 W KR2016011603 W KR 2016011603W WO 2018030578 A1 WO2018030578 A1 WO 2018030578A1
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seq
microorganism
biofilm formation
formation
composition
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Korean (ko)
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이영훈
박근우
이정민
석신애
김다운
이지영
김광선
최병석
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Korea Advanced Institute of Science and Technology KAIST
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention provides a composition for regulating biofilm formation of a microorganism comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism, a recombinant vector comprising the nucleotide, a transformed cell transformed with the recombinant vector, and the recombinant vector It relates to a method for regulating the biofilm formation of a microorganism comprising the step of transforming.
  • Biofilm refers to a three-dimensional form of microbial community formed by microorganisms to protect themselves from various environmental factors. Usually, a polysaccharide matrix is produced and secreted so that it sticks well to the solid surface and microorganisms. It is resistant to various environmental factors, including antibiotics, and is difficult to remove, causing a wide range of problems and repeating the cycle of adhesion-growth-deletion.
  • biofilm is a structure formed by microorganisms sticking to the surface, which can occur in most environments, and tends to increase viability in an environment that inhibits growth such as undernourishment or antibiotics.
  • Bacteria secrete multimeric extracellular matrix components to protect themselves from various environmental factors, forming three-dimensional structures called biofilms on solid surfaces or living tissues.
  • biofilms are reported to occur in about 80% of the body's microbial infections, and problems arise because of biofilms that allow microorganisms to survive for a long time in the medical device or food industry, such as catheters and dialysis devices.
  • biofilms are difficult to remove and cause a wide range of problems such as growth of bacteria in living organisms, tartar, contamination of the surface of medical devices, contamination of various industrial facilities such as water pipes, water purifiers, etc.
  • the market focuses on the removal of microorganisms with antibiotics to block the production of biofilms or to coat the surfaces on which biofilms are to be produced so that bacteria do not stick (J Intern Med. 2012, 272: 541-561).
  • sRNAs are RNA molecules that do not encode proteins but play a central role in regulating various cellular metabolism in the cell. About 100 species of bacterial sRNA are known, which are composed of about 100 bases on average, and mainly target specific mRNA populations to control protein expression so that cells can respond quickly to various stresses.
  • the present inventors construct sRNA expressing plasmid libraries by constructing plasmids capable of expressing sRNA, respectively, and utilizing the aggregates to form biofilms and cause ciliary formation, herd mobility and / or swimming mobility, etc.
  • the sRNAs that can regulate the were found.
  • composition for controlling biofilm formation of a microorganism comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism.
  • Still another object of the present invention is to provide a method for controlling biofilm formation of a microorganism, the method comprising transforming the microorganism into a recombinant vector comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism.
  • the present invention does not encode a protein, but the production of plasmids that can express sRNAs that play a pivotal role in the regulation of various metabolism in cells and the formation of sRNAs and biofilms that can control the formation of biofilms using the same.
  • the present invention relates to an sRNA capable of regulating physiological changes related to physiological changes (bacterial motility, type 1 cilia formation, and curly cilia formation).
  • Biofilms are three-dimensional clusters of bacteria formed by bacteria to protect themselves from various environmental factors. Usually, they form and secrete polysaccharide matrix materials that can adhere to solid surfaces and microorganisms. It also plays a role in strengthening environmental resistance. Because of this, biofilms cause antibiotic resistance, chronic diseases, contamination of medical devices, and corrosion of industrial piping.
  • biofilms in microorganisms occurs through very complex physiological processes.
  • bacterial motility floating mobility, swimming mobility
  • type 1 cilia formation a type 1 cilia formation
  • curly cilia formation a type 1 cilia formation
  • sRNAs that affect the swimming mobility, herd mobility, type 1 cilia formation, and curly cilia formation are known.
  • One embodiment of the present invention relates to a composition for controlling biofilm formation of a microorganism, including a nucleotide expressing a sRNA capable of regulating biofilm formation of the microorganism.
  • sRNA expression plasmid aggregates were constructed to utilize the action of sRNA for biofilm inhibition. 99 sRNAs were selected from the genomic DNA of E. coli, and the beginning and the end of the sequences were identified to ensure the overexpression of each sRNA. If each sequence was not identified, the plasmid was further included as a plasmid. The sequence to be cloned was determined.
  • the sequence was amplified using PCR, the plasmid was digested with restriction enzymes, the amplified sequence was cloned, and transformed into E. coli to obtain a plasmid aggregate. Then, the expression efficiency of the plasmid aggregate was verified by confirming the expression level of the sRNA whether the sRNA overexpression by the plasmid works well under the set conditions.
  • biofilm-related phenotypic changes such as bacterial biofilm formation, swarm mobility, swimming mobility, type 1 cilia formation, and curly cilia formation are analyzed to determine biofilm formation or related SRNAs that control phenotypes were identified.
  • Composition for controlling the biofilm formation of the microorganism is SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 77, and one or more nucleotides consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 94 Can be.
  • composition for controlling biofilm formation of the microorganism is SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 55, It may be for inhibiting the biofilm formation of a microorganism comprising one or more nucleotides consisting of a nucleotide sequence selected from the first group consisting of SEQ ID NO: 57, SEQ ID NO: 59 and SEQ ID NO: 77.
  • composition for controlling biofilm formation of the microorganism includes at least one nucleotide consisting of a base sequence selected from the second group consisting of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 47, SEQ ID NO: 63, and SEQ ID NO: 94 It may be, for promoting the biofilm formation of microorganisms.
  • Control of biofilm formation of microorganisms means that biofilm formation is increased or decreased 1.5 times or more compared to wild-type microorganisms. Specifically, inhibition of biofilm formation is at least 1.5-fold reduction of biofilm formation compared to wild-type microorganisms. This means that the biofilm formation is increased by 1.5 times or more compared to wild type microorganisms.
  • the nucleotide may be to control the biofilm formation of the microorganism by controlling one or more selected from the group consisting of factors related to the biofilm formation of the microorganism, I-type cilia formation, curly cilia formation, swimming mobility and herd mobility.
  • a nucleotide consisting of a nucleotide sequence selected from the third group consisting of SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 50 has an effect of controlling the formation of type I cilia of the microorganism.
  • the nucleotide consisting of the nucleotide sequence selected from the fourth group consisting of SEQ ID NO: 25, SEQ ID NO: 47, SEQ ID NO: 57, and SEQ ID NO: 63 has an effect of controlling the formation of the cilia of the microorganism.
  • the nucleotide consisting of the nucleotide sequence selected from the fifth group consisting of SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 36, and SEQ ID NO: 94 has an effect of controlling the migration mobility of the microorganism.
  • SEQ ID NO: 1 SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, sequence A nucleotide consisting of a nucleotide sequence selected from the sixth group consisting of SEQ ID NO: 63 and SEQ ID NO: 94 has an effect of controlling the micromigration of microorganisms.
  • the microorganisms may be prokaryotic or eukaryotic, for example, Escherichia coli (escherichia coli), Rizzo Away (Rhizobium), Bifidobacterium (Bifidobacterium), Rhodococcus (Rhodococcus), candidiasis (Candida), El Winiah ( Erwinia), Enterobacter (Enterobacter), par Stephen Pasteurella (Pasteurella), Mendoza high Mia (Mannheimia), liquid Tino Bacillus (Actinobacillus), Agde cases tee bakteo (Aggregatibacter), janto Monastir (Xanthomonas), Vibrio (Vibrio), Pseudomonas (Pseudomonas), azo Saturday bakteo (Azotobacter), trying Cine Saturday bakteo (Acinetobacter), Lance Estonia (Ralstonia), Agrobacterium (Agrobacterium), Rizzo Away
  • Another example of the present invention relates to a recombinant vector comprising a nucleotide expressing an sRNA capable of regulating the biofilm formation of a microorganism.
  • the recombinant vector is SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 77, may include one or more nucleotides consisting of a nucleotide sequence selected from the group consisting of.
  • vector means a means for expressing a gene of interest in a host cell.
  • viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors are included.
  • Vectors that can be used as recombinant vectors are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phage (e.g., ⁇ gt4? B, ⁇ -Charon, ⁇ z1 and M13, etc.) or viruses (e.g., SV40, etc.) can be produced, but not limited thereto. It doesn't work.
  • the recombinant vector can typically be constructed as a vector for cloning or a vector for expression.
  • the expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms.
  • the recombinant vector may be constructed through various methods known in the art.
  • the recombinant vector may be constructed using prokaryotic or eukaryotic cells as hosts.
  • a strong promoter for example, a pL ⁇ promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, and a T7
  • ribosome binding sites for initiation of translation and transcription / detox termination sequences.
  • replication origins that operate in eukaryotic cells included in the vector include f1 origin, SV40 origin, pMB1 origin, adeno origin, AAV origin and BBV origin.
  • promoters derived from the genome of mammalian cells eg, metallothionine promoters
  • promoters derived from mammalian viruses eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus promoter and tk promoter of HSV
  • adenovirus late promoters e.g., vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus promoter and tk promoter of HSV
  • Vectors of the present invention may include antibiotic resistance genes commonly used in the art as optional markers, for example ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.
  • antibiotic resistance genes commonly used in the art as optional markers, for example ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.
  • Another example of the invention relates to a cell transformed with a recombinant vector comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism.
  • the transformed cells are SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 55, SEQ ID NO: 57, sequence No. 59 and SEQ ID NO: 77 is transformed with a recombinant vector comprising one or more nucleotides consisting of a nucleotide sequence selected from the group consisting of, the transformed cells may be inhibited the formation of a biofilm.
  • the transformed cells are transformed with a recombinant vector comprising one or more nucleotides consisting of nucleotide sequences selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 47, SEQ ID NO: 63, and SEQ ID NO: 94
  • the transformed cell may be one that promotes the formation of a biofilm.
  • Cells used in the present invention may include E. coli, yeast, animal cells, plant cells, insect cells and the like, prokaryotic cells, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E.
  • coli W3110 Bacillus subtilis, Bacillus genus strains, such as Bacillus thuringiensis, and Salmonella typhimurium, Serratia martensons and various Enterobacteria and strains such as Pseudomonas species, and when transforming eukaryotic cells, as a host cell, yeast (Saccharomyce cerevisiae), insect cells, plant cells and animal cells, such as Sp2 / 0, CHO (Chinese hamster) ovary) K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell line, etc. may be used, but is not limited thereto.
  • the transport (introduction) of the polynucleotide or the recombinant vector including the same into cells may be carried by a transport method well known in the art.
  • a transport method well known in the art.
  • the host cell is a prokaryotic cell
  • a CaCl 2 method or an electroporation method may be used.
  • the host cell is a eukaryotic cell
  • a micro-injection method, calcium phosphate precipitation method, electroporation method, Liposome-mediated transfection and gene bombardment may be used, but is not limited thereto.
  • the method for selecting the transformed cells can be easily carried out according to methods well known in the art using a phenotype expressed by a selection marker.
  • the selection marker is a specific antibiotic resistance gene
  • the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.
  • Another embodiment of the present invention relates to a method for regulating biofilm formation of a microorganism, the method comprising transforming the microorganism into a recombinant vector comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism.
  • Biofilm formation control method of the microorganism SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 55, sequence Transforming the microorganism into a recombinant vector comprising at least one nucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 77, wherein the microorganism may be inhibited in biofilm formation .
  • the biofilm formation method of the microorganism may include a recombinant vector comprising at least one nucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 63, and SEQ ID NO: 94 It includes the step of transforming the microorganism, the microorganism may be to promote the biofilm formation.
  • nucleotides, recombinant vectors and transformations are as described above.
  • the present invention provides a composition for regulating biofilm formation of a microorganism comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism, a recombinant vector comprising the nucleotide, a transformed cell transformed with the recombinant vector, and the recombinant vector
  • the present invention relates to a method for regulating biofilm formation of a microorganism, the method comprising transforming the microorganism into a biofilm, and controlling the motility of the biofilm, the fimbriae, and the bacteria formed by the microorganism.
  • 1 is a cleavage map of the pHMB1 vector used to prepare an sRNA expression plasmid aggregate according to an embodiment of the present invention.
  • FIG. 2 is a table showing 99 sRNAs according to an embodiment of the present invention.
  • Figure 3 is a graph showing the ratio of the 99 known sRNA function is known and unknown according to an embodiment of the present invention.
  • FIG. 4 to 7 are photographs showing the results of observing the degree of biofilm formation in each strain when overexpressing each sRNA in E. coli with 99 sRNA expression plasmids according to one embodiment of the present invention.
  • FIGS. 8 to 10 are graphs showing the results of observing the degree of biofilm formation in each strain when overexpressing each sRNA in E. coli with 99 sRNA expression plasmids according to one embodiment of the present invention.
  • 11 to 14 are photographs showing the results of experiments to determine whether the coliform and yeast aggregation by the expression of type I cilia according to an embodiment of the present invention.
  • 15 to 16 are photographs showing the results of experiments to determine the difference in dye uptake according to the expression of the curly cilia in LB medium Petri dish to which Congo red dye is added according to an embodiment of the present invention.
  • 17 to 20 are photographs showing the results of measuring swimming mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • 21 to 23 are graphs showing the results of measuring swimming mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • 24 to 27 are photographs showing the results of measurement of strain mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • 28 to 30 are graphs showing the results of measurement of strain mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • FIG. 31 is a van diagram showing sRNAs affecting biofilm formation, formation of type I cilia, expression of curly cilia, swimming mobility, and herd mobility when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • the cut plasmid 50uL ( ⁇ 2ug) and 10x restriction enzyme reaction solution D (Promega, USA, R9921) 10.0 uL, distilled water (Nuclease-free water, NFW) 38.0 uL, EcoRI restriction enzyme (Promega, USA, R6011) 1.0
  • 100.0 uL of reaction solution containing uL (10 U) and 1.0 uL (10 U) of XbaI restriction enzyme (Promega, USA, R6181), react at 37 ° C for 2 to 3 hours, and then transfer to 65 ° C. Reaction was inactivated for a minute.
  • the reaction solution in which the restriction enzyme was inactivated was electrophoresed on a 1% agarose gel, and a DNA band of about 4300 bp was cut out of the gel to obtain a spin column type gel extraction kit (Intron Biotechnology, Korea, 17288).
  • DNA was purified, dissolved in 30-50 uL of distilled water, and the purity and amount of DNA were measured using Nanodrop (Thermo Scientific, USA). As a result, it was confirmed that the purity is about 2.0 with an OD 260 / OD 280 value and the linear DNA concentration is 30 ng / uL or more.
  • the whole DNA was purified using a spin column type E. coli whole DNA extraction kit (Intron Biotechnology, Korea, 17046), and the purity and amount of DNA were measured using Nanodrop (Thermo Scientific, USA). As a result, the purity was OD 260 / OD 280 value of about 2.0 and the concentration was confirmed to be more than 1 ug / uL.
  • PCR was performed based on the whole DNA of E. coli extracted in Example 2-1.
  • reaction solution as shown in Table 1 using a PCR equipment at 95 ° C. for 2 minutes
  • reaction of 95 ° C. 30 sec, 58 ° C. 60 sec, and 72 ° C. 30 sec was repeated 35 times, followed by 72 ° C. 2.
  • Reaction was carried out for 25 minutes to 25 minutes to obtain a reaction product.
  • the resulting reaction product was then analyzed using electrophoresis on an agarose gel, followed by electrophoresis on a 1% agarose gel if it matched the expected product size, and DNA bands matching the expected size were cut out of the gel.
  • DNA was purified using a spin column type gel extraction kit (Intron Biotechnology, Korea, 17288), dissolved in 20 to 30 uL of distilled water, and then measured for purity and amount of DNA using Nanodrop (Thermo Scientific, USA). .
  • the purity was OD260 / OD280 value of about 2.0 and the linear DNA concentration was confirmed to be more than 50 ng / uL.
  • DNA sequence fragments are treated with restriction enzymes before introduction into the vector
  • the PCR reaction product obtained in Example 2-2 was digested using EcoRI restriction enzyme (Promega, USA, R6011) and XbaI restriction enzyme (Promega, USA, R6181). 20 uL ( ⁇ 1 ug) of the DNA fragment to be digested and 5 uL of 10x restriction enzyme D (Promega, USA, R9921), 25 uL of distilled water (Nuclease-free water, NFW), EcoRI restriction enzyme (Promega, USA, R6011) 1 uL (10 U), XbaI restriction enzyme (Promega, USA, R6181) A total of 50 uL of a reaction solution prepared by mixing 1 uL (10 U) was prepared, and reacted at 37 °C for 1 to 2 hours, 65 The restriction enzyme was inactivated by transferring to °C and reacting for 20 minutes.
  • EcoRI restriction enzyme Promega, USA, R6011
  • XbaI restriction enzyme Promega, USA, R6181
  • RNA expression plasmid DNA prepared in Example 1 and the DNA fragment prepared in Example 2-3 were mixed so that the final volume was 4 uL or less and the molar concentration was 1: 150. Then, 5 uL 2 ⁇ Rapid Ligation buffer (Promega, USA, C6711; 60 mM Tris-HCl, pH 7.8, 20 mM MgCl 2 , 20 mM DTT, 2 mM ATP and 10% PEG), 1 uL T4 ligase ( Enzynomics, Korea, M019) was added and distilled water was charged up to 10 uL, and reacted at 4 °C for 10 hours to bind the DNA fragment to the plasmid.
  • Rapid Ligation buffer Promega, USA, C6711; 60 mM Tris-HCl, pH 7.8, 20 mM MgCl 2 , 20 mM DTT, 2 mM ATP and 10% PEG
  • 1 uL T4 ligase Enzynomics, Korea, M019 was added
  • a reaction solution containing E. coli transformed in Example 3-1 1.0 mL of LB medium prepared by dissolving 1.0 g of NaCl, 0.5 g of yeast extract, and 1.0 g of tryptone in 100 mL distilled water was added, and 100 ml LB.
  • LB / Ap agar medium plate prepared by adding 1.5 g of micro agar (Duchefa, Nederland, M1002) and ampicillin concentration to 100 ug / mL in a medium, in a 37 ° C shaking incubator rotating at 250 rpm. 300 uL of solution was applied and further incubated overnight in a 37 ° C. incubator.
  • Plasmid DNA in cells of colonies obtained from Example 3-2 was obtained according to the manufacturer's protocol using a plasmid DNA purification kit (Intron Biotechnology, Korea, 17096), followed by nucleotide sequence using pBAD-reverse sequence (SEQ ID NO: 100). Verified through analysis.
  • the obtained plasmid DNA is a plasmid aggregate which can overexpress 99 ncRNAs inserted respectively.
  • the sRNA overexpression plasmid obtained in Example 3-3 was introduced into E. coli by electroporation and transformed.
  • 1 mL of LB medium 1.0 g of NaCl, 0.5 g of yeast extract, and 1.0 g of tryptone were dissolved in 100 mL of distilled water
  • LB / Ap agar medium Duchefa, Nederland in 100 ml LB medium
  • M1002 1.5 g, ampicillin concentration added to 100 ug / mL) in a 90 mm Petri dish containing 20 ml each of 500 ⁇ L shaken solution in a 37 °C shake incubator rotating at 250 rpm each, Further incubation overnight at 37 ° C. incubator.
  • storage strains were made with the transformed colonies, and each plate was used after linearly inoculating the storage strain on a new LB / Ap agar medium plate.
  • ncRNAs that inhibit bacterial biofilm formation upon overexpression 100 ul LB medium containing 100 ug / ml ampicillin was added to 96-well cell culture plates (SPL, Korea, 34296), respectively. Colonies of Escherichia coli strains containing each of the sRNA overexpressing plasmids prepared in 1 were inoculated into each well, followed by shaking culture for 16 hours in a 37 ° C. incubator, followed by 100 ug / ml amplification in 96-well cell culture plates of the same type. The culture was diluted 1: 100 in 100 ul LB medium containing silin, 1 mM IPTG, and cultured at 30 ° C. for 12 hours.
  • FIGS. 8 to 10 are graphs showing the results of observing the degree of biofilm formation in each strain when overexpressing each sRNA in E. coli with 99 sRNA expression plasmids according to one embodiment of the present invention.
  • 11 to 14 are photographs showing the results of experiments to determine whether the coliform and yeast aggregation by the expression of type I cilia according to an embodiment of the present invention.
  • 15 to 16 are photographs showing the results of experiments to determine the difference in dye uptake according to the expression of the curly cilia in LB medium Petri dish to which Congo red dye is added according to an embodiment of the present invention.
  • 17 to 20 are photographs showing the results of measuring swimming mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • 21 to 23 are graphs showing the results of measuring swimming mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • 24 to 27 are photographs showing the results of measurement of strain mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • 28 to 30 are graphs showing the results of measurement of strain mobility by strain when overexpressing sRNA with an sRNA expression plasmid according to an embodiment of the present invention.
  • FIG. 31 is a van diagram showing an sRNA that affects biofilm formation, type I cilia, expression of curly cilia, swimming mobility, and herd mobility when sRNA is overexpressed with the sRNA expression plasmid according to the experimental results.
  • SRNAs that increase biofilm formation are indicated in squares, and decreasing sRNAs are indicated in ellipses.

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Abstract

La présente invention concerne une composition permettant de réguler la formation de biofilms microbiens, ladite composition contenant un nucléotide exprimant un petit ARN capable de réguler la formation de biofilms microbiens ; un vecteur recombiné comprenant le nucléotide ; une cellule transgénique transformée par le vecteur recombiné ; et un procédé permettant de réguler la formation de biofilms microbiens et comprenant une étape de transformation du vecteur recombiné en un micro-organisme.
PCT/KR2016/011603 2016-08-11 2016-10-17 Composition de régulation de la formation de biofilms, et procédé de régulation de la formation de biofilms au moyen de la composition Ceased WO2018030578A1 (fr)

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WO2004056996A2 (fr) * 2002-12-20 2004-07-08 University Of North Texas Health Science Center At Fort Worth Polynucleotides de csrc et utilisations associees pour la modulation de film biologique
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WO2004056996A2 (fr) * 2002-12-20 2004-07-08 University Of North Texas Health Science Center At Fort Worth Polynucleotides de csrc et utilisations associees pour la modulation de film biologique
WO2015118407A2 (fr) * 2014-01-29 2015-08-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Oligonucléotides et procédés d'inhibition ou de réduction de biofilms bactériens

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