KR20040028205A - Methods for high level transcription from Bacillus subtilis using physiological stimuli - Google Patents

Abstract

PURPOSE: A method for high expression of DGKase(diacylglycerol kinase) from Bacillus subtilis using physiological stimuli is provided, thereby accomplishing transcriptional regulation of the expression level of DGKase, and preparing high expression of mRNA for mass-producing a specific protein. CONSTITUTION: A method for increasing the expression of nucleotide fragments containing DGKase gene having the nucleotide sequence set forth in SEQ ID NO: 1 from Bacillus subtilis MI113 comprises applying physiological stimuli such as changes of culture conditions to Bacillus subtilis MI113, wherein the nucleotide fragments containing DGKase gene has the nucleotide sequence set forth in SEQ ID NO: 2; the changes of culture conditions include osmosis increase, pH reduction and temperature reduction.

Description

Method for increasing D. gene expression in Bacillus subtilis using physiological stimulation {Methods for high level transcription from Bacillus subtilis using physiological stimuli}

The present invention relates to a method of increasing the expression of the DGKase gene of Bacillus subtilis. More specifically, the present invention relates to a method of increasing the expression of the DGKase gene of Bacillus subtilis by physiological stimulation by exposure to media with varying osmotic pressure, pH, temperature.

Staphylococcus mutans, a bacterium that causes tooth decay,Staphylococcus mutans) Has been recognized as an important dental strain and various studies have been conducted on its physiological characteristics. Represented by Staphylococcus mutans Two important physiological characteristics, particularly related to the induction of tooth decay, are the ability to inhabit the tooth surface and the strong acid resistance that destroys the enamel surface of the tooth. Staphylococcus mutans is known as one of the most acid resistant bacteria in the oral cavity, and its strong acid tolerance is proton permeable through proton-extruding through cell membranes. is achieved by proton permeability, and the decrease in the pH of the environment is due to the production of lactate dehydrogenase and glycolytic acitivity, including proton extruded ATPase in Staphylococcus mutans cells. Induce. therefore The acid resistance of Staphylococcus mutans is closely related to the adaptation of the strain to environmental stress caused by acidic hydrogen ion concentration (pH). For this reason, Yamashita et al. (1993) reported that Staphylococcus mutans lacking acid resistance using conjugative transposon Tn916 to molecularly identify the phenomena of environmental adaptability of Staphylococcus mutans. of Mutant strains were produced. Staphylococcus mutans was identified by analysis of genes related to acid deficiency in this mutant strain. Staphylococcus mutans is expressed in various environmental stress conditions, including acid resistance. Gene regions encoding DGKase activity were identified as genes that play an important role in adapting to environmental stress. Subsequently, various studies on DGKase have been carried out and found that DGKase is involved in environmental stress adaptation and signal transduction and has many other important biological activities.

In the present invention, based on the experimental background, Bacillus subtilis MI113 is identified by identifying related genes and transcription units including DGKase genes involved in environmental stress adaptation and analyzing physiological factors for expression amplification. It was intended to utilize industrially useful characteristics of the strain.

Accordingly, an object of the present invention is to provide a method of controlling the expression level of DGKase gene by giving a physiological stimulus to change the osmotic pressure, pH, temperature during the culturing of Bacillus subtilis.

The object of the present invention was achieved by exposing the Bacillus subtilis MI113 strain to media prepared differently with osmotic pressure, pH, and temperature, and then extracting total RNA from the collected cells and analyzing the expression pattern of the DGKase gene.

1 is a schematic diagram of the dgk gene region of Bacillus subtilis MI113 strain.

Figure 2a is a photograph showing the analysis results according to the osmotic pressure change in the dot hybridization results using the dgk probe of the total RNA isolated from the Bacillus subtilis MI113 strain.

Figure 2b is a photograph showing the analysis results according to the hydrogen ion concentration change in the dot hybridization results using the dgk probe of the total RNA isolated from the Bacillus subtilis MI113 strain.

Figure 2c is a photograph showing the analysis results according to the temperature change of the dot hybridization results using the dgk probe of the total RNA isolated from Bacillus subtilis MI113 strain.

Figure 3a is a photograph showing the analysis results of the expression level of DGKase gene transcription according to the osmotic pressure change. The total RNA attached to the nylon membrane (nylon membrane) stained with 0.4% methylene blue.

Figure 3b is a photograph showing the analysis results of the expression level of DGKase gene transcription according to the osmotic pressure change. The results of northern hybridization using a dgk probe are shown.

Figure 4a is a photograph showing the analysis results of the expression level of DGKase gene transcription according to the change in hydrogen ion concentration. The total RNA attached to the nylon membrane (nylon membrane) stained with 0.4% methylene blue.

Figure 4b is a photograph showing the analysis results of the expression level of DGKase gene transcription according to the change in hydrogen ion concentration. The results of northern hybridization using a dgk probe are shown.

Figure 5a is a photograph showing the analysis results of the expression level of DGKase gene transcription according to the osmotic pressure change. The total RNA attached to the nylon membrane (nylon membrane) stained with 0.4% methylene blue.

Figure 5b is a photograph showing the analysis results of the expression level of DGKase gene transcription according to the osmotic pressure change. The results of northern hybridization using the ORF2 probe are shown.

The present invention comprises the steps of culturing Bacillus subtilis MI113 strain and exposing the osmotic pressure, pH, temperature to a differently prepared medium; Collecting cells of each cultured strain and extracting total RNA; Northern hybridization and dot blot hybridization are used to measure the expression of the DGKase gene.

The present invention provides a physiological stimulation method including osmotic pressure, pH and temperature conditions to increase the expression of the Bacillus subtilis DGKase gene.

In another aspect, the present invention provides an integral transcription unit comprising a genetic regulatory region associated with the DGKase gene that can increase the production of the gene product in response to physiological stimulation.

In addition, the present invention can be used in parts and methods for mass production of materials contained in foods, pharmaceuticals, animal drugs and various biotechnology products.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

Example 1; Bacillus subtilis Culture of MI113 Strains and Exposure to Physiological Stimulation of Osmotic Pressure, pH, and Temperature

To investigate the expression patterns of physiological stimulation of Bacillus subtilis DGKase gene, Bacillus subtilis 168 (trpC2) Bacillus subtilis from strains MI113 (arg,trpC2) Strain was used. Bacillus subtilis to evaluate the expression level of DGKase gene in the transcriptional phase The MI113 strain was inoculated in Luria-Broth medium (1.0% Bacto-tryptone, 0.5% Bacto-yeast extract, 1.0% NaCl, pH 7.0), shaken at 37 ° C, and incubated overnight. The next day, the culture was inoculated in 5 mL of fresh LM medium and shaken at 37 ° C. for 4 hours, and then exposed to LB medium prepared with different osmotic pressure, temperature, and pH as follows. The osmotic pressure test group used LB medium with different NaCl concentrations of 0.05 M, 0.23 M, 0.5 M, 1 M and 1.5 M, and pH test groups were adjusted to pH 5, pH 6, pH 6.7, pH 7 and pH 8, respectively. LB medium was used, and the temperature test zone was inoculated with the strain in the LB medium and experimented differently at 25 ℃, 30 ℃, 35 ℃, 37 ℃ and 42 ℃.

Example 2; Bacillus subtilis ( Bacillus subtilis ) Extraction of Total RNA from MI113

Bacillus subtilis Extraction of total RNA for analysis of expression patterns according to physiological stimuli represented by the DGKase gene of the MI113 strain was performed as follows. Bacillus subtilis under the culture conditions of the above-described NaCl, pH and temperature respectively After incubating the MI113 strain for 14 hours, 5 mL of the culture solution was collected from each experimental zone, and the cells were collected by centrifugation at 7000 rpm for 5 minutes at 4 ° C. The collected cells were suspended in 2 mL suspension (10 mM Tris-HCl, 1 mM EDTA, 0.9% NaCl, 0.5 mg / mL lysozyme) treated with diethyl pyrocarbonate (DEPC). To this suspension was added 0.24 mL of a 10% SDS solution and treated several times with saturated phenol. The water-soluble portion of the treatment solution was then retreated with phenol / chloroform and 2 volumes of cold ethanol were added and allowed to stand overnight at -20 ° C to precipitate RNA. The next day, the precipitated RNA was collected by centrifugation at 15,000 rpm for 30 minutes at 4 ° C, washed with 70% (v / v) ethanol and dried. Purified RNA was suspended in sterile water treated with DEPC and stored at -70 ℃.

Example 3; Measurement of RNA Expression of Bacillus subtilis MI113 DGKase by Physiological Stimulation by Northern Hybridization and Dot Blot Hybridization

Northern hybridization experiments for measuring RNA expression according to physiological stimuli were conducted with a slight modification of Farrell et al. The collected total RNA was denatured by heating at 65 ° C. for 10 minutes and iced in the presence of 50% formamide, 2.2 M formaldehyde and 1 × MOPS buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA, pH 8.0). It was left for 1 minute at. Electrophoresis of total RNA was performed for 4 hours using 1.0% agarose / formamide gel as 1 × MOPS electrophoresis buffer. Northern hybridization of electrophoretically separated RNAs was carried out from the gel to the nylon membrane using capillary action in solution and baked for 2 hours in a vacuum at 80 ℃. Thereafter, the nucleic acid-attached nylon membrane was prehybridized for 4 hours, and then hybridized with a radioisotope probe for 20 hours at 42 ° C. The probes used for northern hybridization and dot blot hybridization were dgk probes and ORF2 probes (FIG. 1). The prehybridization and hybridization solutions used were 5XSSPE (3.6 M NaCl, 20 mM EDTA, 0.2 M sodium phosphate, pH 7.7), 50% formamide, 5X Denhard's solution, 100 μg / mL denatured herring pump DNA and It consisted of 0.5% SDS. After hybridization, the membrane was washed twice at room temperature for 10 minutes with 1X SSC / 0.1% SDS and then twice at 45 ° C. for 30 minutes with 0.2X SSC / 0.1% SDS. The base sequence (ORF2, ORF3, ORF4, dgk gene in Fig. 1) of the DGKase gene and related gene sites including the dgk probe and the ORF2 probe are described in SEQ ID NO: 1.

The dot blot hybridization was performed as follows. First, total RNA was dissolved in 1XSSC solution containing 50% formamide and 7% formaldehyde, denatured at 68 ° C. for 15 minutes, and cooled on ice. Denatured RNA was spotted on the nylon membrane at concentrations of 35 μg, 20 μg and 5 μg, respectively, and the remaining dot blot hybridization experiments were performed in the same manner as the northern hybridization described above.

Experimental Example 1; Analysis of DGKase Gene Expression Pattern by Osmotic Pressure Change

DGKase with Osmotic Pressure The change in the amount of transcription of the gene was measured using the dot blot hybridization and the northern hybridization described above. Dot hybridization was performed on Bacillus subtilis in LB medium containing NaCl at 0.05 M, 0.5 M, 1 M and 1.5 M concentrations, respectively. After exposure to the MI113 strain for 30 minutes, the total RNA of the cells collected from each experimental section was extracted, and 30 μg, 20 μg and 10 μg of RNA were attached onto the blot and hybridized with a dgk probe to which a radioisotope was attached. Expression of the DGKase gene according to the intensity of osmotic stress measured by dot blot hybridization is shown in FIG. 2A. As a result of dot blot hybridization, the level of transcription of the DGKase gene increased to 1 M NaCl concentration in response to osmotic stress, but decreased at 1.5 M NaCl concentration. The highest level of transcription was observed at 1 M NaCl concentration. As a result of analyzing the molecular weight of the DGKase gene by Northern hybridization, it was shown in FIG. 3 and appeared as a single band of about 2.5 kb. The change in the amount of transcription associated with osmotic stress was similar to that of dot blot hybridization, and the highest level of increase in transcription was 1M NaCl.

Experimental Example 2; Analysis of DGKase Gene Expression Patterns by pH Change

Bacillus subtilis in LB medium adjusted to pH 5, pH 6, pH 6.7, pH 7 and pH 8, respectively After exposing the MI113 strain for 30 minutes, total RNA was extracted from the cells collected in each experiment, and 30 ㎍, 20 ㎍ and 10 ㎍ of RNA were attached onto the blot and hybridized with a dgk probe with radioisotope attached thereto. Expression of the DGKase gene according to the hydrogen ion concentration measured by dot blot hybridization is shown in FIG. 2B. Dot Hybridization Results DGKase Gene transfer levels were detected at high concentrations at low pH (pH 5) and at low concentrations at neutral (pH 6.7 and pH 7) (Figure 2b). The highest level of transcription was observed under acidic conditions (pH 5). Northern hybridization of the molecular weight of the DGKase gene generated in response to the hydrogen ion concentration is shown in FIG. 4, and a single band of about 2.5 kb appeared. DGKase Associated with Osmotic Stress The molecular weight was similar to that of the gene transcript, and the change in the amount of transcription detected in response to the hydrogen ion concentration was the same as that of dot blot hybridization.

Experimental Example 3; Analysis of DGKase Gene Expression Pattern by Temperature ChangeDGKase with Temperature Changes in the amount of transcription of the gene were measured using dot hybridization. Bacillus subtilis in LB medium Inoculated with the same amount of MI113 strain and incubated for 30 minutes in an incubator adjusted to 25 ℃, 30 ℃, 35 ℃, 37 ℃ and 42 ℃, respectively, and extracted the total RNA from the cells collected in each experiment and 30 ㎍, 20 Μg and 10 μg of RNA were attached onto the blots and hybridized with dgk probes to which radioisotopes were attached. Expression of the DGKase gene according to the temperature change measured by dot blot hybridization is shown in FIG. 2C. Dot blot hybridization results DGKase Transcription was increased at low temperatures but decreased at high temperatures (42 ° C). The highest level of transcription was observed in cells incubated at 25 ° C. (FIG. 2C).

As a result of the analysis of Northern hybridization performed by the dgk probe in Experimental Examples 1 and 2, an mRNA of about 2.5 kb in length, including the DGKase gene, was detected. ), And the hybridization of the DGKase gene to polycistronic mRNA was confirmed by Northern hybridization using the ORF2 probe of Figure 1 to determine the total RNA expression according to NaCl concentration. As a result of the analysis, mRNA of the same size as 2.5kb was observed and the expression pattern was also the same as the results of Northern hybridization experiments using the dgk probe (Fig. 3 and 5).

Based on these results, it can be seen that the DGKase gene of Bacillus subtilis MI113 strain is transcribed into polycistronic mRNA, and the 2.5 kb mRNA fragment containing the DGKase gene detected in the present invention responds to various environmental stresses. Was found to be a gene whose expression level is changed. In addition, the difference in DGKase gene expression of Bacillus subtilis MI113 strain against various physiological stimuli was investigated by dot blot and northern blot analysis. As a result, the transcript of DGKase gene was increased at low pH, high osmotic pressure and low temperature. In addition, it can be widely used for the production of high-expression mRNA for mass production of specific protein and development of components and environmental stress response factors for measuring environmental pollution.

As described above, the method of controlling the expression of the DGKase gene of the Bacillus subtilis of the present invention by using the characteristic that the transcript of the DGKase gene increases at high osmotic pressure, low pH, and low temperature, for mass production of a specific protein. It is a useful invention for the bioenvironmental industry because it has a remarkable effect that can be used to develop high-expression mRNA and development of components and environmental stress response factors for measuring environmental pollution.

Claims (6)

Bacillus subtilis (Bacillus subtilis)of A method of increasing the expression of a base fragment comprising the DGKase gene of SEQ ID NO: 1 by giving a physiological stimulus to change the culture conditions. 2. The method of claim 1, wherein Bacillus subtilis is a Bacillus subtilis MI113 strain. 3. The method of increasing the expression of a base fragment comprising a DGKase gene according to claim 1, wherein the base fragment comprising the DGKase gene is a base fragment as set forth in SEQ ID NO: 2. The method of claim 1, wherein the change in culture conditions increases the osmotic pressure, the method of increasing the expression of the base fragment containing the DGKase gene. [Claim 2] The method of claim 1, wherein the change in culture conditions lowers the pH of the base fragment containing the DGKase gene. The method of claim 1, wherein the change in culture conditions decreases the temperature.