WO2018030578A1 - Composition for regulating biofilm formation and method for regulating biofilm formation by using same - Google Patents

Abstract

The present invention relates to: a composition for regulating microbial biofilm formation, containing a nucleotide expressing an sRNA capable of regulating microbial biofilm formation; a recombinant vector comprising the nucleotide; a transgenic cell having been transformed by the recombinant vector; and a method for regulating microbial biofilm formation, comprising a step of transforming the recombinant vector into a microorganism.

Description

Biofilm formation control composition and biofilm formation control method using the same

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.

In addition, the 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.

In addition, 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.

These 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. To this end, 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).

However, since these methods have many problems such as antibiotic abuse and limitation of application, a new biofilm suppression methodology that naturally inhibits the biological formation of bacteria is required.

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.

Although it is known that certain sRNAs in bacteria control cilia formation or mobility, the phenotype associated with biofilm formation (Trends Microbiol. 2013, 21: 39-49), they have not been systematically investigated. However, considering that sRNA function is closely related to environmental stress response, a significant number of sRNAs are expected to regulate biofilms and related phenotypes.

Accordingly, 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.

Accordingly, it is an object of the present invention to provide a composition for controlling biofilm formation of a microorganism comprising a nucleotide expressing a sRNA capable of controlling the biofilm formation of the microorganism.

It is still another object of the present invention to provide a recombinant vector comprising a nucleotide expressing sRNA capable of regulating the formation of a biofilm of a microorganism.

It is another object of the present invention to provide a cell transformed with a recombinant vector 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.

The formation of biofilms in microorganisms occurs through very complex physiological processes. In general, bacterial motility (floating mobility, swimming mobility), type 1 cilia formation, and curly cilia formation are known to be important factors for the formation of biological membranes.

In the present invention, as well as sRNA that directly affects the formation of biofilms, sRNAs that affect the swimming mobility, herd mobility, type 1 cilia formation, and curly cilia formation are known.

Hereinafter, the present invention will be described in more detail.

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.

Therefore, in the present invention, it was intended to utilize the function of sRNA known as a key regulatory factor of cell metabolism in recent years. First, 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.

Then, 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.

Then, when sRNA is overexpressed using sRNA-expressing plasmid aggregates, 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.

In addition, the 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.

In addition, the 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 (Rhizobium), Rhodobacter (Rhodobacter), Eisai Momo Nas (Zymomonas ), Bacillus (Bacillus), Lactococcus as stearyl pliers (Staphylococcus), Lactococcus (Lactococcus), sat strap Lactococcus (Streptococcus), Lactobacillus bacteria (Lactobacillus), Clostridium (Clostridium), Corynebacterium (Corynebacterium), strap Sat My process (Streptomyces), may be the intended Bifidobacterium (Bifidobacterium) or cycloalkyl tumefaciens (Cyclobacterium), preferably, but may be an E. coli, and the like.

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.

The term "vector" means a means for expressing a gene of interest in a host cell. For example, 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., λgt4? B, λ-Charon, λΔz1 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. For example, when the vector used is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, and a T7) capable of promoting transcription Promoters, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. In the case of eukaryotic cells as hosts, 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. It is not limited. In addition, promoters derived from the genome of mammalian cells (eg, metallothionine promoters) or promoters derived from mammalian viruses (eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

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.

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. For example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method may be used. When 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. For example, when 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.

The 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.

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.

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.

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.

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

Example  One. ncRNA  Extraction and Restriction Enzyme Processing of Overexpression Vectors

Obtain the pHMB1 plasmid (Sci Rep 2015 Oct 15, 5: 15287) with the cleavage map of FIG. Cut).

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 Prepare a total of 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.

Then, 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.

Example  2. Preparation of synthetic DNA to be inserted into vector and restriction enzyme treatment

2-1. Complete DNA Purification of Escherichia Coli

First, 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.

2-2. Construct DNA Sequence Fragments to Insert into Plasmid Vectors

In order to prepare an sRNA sequence to be overexpressed by inserting it into the plasmid vector digested with the restriction enzyme obtained in Example 1, PCR was performed based on the whole DNA of E. coli extracted in Example 2-1.

Specifically, after reacting the reaction solution as shown in Table 1 using a PCR equipment at 95 ° C. for 2 minutes, the 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). . As a result, the purity was OD260 / OD280 value of about 2.0 and the linear DNA concentration was confirmed to be more than 50 ng / uL.

division sheep Forward primer 1 ul (10 pmol / ul) Reverse primer 1 ul (10 pmol / ul) E. coli whole DNA 0.5 ul (1 ug / ul) 2X TOPsimple (TM) PreMIX-Forte (Enzynomics, Korea, P610F) 25 ul NFW 22.5 ul total 50 ul

2-3. 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 ℃ for 1 to 2 hours, 65 The restriction enzyme was inactivated by transferring to ℃ and reacting for 20 minutes.

Example  3. ncRNA  Overexpression Plasmid Construction

3-1. Linking and Transforming Plasmids and DNA Fragments

The 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 ℃ for 10 hours to bind the DNA fragment to the plasmid.

Next, 1 to 2 uL of the reaction solution containing the DNA fragment bound plasmid is mixed with 50 uL of DH5a competent cells (Enzynomics, Korea, CP010), placed on ice for 20 minutes, and then in a 42 ° C. constant temperature bath. Heat shock was applied for 5 minutes and transformed by standing on ice for 2 minutes.

3-2. Ligation efficiency and inserted DNA size confirmation

In 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. Incubated in 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.

Then, randomly picked 15 transformed E. coli colonies randomly mixed with the solution in the PCR test tube under the conditions shown in Table 2, and then randomly picked colonies one by one, using a PCR equipment After the reaction at 95 ° C. for 5 minutes, the reaction of 95 ° C. 30 seconds, 58 ° C. 60 seconds, and 72 ° C. 30 seconds was repeated 30 times, followed by reaction at 72 ° C. for 5 minutes and 25 ° C. for 5 minutes to obtain a reaction product. The reaction product was analyzed by electrophoresis on agarose gel.

division content Forward primer 0.5 ul (10 pmol / ul) Reverse primer 0.5 ul (10 pmol / ul) 2X TOPsimple (registered trademark) PreMIX-nTaq (Enzynomics, Korea, P610T) 10 ul NFW 9 ul total 20 ul

3-3. Collected Colonies  Purifying Plasmid DNA from Cells

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.

Example  4. Escherichia coli strains sRNA  Overexpression plasmid introduction

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) was added to the reaction solution, and LB / Ap agar medium (Duchefa, Nederland in 100 ml LB medium) was added. , M1002) 1.5 g, ampicillin concentration added to 100 ug / mL) in a 90 mm Petri dish containing 20 ml each of 500 μL shaken solution in a 37 ℃ shake incubator rotating at 250 rpm each, Further incubation overnight at 37 ° C. incubator. Then, 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.

Example  5. Bacteria on Overexpression Biofilm  Forming degree measurement

To screen 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. Then, after measuring the OD ₅₉₅ of the culture medium of the culture plate in which the cells were cultured, soaked in distilled water and then washed twice by shaking, and 125 ul of 0.1% (w / v) crystal violet solution was added to each well. Stain for 10 minutes. After dyeing, the culture plate was immersed in distilled water and washed four times by shaking. After adding dyed acetic acid, 30% (w / v) acetic acid was extracted, the OD ₅₅₀ was measured and the value was divided by OD ₅₉₅ . The amount of biofilm formed when each of the sRNAs was overexpressed and the percentage of cells in the culture medium were compared with each other. The results are shown in Figure 3 and Table 3.

sRNA Biofilm Formation a sRNA Biofilm Formation a sRNA Biofilm Formation a Vector 100 ± 6% RybA 112 ± 13% SraA 122 ± 13% Arcz 18 ± 4% RybB 10 ± 2% SroA 96 ± 13% C0067 91 ± 17% RybD 113 ± 10% SroC 63 ± 6% C0293 83 ± 10% RydB 114 ± 3% SroD 87 ± 14% C0299 81 ± 14% RydC 99 ± 5% SroE 98 ± 10% C0343 73 ± 7% Ryea 108 ± 4% SroG 115 ± 10% C0362 78 ± 16% SdsR 57 ± 11% SroH 112 ± 16% C0465 90 ± 17% Ryef 119 ± 10% SsrA 120 ± 8% C0614 87 ± 15% RyfA 56 ± 22% SsrS 134 ± 9% C0664 99 ± 17% RyfB 127 ± 2% SymR 98 ± 9% C0719 99 ± 15% RyfD 66 ± 13% T44 106 ± 7% CsrB 145 ± 13% RyhB 92 ± 11% Tp2 109 ± 8% CsrC 182 ± 24% RyjA 88 ± 10% Tpke11 120 ± 7% CyaR 80 ± 16% RyjB 94 ± 15% Tpke70 119 ± 10% DicF 11 ± 5% SgrS 160 ± 18% pRNA 118 ± 8% DsrA 2 ± 1% SibA 99 ± 13% Och1 109 ± 9% EyeA 100 ± 10% SibB 88 ± 15% Och2 92 ± 10% Ffs 90 ± 12% SibC 86 ± 14% Och3 107 ± 10% FnrS 165 ± 5% SibD 99 ± 11% Och4 99 ± 11% GadY 116 ± 13% SibE 104 ± 14% Och5 229 ± 10% GcvB 150 ± 10% SokA 101 ± 14% Och6 115 ± 10% Glmy 122 ± 18% SokB 107 ± 14% Och7 108 ± 7% Glmz 139 ± 10% SokC 87 ± 14% Och8 98 ± 6% IS118 66 ± 10% SokE 96 ± 11% Och9 112 ± 10% RseX 174 ± 12% SokX 87 ± 7% Och10 119 ± 2% RttR 97 ± 9% Spf 110 ± 12% - -

As can be seen in Figure 3 and Table 3, when overexpressed the biofilm formation of the six sRNA of CsrC, FnrS, GcvB, RseX, SgrS, Och5 more than 1.5 times increased compared to the control and ArcZ, DsrA more than 1.5 times reduced 13 sRNAs of DicF, IS118, McaS, MicA, MicM, OmrA, RybB, RyfA, RyfD, SdsR, SroC were identified.

Example  6. Measuring Physiological Changes

6-1. Measurement of Type I Cilia Formation

Colonies of E. coli strains containing 100 ul LB medium containing 100 ug / ml ampicillin in a 96-well cell culture plate (SPL, Korea, 34296) containing each sRNA overexpressing plasmid prepared in Example 4-1 After inoculating each well, incubated for 16 hours in a 37 ° C. incubator, and then cultured in 100 ul LB medium containing 100 ug / ml ampicillin and 1 mM IPTG in 96-well cell culture plates of the same type. Dilute to 100 and stop culture overnight at 37 ° C.

Then, 100 ul of dry yeast (Sigma-Aldrich, USA, YSC2) dissolved in 0.5% PBS solution was mixed with the culture medium and mixed with a microplate shaker at room temperature for 20 minutes to induce the yeast and E. coli to aggregate with each other. In order to better observe the aggregation of yeast and E. coli, 0.1% (w / v) crystal violet was added in a ratio of 1: 100 and ul was observed. The results are shown in FIGS. 4 to 7.

As can be seen in Figures 4 to 7, when overexpressed sRNA to reduce the expression of type I cilia to prevent the aggregation of yeast and E. coli DsrA, IS118, MicA, MicM, RybB five species were identified.

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.

6-2. Curly  Measuring the degree of cilia formation

Yeast extract 5 mg / mL, Tryptone 10 mg / mL, Micro agar (Duchefa, Nederland, M1002) 15 mg / mL, Ampicillin 100 ug / mL, Congo red (Sigma-Aldrich, USA, C6767) 40 ug / mL , Coomassie Brilliant Blue G (Sigma-Aldrich, USA, B0770) linear inoculation of the overexpressing plasmid strain of Example 4-1 in a 90 mm Petri dish containing 20 ml Congo red medium prepared at a ratio of 20 ug / mL After 48 hours incubation at 28 ℃ incubator. The results are shown in FIGS. 11 to 14 and 15 to 16.

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.

As can be seen in Figures 11 to 14 and 15 to 16, the sRNA that does not develop the coli cilia during the overexpression, so that the absorption of Congo red shows a light color CyaR, McaS, OmrA, OmrB, OxyS, RdlB, RprA , RseX, RydC, RyfA, RyhB, SgrS 12 species were identified.

6-3. Streaming mobility measurement

In order to measure swimming mobility, 100 ul LB medium containing 100 ug / ml ampicillin was placed in a 96-well cell culture plate (SPL, Korea, 34296), with each sRNA overexpression plasmid prepared in Example 4-1. Colonies of E. coli strains were inoculated into each well and cultured for 16 hours in a 37 ℃ incubator.

Next, a 150 mm Petri dish containing 50 mg of micro agar (Duchefa, Nederland, M1002) 3 mg / mL, tryptone 10 mg / mL, NaCl 5 mg / mL, ampicillin 100 ug / mL, IPTG 1 mM medium Cultures were inoculated for each strain. The plates were then packaged using plastic wrap to prevent moisture evaporation and placed incubator at 30 ° C. for 12 hours to incubate. The results are shown in FIGS. 17 to 20 and 21 to 23.

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.

As can be seen in FIGS. 17 to 20 and 21 to 23, nine species of ArcZ, DicF, DsrA, GlmY, IS118, OmrA, OxyS, RyfB, and Och5, which are sRNAs that reduce swimming mobility by 1.5 times or more, compared to the control group when overexpressed. Confirmed.

6-4. Herd mobility  Measure

100 ul LB medium containing 100 ug / ml ampicillin was added to the 96-well cell culture plate (SPL, Korea, 34296) to determine the herd mobility, with each sRNA overexpression plasmid prepared in Example 4-1. Colonies of E. coli strains were inoculated into each well and cultured for 16 hours in 37 incubators.

Then, 6 mg / mL Eiken Agar (Eiken Chemical, Japan, E-MJ00), glucose 5 mg / mL, tryptone 10 mg / mL, yeast extract 5 mg / mL, NaCl 5 mg / mL, ampicillin 100 ug The culture solution was inoculated in each strain into a 150 mm Petri dish containing 50 mL of / mL and 50 mL of IPTG 1 mM composition. The plates were then packaged using plastic wrap to prevent evaporation of the water and incubated at 37 ° C. for 16 hours in the incubator. The results are shown in FIGS. 24 to 27 and 28 to 30.

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.

As can be seen from Figures 24 to 27 and 28 to 30, ArcZ, CsrB, CsrC, DicF, DsrA, GadY, GcvB, IS118, MicA, MicC, which are sRNAs that reduce the herd mobility more than 1.5 times compared to the control group when overexpressed 25 sRNAs of OmrA, OxyS, RdlB, RdlC, RprA, RseX, RydC, RyeF, RyfA, RyfB, RyfD, RyhB, SdsR, SgrS and Och5 were identified.

SEQ ID NO: sRNA Yooyoung a Bunch b Type 1c Curly d SEQ ID NO: 1 Arcz ↓ ↓ - - SEQ ID NO: 12 CsrC - ↓ - ↑ SEQ ID NO: 14 DicF ↓ ↓ - NDe SEQ ID NO: 15 DsrA ↓ ↓ ↓ - SEQ ID NO: 18 FnrS - - - - SEQ ID NO: 20 GcvB - ↓ - - SEQ ID NO: 23 IS118 ↓ ↓ ↓ ↓ SEQ ID NO: 25 McaS - ↑ - ↓ SEQ ID NO: 31 MicA ↑ ↓ ↓ - SEQ ID NO: 34 MicM - - ↓ ↓ SEQ ID NO: 36 Omra ↓ ↓ - ↓ SEQ ID NO: 47 RseX - ↓ - ↓ SEQ ID NO: 50 RybB - - ↓ - SEQ ID NO: 55 SdsR - ↓ - - SEQ ID NO: 57 RyfA - ↓ - ↓ SEQ ID NO: 59 RyfD - ↓ - - SEQ ID NO: 63 SgrS - ↓ - ↓ SEQ ID NO: 77 SroC - - - - SEQ ID NO: 94 Och5 ↓ ↓ - ND

a change in relative biofilm formation.

more than 1.5-fold reduction compared to the b ↓ pHMB1 vector; mobility similar to the pHMB1 vector; ↑ More than 1.5 times greater than the pHMB1 vector.

c ↓, not conjugated with yeast cells; -Conjugated with yeast cells.

d ↓, white-pink colony formation in Congo red plate; a color comparable with the pHMB1 vector; ↑ Dark red colony formation in Congo red plate.

eND; Toxic to growth, not experimental.

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.

As can be seen in Figure 31, sRNAs affecting the formation of type I cilia, expression of curly cilia, swimming mobility, and herd mobility all reduced their phenotype.

Claims (16)

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, SEQ ID NO: 94 composition for regulating biofilm formation of a microorganism comprising one or more nucleotides consisting of a nucleotide sequence. According to claim 1, wherein the composition for controlling biofilm formation 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, SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: Comprising one or more nucleotides consisting of a base sequence selected from the first group consisting of 77, which is for inhibiting the biofilm formation of microorganisms. According to claim 1, wherein the composition for controlling biofilm formation of the microorganism, It comprises at least one nucleotide consisting of a nucleotide 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, and promotes biofilm formation of microorganisms. Will, composition. According to claim 1, wherein the composition for controlling biofilm formation of the microorganism, Composition that controls the biofilm formation of the microorganism by controlling at least one selected from the group consisting of type I cilia formation, curly cilia formation, swimming mobility and herd mobility, which are factors related to biofilm formation of a microorganism. According to claim 4, wherein the composition for controlling biofilm formation of the microorganism, It comprises one or more nucleotides 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, and controls the type I cilia formation of the microorganism , Composition. According to claim 4, wherein the composition for controlling biofilm formation of the microorganism, A composition comprising one or more nucleotides consisting of a 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, and controls the formation of microciliar cilia. According to claim 4, wherein the composition for controlling biofilm formation of the microorganism, It comprises one or more nucleotides consisting of a 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, and regulates the mobility of microorganisms To, composition. According to claim 4, wherein the composition for controlling biofilm formation 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, SEQ ID NO: A composition comprising one or more nucleotides consisting of a nucleotide sequence selected from the sixth group consisting of 63 and SEQ ID NO: 94, and controls the microbial mobility. The composition of claim 1, wherein the microorganism is a prokaryote or a eukaryote. 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: A recombinant vector comprising at least one nucleotide consisting of a nucleotide sequence selected from the group consisting of 50, SEQ ID 55, SEQ ID 57, SEQ ID 59, SEQ ID 63, SEQ ID 77, SEQ ID 94. A transformed cell in which formation of a biofilm transformed with the recombinant vector of claim 10 is controlled. The method of claim 11, wherein the transformed cells, 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, SEQ ID NO: 59, and SEQ ID NO: Transformed with a recombinant vector comprising one or more nucleotides consisting of nucleotide sequences selected from the group consisting of 77, The transformed cell is a transformed cell that is inhibited the formation of a biofilm. The method of claim 11, wherein the transformed cells, 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 is to facilitate the formation of the biofilm, the transformed cell. 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: Transforming a microorganism into a recombinant vector comprising at least one nucleotide consisting of a nucleotide sequence selected from the group consisting of 50, SEQ ID 55, SEQ ID 57, SEQ ID 59, SEQ ID 63, SEQ ID 77, SEQ ID 94 Biofilm formation control method of microorganisms comprising a. The method according to claim 14, wherein the microbial biofilm formation control method 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, SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: And transforming the microorganism into a recombinant vector comprising at least one nucleotide consisting of a nucleotide sequence selected from the group consisting of 77, wherein the microorganism is inhibited in biofilm formation. The method according to claim 14, wherein the biofilm formation establishment method of the microorganism is 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: 12, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 47, SEQ ID NO: 63, and SEQ ID NO: 94 Wherein the microorganism is promoted biofilm formation.

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