KR100987259B1 - Expression vectors for fission yeast, construction method thereof, and medium composition for culturing the fission yeast - Google Patents

Abstract

The present invention is an expression vector for cleavage yeast comprising a hygromycin resistance gene as a marker gene and a method for producing the same, a strain transformed by the expression vector, a complex medium composition for cleavage yeast culture, and a minimal medium composition for cleavage yeast culture To provide. The expression vector of the present invention, the cleavage yeast transformed by the expression vector, and the culture composition for cleavage yeast are suitable for the selection of cleavage yeast containing an antibiotic resistance gene. In addition, the minimum medium composition of the present invention has the advantage that the activity against the hygromycin does not lose.

Hygromycin, cleavage yeast, expression vector

Description

Expression vectors for fission yeast, preparation method and medium composition for fission yeast culture {Expression vectors for fission yeast, construction method etc, and medium composition for culturing the fission yeast}

The present invention relates to an expression vector for cleavage yeast comprising a drug resistance marker gene.

Yeasts are the simplest model cells of eukaryotic cells that can be cultured at low cost, are easy to genetically research and manipulate, and have a well established biochemical and physiological function analysis system. It is used as a model cell for development.

In particular, in fission yeast, if genes related to cell cycle and signal transduction are missed or are expressed in cells, abnormality in regulation of signaling system is caused. Thus, growth rate inhibition and morphological changes of cells due to abnormal cell cycle regulation or metabolic abnormalities are caused. Caused. Thus, by exploring these phenotype changes, we can infer the function of the gene.

Marker genes are used as an important tool in various studies, such as making mutant strains in fission yeast or inserting and expressing specific genes in strains to analyze their function. The most ideal marker gene does not affect any function in the cell, and since the sequence similarity with the cell line is small, the efficient selection should be possible with minimal insertion or recombination into random chromosomes. To date, cleavage yeasts use a number of nutritional marker genes such as ura4, his3, arg6, and LEU2 as expression markers.However, vectors with nutritional marker genes must be strains with mutations in the nutritional marker. In addition to the limitations, strains with mutant markers of nutritional markers are not ideal because they have a slight inhibition of cell growth and other signaling effects compared to normal strains.

In addition to the nutritional marker gene, the drug resistance marker gene is used as a marker gene, but only the vector containing the antibiotic resistance marker gene is used in germination yeast, and the expression vector with the gene gene KanMX, which is the gene gene resistance, is the only one in cleavage yeast. .

However, fission yeast has a low antibiotic activity against geneticin, so there is a problem that the sensitivity to geneticin is not high when geneticin resistance gene is used as a marker gene.

In addition, the nutrition marker gene is usually used in a minimal medium to screen for nutritionally demanding mutant strains, but since geneticin loses its activity in the minimal medium, the gene marker gene can be used as a marker gene simultaneously with the nutrition marker gene. There is a disadvantage. In other words, if you want to use vectors with both nutritional markers and antibiotic resistance markers, it is difficult to use geneticin resistance genes as antibiotic marker genes. It was difficult to use without restriction of markers in the functional studies and the protein interaction studies of various genes using the mutants generated using.

Therefore, there is a need to develop an expression vector for cleavage yeast comprising a gene of resistance to antibiotics having sufficient activity against cleavage yeast as a marker gene.

There is also a need to develop a method that does not lose activity against antibiotics that lose activity in minimal media.

Therefore, the first technical problem to be solved by the present invention is an expression vector for cleavage yeast comprising a resistance gene for antibiotics with sufficient activity against cleavage yeast, a strain transformed by the expression vector, and for cleavage yeast culture To provide a composite medium composition.

The second technical problem to be solved by the present invention is to provide a method for producing the expression vector.

The third technical problem to be solved by the present invention is to provide a minimal medium composition for fission yeast culture.

The present invention provides an expression vector for cleavage yeast comprising a hygromycin resistance gene as a marker gene.

In the present invention, the marker gene refers to a gene that can be used as an important tool for various studies, such as making a mutant strain in cleaved yeast or inserting and expressing a specific gene into the strain to analyze its function.

In the present invention, a vector refers to a DNA molecule that is cloned by itself that is used to carry a clone gene (or another piece of clone DNA).

Since hygromycin is an antibiotic highly sensitive to the cleavage yeast, it is possible to use a resistance gene for the antibiotic as a marker gene.

The base sequence of the hygromycin resistance gene may be SEQ ID NO: 3.

In addition, the present invention provides a strain transformed by the expression vector.

The strain may be a bacterium.

The bacterium may be DH5a.

The bacterial strain may be accession number KCTC11230BP. The bacterial strain of Accession No. KCTC11230BP is a bacterial strain DH5a capable of being transformed by an expression vector for cleavage yeast containing a hygromycin resistance gene.

In addition, the strain may be a cleavage yeast.

In another aspect, the present invention provides a hybrid medium composition for cleavage yeast culture, comprising hygromycin. It is possible to select a cleavage yeast transformed with a vector comprising a hygromycin resistance gene according to the present invention by the complex medium composition for cleavage yeast culture containing the hygromycin.

The complex medium means a medium rich in components for the growth of fission yeast.

The cleavage yeast culture complex medium composition comprises glucose, galactose, fructose, lactose, maltose, or sucrose as a hygromycin and a carbon source, peptone, tryptone, yeast extract, malt extract, beef extract as a nitrogen source , Casein, soybean mill, NaNO ₃ , NH ₄ Cl, or (NH ₄ ) ₂ SO ₄ and the like, MgSO ₄ , K ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, CaCl ₂ , FeSO ₄ , ZnSO ₄ or MnSO ₄ and the like, and may include L-glutamic acid, L-cysteine, glycine, L-methionine, L-cystine, or taurine as an amino acid source.

The concentration of hygromycin may be 100-300 µg / ml.

In addition, the present invention is a split yeast comprising the step of culturing the split yeast according to the present invention in a complex medium containing hygromycin and the use of a complex medium containing hygromycin according to the present invention. Provide a culture method.

In order to achieve the second technical problem, (a) removing the geneticin resistance gene readout frame from the vector containing the geneticin resistance gene cassette, the hygromycin resistance gene readout frame Cloning into a vector from which the netisin resistance gene readout frame has been removed, thereby preparing a vector comprising a hygromycin resistance gene cassette; And (b) amplifying the hygromycin resistance gene cassette from the vector prepared in step (a) by PCR and cloning the expression vector for cleavage yeast in which the nutrient marker is removed. To provide.

In step (a), the hygromycin resistance gene can be amplified by PCR from the plasmid pRS-HYG for bacteria having hygromycin B phosphotransferase (Streptomyces hygroscopicus) gene (Fig. 3). The primers used for the PCR may be SEQ ID NO: 1 (5 'terminal primer for the hygromycin resistance gene open reading frame), and SEQ ID NO: 2 (3' terminal primer for the hygromycin resistance gene open reading frame).

In addition, a promoter of the translation elongation factor of eukaryotic fungus (Ashbya gossypii) and a terminator of the bacterium Tn903 are coupled to the N-terminus and C-terminus of the open reading frame of geneticin, respectively. The open reading frame of the gene gene resistance gene was removed from pFA6A, which is a bacterial plasmid with the gene gene resistance gene cassette (KanMX4), produced using restriction enzymes (Nco I / Sca I) and amplified by the PCR. A bacterial cloning vector (pFA6) having a hygromycin resistance gene (hygMX) cassette inserted into the pFA6A plasmid from which the open reading frame of mycin resistance gene (HYG) was removed. -hygMX) (FIG. 3). The base sequence of the hygromycin resistance gene open reading frame may be SEQ ID NO.

In step (b), the hygromycin resistance gene cassette (HygMX) can be amplified by PCR from the bacterial cloning vector pFA6-hygMX prepared in step (a). Primers used for PCR may be SEQ ID NO: 4 (5 'terminal primer for the hygromycin resistance gene cassette), SEQ ID NO: 5 (3' terminal primer for the hygromycin resistance gene cassette).

In order to eliminate the possibility of a base mutation by PCR, the amplified PCR product was sequenced to confirm the nucleotide sequence, and then the ends were cleaned up using restriction enzymes (Sph I / Nsi I). An expression vector for cleavage yeast having a hygromycin resistance gene (HYG) was inserted into the expression vector for cleavage yeast (pSLF173, pSLF273, pSLF373) from which the nutrient marker Ura4 gene was removed using restriction enzymes (Sph I / Nsi I). Can be made (FIG. 4). The base sequence of the hygromycin resistance gene cassette (hygMX) may be SEQ ID NO: 6.

The cleavage yeast expression vector comprising the hygromycin resistance gene prepared in step (b) is transformed into cleavage yeast by a known transformation method (for example, the method disclosed in Methods in Enzymology Vol. 194). Can be.

The present invention provides a minimal medium composition for cleavage yeast culture transformed by an expression vector comprising the hygromycin resistance gene, including monosodium glutamate as a nitrogen source, in order to achieve the third technical problem.

Minimal medium (minimal medium) in the present invention is usually a medium for selecting a nutritionally constitutive mutant strain, means a medium containing only the minimum components necessary for the growth of wild-type bacteria, and generally consists of a single carbon source and minerals. This minimal medium is used for the purpose of screening for nutritionally demanding mutants, and therefore must be a marker gene for an antibiotic that maintains activity in the minimal medium. It can be used without marker limitations in the functional studies and protein interaction studies of various genes.

Minimal media using ammonium chloride (NH ₄ Cl) as a conventional nitrogen source (e.g., Potassium Hydrogen Phthallate, Na ₂ HPO ₄ , NH ₄ Cl, Glucose, MgCl ₂ , CaCl ₂ , KCl, Na ₂ SO ₄ , pantothetic acid, nicotinic acid, Inositol, Biotin, Boric acid, MnSO ₄ , ZnSO ₄ , FeCl ₂ , molybdic acid ), KI, CuSO ₄ , minimal medium consisting of citric acid), but when hygromycin is used, hygromycin loses its antibiotic activity, but the minimum according to the present invention uses monosodium glutamate as the nitrogen source. By using a medium composition, hygromycin antibiotic activity is exhibited even in minimal medium. Therefore, it is possible to simultaneously use the nutritional marker gene in the expression vector containing the hygromycin resistance gene according to the present invention, and simultaneously the nutritional marker gene and the hygromycin resistance gene in the split yeast transformed with the expression vector. It is possible to select a split yeast that is nutritionally constitutive and resistant to hygromycin in minimal media.

In addition, the present invention is the use of a minimal medium containing monosodium glutamate as a nitrogen source for the cultivation of fission yeast according to the present invention and the step of culturing the cleavage yeast according to the present invention in a minimal medium containing monosodium glutamate as a nitrogen source Provides a cleavage yeast culture method comprising a.

It is apparent to those skilled in the art that the hygromycin resistance gene, the open reading frame of the resistance gene, and the sequence of the resistance gene cassette are exemplary and not limited thereto. Sequences having substantial sequence identity or substantial sequence homology to those sequences are also within the scope of the present invention. As used herein, the term "substantial sequence identity" or "substantial sequence homology" is used to express that a sequence represents substantial structural or functional identity with another sequence. This difference is due, for example, to inherent variations in codon usage between different species. If there is a significant amount of sequence overlap or similarity between two or more different sequences, the structural differences are negligible if they have similar physical properties, even if their lengths or structures differ.

Matters related to genetic engineering in the present invention are described in Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).

The expression vector of the present invention, the split yeast transformed by the expression vector, and the split yeast complex medium are suitable for the selection of split yeast comprising an antibiotic resistance gene, the minimum medium composition of the present invention is active in the minimum medium It has the advantage of not losing its activity against antibiotics that lose weight.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Example 1 Preparation of Expression Vector for Fission Yeast Including Hygromycin Resistance Gene

1-1. Preparation of Vectors Containing Hygromycin Resistance Gene Cassettes (hygMX)

As shown in FIG. 3, first, an open reading frame of the gene gene resistance gene from pFA6A (Wach et al., 1994, Yeast 10: 1793), which has a gene cassette of gene gene resistance, was restriction enzyme NcoI / ScaI. It was removed using. The removed DNA fragments were separated by electrophoresis and purely separated using a DNA fragment purification solution (Gel Purification Kit, Bioneer Co.) and a column (Gel Purification Kit, Bioneer Co.). In addition, the open reading frame of the hygromycin resistance gene was also purified after amplification by PCR from pRS-HYG (Taxis C and Knop M. Biotechniques 2006, 40 (1): 73-78). These PCR products were designed to have an NcoI site on one side and no restriction enzyme sites on the other.

PCR amplification was thermally denatured at 94 ° C. for 5 minutes, thermal denaturation at 94 ° C. for 1 minute, primer binding at 54 ° C. for 30 seconds, and extension at 72 ° C. for 90 seconds. ) Was performed 30 times, and the final length extension reaction was performed at 72 ° C. for 5 minutes.

DNA fragments from the pFA6A vector prepared above and PCR products having an open reading frame of the hygromycin resistance gene were respectively combined by DNA ligation to obtain a pFA6-hygMX plasmid having a hygromycin resistance gene cassette. made.

1-2. Preparation of Expression Vector for Fission Yeast Including Hygromycin Resistance Gene Cassette

As shown in FIG. 4, the hygromycin resistance gene cassette region of the pFA6-hygMX plasmid was amplified by PCR. These PCR products were designed to have Sph I sites on one side and Nsi I sites on the other. PCR amplification was performed once with thermal denaturation at 94 ° C. for 5 minutes, thermal denaturation at 94 ° C. for 1 minute, primer binding at 54 ° C. for 30 seconds, and length extension at 72 ° C. for 120 seconds. The extension was carried out 30 times and the final length extension reaction was carried out at 72 ° C. for 10 minutes. PCR products were digested with restriction enzymes (Sph I, Nsi I) and electrophoresed to separate the hygromycin resistance gene cassette from agarose gels and purified using a DNA purification kit (Bioneer Co).

Restriction enzyme SphI / NsiI to remove Ura4 markers of pSLF173 (strong promoter strength), pSLF273 (medium promoter strength), pSLF373 (weak promoter strength) (ATCC), the global bioresource center (ATCC) Was used. Thus, the DNA fragment from which Ura4 was removed was separated by electrophoresis.

Bacterium that binds to the expression vector pSLF173, pSLF273, pSLF373 and PCR-amplified hygromycin resistance gene cassette with Ura4 marker removed by DNA ligation, transformed to bacteria and was correctly bound to the Ura4 position from which the hygromycin resistance gene cassette was removed Colonies were selected to amplify DNA. As a result, fission yeast expression vectors pSLF173-hygMX, pSLF273-hygMX, and pSLF373-hygMX having a hygromycin resistance gene cassette were obtained. Cassette portion of the obtained expression vector was confirmed that the mutation did not occur through sequencing.

Among the cleavage yeast expression vectors, the pSLF173-hygMX vector having the highest promoter strength was transformed into a bacterial strain DH5a capable of amplification (using RbCl ₂₎ . Frozen Stock Method) was named 'DH5a / pSLF173-hygMX' and was deposited on November 8, 2007 to the Korea Biotechnology Research Institute Gene Bank (Accession No. KCTC11230BP).

Test Example 1 Sensitivity Test of Cleavage Yeast Strains against Geneticin or Hygromycin in Complex Medium

Representative fission yeast strains ED665 (haploid, Fanter PA laboratories, UK.) And SP286 (diploid, Paul Nurse laboratories, UK) were prepared in liquid complex medium (5 g / l yeast extract (Difco), 30 g / l glucose (Duksan), 225 mg. / l adenine (Sigma)) incubated for 30 days in an incubator at 30 ℃ to form 5 × 10 ⁷ / ml cells and diluted four times in sequence to add the antibiotics genetisin (Promega), hygromycin (Invitrogen) The mixed medium (the liquid complex medium + 20g / l agar) was spotted (spotting) and incubated for 2 days at 30 ℃ incubator and compared the growth degree. The results are shown in Figure 1, the concentration of Figure 1 refers to the concentration of each antibiotic, the control (control) means the result in a complex medium does not contain antibiotics. As a result, it was found that the cleavage yeast strain was more sensitive in the medium to which hygromycin was added as compared to geneticin. Therefore, when transforming cleavage yeast with an expression vector containing a hygromycin resistance gene as a marker gene according to the present invention, it can be seen that the selectivity of antibiotic resistance strains is higher than that of geneticin resistance genes.

Test Example 2 Sensitivity Test of Cleavage Yeast to Geneticin or Hygromycin in Minimal Medium

Minimal medium instead of complex medium (3g / l Potassium Hydrogen phthallate, 2.2g / l Na ₂ HPO ₄ , 5g / l NH ₄ Cl, 20g / l glucose, 0.26M MgCl ₂ , 4.99mM CaCl ₂ , 0.67M KCl, 14.1mM Na ₂ SO ₄ , 4.2mM pantothetic acid, 81.2mN Nicotinic acid, 55.5mM Inositol, 40.8uM Biotin, 80.9mM Boric acid, 23.7mM MnSO ₄ , 13.9 mM ZnSO ₄ , 7.4 mM FeCl ₂ , 2.47 mM molybdic acid, 6.02 mM KI, 1.6 mM CuSO ₄ , 47.6 mM Citric acid; Sigma) and spotting Spotting) was carried out in the same manner as in Test Example 1, except that the culture was carried out for 3 days at 30 ℃ incubator. The results are shown in Figure 2, the concentration of Figure 2 means the concentration of each antibiotic, the control (control) means the result in a complex medium containing no antibiotics. As shown in FIG. 2, in the case of geneticin and hygromycin, there was a slight growth inhibition as compared to the medium without antibiotics, but over time, the cell growth was almost the same as the control. Indicates.

Comparative Example 1 Preparation of Composite Medium

Complex medium (5 g / l yeast extract (Difco), 30 g / l glucose (Duksan), 225 mg / l adenine (Sigma)) was prepared in Methods in Enzymology Vol. Prepared by the method of 194.

Example 2 Preparation of Hygromycin-Containing Medium

A composite medium was prepared in the same manner as in Comparative Example 1 except that the concentration of hygromycin was changed to 100, 200, and 300 µg / ml.

Test Example 3 Antibiotic Resistance Confirmation Test of Yeast Expression Vector Containing Antibiotic Resistance Gene

PSLF173-hygMX, a representative vector of cleavage yeast expression vectors prepared by amplification in Example 1, and pSLF173, a mother vector of the expression vectors, were respectively transformed into lithium acetate in a cleavage yeast ED665 (Fantes PA laboratory). The cells were transformed (Methods in Enzymology Vol. 194), diluted about 10-fold, and plated in the complex medium of Comparative Example 1 and the complex medium of Example 2 (300 g / ml of hygromycin), respectively, and incubated at 30 ° C for 2 days. After the growth of cleavage yeast was observed. The results are shown in Fig. 5 is a photograph showing the results of assaying whether colonies of strains transformed with the vector were transformed by transforming the pSLF173-hygMX vector into fission yeast and spreading on a complex medium to which antibiotic hygromycin was added. As a result, fission yeast transformed with the pSLF173 vector without antibiotic markers showed growth only in complex medium containing no antibiotics, and fission yeast transformed with pSLF173-hygMX showed even growth in complex medium containing antibiotics. . These results indicate that the hygromycin resistance gene included in the cleavage yeast expression vector according to the present invention can be used as an antibiotic marker gene. In addition, it can be seen that antibiotic activity occurs effectively at a concentration of 300 ㎍ / ml hygromycin.

Test Example 4 Antibiotic Resistance Test of Vector

The expression vector prepared by the amplification in Example 1 was transformed into fission yeasts in the same manner as in Test Example 3, and screened in a complex medium containing hygromycin (300 µg / ml), and a pSLF173 / 273/373 vector having a nutritional marker Were screened in minimal medium without uracil and liquid culture (5 g / l yeast extract (Difco), 30 g / l glucose (Duksan), 225 mg / l adenine (Sigma); 30 ° C.).

Each culture solution was diluted to 1 × 10 ⁷ cells / ml, sequentially diluted 4 times to make 5 stages, and then to the complex medium prepared in Comparative Example 1 and the complex medium of Example 2 (Hygromycin 200㎍ / ml). Spotting was performed. After incubation at 30 ° C. for 2 days, the degree of resistance was determined from the degree of growth of each vector. The results are shown in FIG. Figure 6 is transformed pSLF173-hygMX, pSLF273-hygMX, pSLF373-hygMX vector to fission yeast cultured in liquid medium and then diluted in spotted (spotting) in a complex medium with antibiotic hygromycin added As a photograph showing the results of assaying the antibiotic resistance of the transformed strain by strain concentration, column 1 of Figure 6 is the result of transforming the pSLF173, pSLF173-hygMX vector in the complex medium prepared in Comparative Example 1, Rows 2 to 4 show the results in the composite medium of Example 2, respectively. As a result, all the cleavage yeast transformed by the vector containing the hygromycin resistance gene prepared in Example 1 was found to be grown regardless of the concentration of the cells. In addition, it can be seen that the antibiotic activity occurs effectively at a concentration of 200 ㎍ / ml hygromycin.

<Test Example 5>

Except for spotting on the composite medium of Example 2 (100 g / ml of hygromycin), the test was carried out in the same manner as in Example 4, and as a result, it was found that Only fission yeasts with mycin resistance genes were grown in complex media containing antibiotics.

From the above results and the results of Test Example 3 and Test Example 4, the composite medium composition for cleavage yeast according to the present invention exhibits sufficient antibiotic activity at a hygromycin concentration of 100 to 300 µg / ml, thereby indicating a hygromycin resistance gene. It can be seen that the selectivity of cleavage yeast used as a gene is excellent.

<Test Example 6> Usability test in the minimum medium of the yeast expression vector

The expression vector prepared by amplification in Example 1 was transformed into a fission yeast ED665 by a lithium acetate transformation method (Methods in Enzymology Vol. 194), diluted about 10-fold, and then a minimal medium containing 200 g / ml of hygromycin. (3g / l Potassium Hydrogen phthallate, 2.2g / l Na ₂ HPO ₄ , 5g / l NH ₄ Cl, 20g / l Glucose, 0.26M MgCl ₂ , 4.99mM CaCl ₂ , 0.67M KCl, 14.1 mM Na ₂ SO ₄ , 4.2 mM pantothetic acid, 81.2 mM Nicotinic acid, 55.5 mM Inositol, 40.8 uM Biotin, 80.9 mM Boric acid, 23.7 mM MnSO ₄ , 13.9 mM ZnSO ₄ , 7.4mM FeCl ₂ , 2.47mM molybdic acid, 6.02mM KI, 1.6mM CuSO ₄ , 47.6mM Citric acid; Sigma) and incubated for 3 days at 30 ℃ The growth in the minimal medium of the strain with the marker was examined. Cleavage yeast ED665 transformed with a pSLF173 vector with nutrition markers was used as a control, and the results are shown in FIG. 7. Figure 7 transforms pSLF173-hygMX, pSLF273-hygMX, or pSLF373-hygMX vector to cleavage yeast, diluted in minimal medium with antibiotic hygromycin (200 µg / ml) and plated, and then transformed into vector. As a result of assaying the antibiotic resistance of each strain concentration, as a result, in the minimal medium containing hygromycin, only nutrition markers in addition to the cleavage yeast transformed with pSLF173-hygMX, pSLF273-hygMX, or pSLF373-hygMX vector The branched yeast ED665 transformed with pSLF173 vectors also showed normal growth. In other words, it was confirmed that hygromycin does not have proper activity in minimal medium of cleavage yeast.

Example 5 Preparation of Modified Minimum Medium

A minimum medium containing hygromycin was prepared in the same manner as the minimum medium of Test Example 6 except that 1 g / l of monosodium glutamate (Sigma) was used instead of ammonium chloride as a nitrogen source.

Hygromycin availability test in modified minimal media

Smeared yeast strain ED665 in a minimal medium prepared above was incubated for 3 days at 30 ℃ growth was examined. The results are shown in Fig. 8B. FIG. 8 (A) shows a case where the fission yeast strain ED665 is plated in a minimal medium containing hygromycin of Test Example 6 for comparison and incubated at 30 ° C. for 3 days. As a result, the cleavage yeast strain grows in the minimal medium having the minimum nitrogen source of ammonium chloride (NH ₄ Cl), which shows no activity of hygromycin, but in the minimum medium using Monosodium Glutamate as the nitrogen source, the hygromycin By cleavage yeast strain can be seen that. Therefore, the minimal medium according to the present invention does not lose the activity of hygromycin, it is possible to use a hygromycin resistance gene as an antibiotic marker gene.

<Survival and Screening Test of Cleaved Yeast Transformed by Vectors Containing Hygromycin Resistance Gene in Modified Minimal Media>

PSLF173-hygMX vector prepared in Example 1 or pSLF173 vector containing Ura4 gene, a nutritional marker, was transformed into cleavage yeast (ED665, Fantes PA Laboratories) by lithium acetate transfer method (Methods in Enzymology Vol. 194) After diluting four times sequentially, the minimum medium of Test Example 2 (medium without hygromycin), the minimum medium of Test Example 6 (including 200 μg / ml of hygromycin), or the minimum medium (hygromycin) Spotted strains, each diluted 4 fold in 200 μg / ml, including monosodium glutamate as a nitrogen source, incubated at 30 ° C. for 2 days, and then grown in a modified minimal medium of the antibiotic marker strain. Investigate. The results are shown in FIG. 9, respectively. Column 1 shows the minimum medium of Test Example 2, column 2 shows the minimum medium of Test Example 6, and column 3 shows the results for the minimum medium. As shown in FIG. 9, the fission yeast transformed with the pSLF173 vector having only the nutritional marker was grown in the minimum medium of Test Example 2 and the minimum medium of Test Example 6, but did not grow in the minimum medium according to the present invention. It can be seen that the cleavage yeast transformed by the vector containing the hygromycin resistance gene according to the growth in all media. Therefore, the minimum medium using the minimum medium composition according to the present invention made by replacing ammonium chloride (NH ₄ Cl) with monosodium glutamate as the nitrogen source in the minimum medium can be used for hygromycin without activity in the minimum medium. It can be seen.

Figure 1 is a photograph showing the sensitivity test results of the cleavage yeast strain for genesis, or hygromycin in the complex medium.

Figure 2 is a photograph showing the sensitivity test results of the cleavage yeast strain for genesis, or hygromycin in the minimal medium.

Figure 3 is a schematic diagram showing the manufacturing process of the vector containing the hygromycin resistance gene cassette.

Figure 4 is a schematic diagram showing the manufacturing process of the expression vector for cleavage yeast containing a hygromycin resistance gene cassette.

Figure 5 is a photograph showing the results of confirming the antibiotic resistance of the yeast expression vector containing the antibiotic resistance gene.

Figure 6 is a photograph showing the antibiotic resistance test results of the yeast expression vector containing the antibiotic resistance gene.

Figure 7 is a photograph showing the results of testing the usability in the minimal medium of the yeast expression vector.

Figure 8 is a photograph showing the results of activity investigation of hygromycin in a minimal medium containing NH ₄ Cl as a nitrogen source (A), and the results of activity investigation of hygromycin in a modified minimum medium (B).

Figure 9 is a photograph showing the results of the survival and screening test of the cleavage yeast transformed by the vector containing the hygromycin resistance gene in modified minimal medium.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Expression vectors for fission yeast, construction method          obvious, and medium composition for culturing the fission yeast <130> P07-055-KRI <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer for ORF of hygromycin resistance gene <400> 1 caaccatggg taaaaagcct gaac 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer for ORF of hygromycin resistance gene <400> 2 tgattattcc tttgccctcg gac 23 <210> 3 <211> 1029 <212> DNA <213> Artificial Sequence <220> <223> ORF of hygromycin resistance gene <400> 3 atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc 60 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc 120 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa 180 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac 240 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg 300 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg 360 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa 420 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg 480 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat 540 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc 600 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag 660 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg 720 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg 780 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt 840 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc 900 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat 960 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca 1020 aaggaataa 1029 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer for cassette of hygromycin resistance gene <400> 4 gcatgcattg tttagcttgc ctcgtccc 28 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer for cassette of hygromycin resistance gene <400> 5 gcgcatgccg ttttcgacac tggatggc 28 <210> 6 <211> 1670 <212> DNA <213> Artificial Sequence <220> <223> cassette of hygromycin resistance gene <400> 6 atgcattgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca tggaggccca 60 gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc 120 gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg 180 cacggcgcga agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc 240 cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta 300 ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag 360 ttctcacatc acatccgaac ataaacaacc atgggtaaaa agcctgaact caccgcgacg 420 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg 480 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg 540 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg 600 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat 660 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc 720 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag 780 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat 840 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc 900 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc 960 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc 1020 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc 1080 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc 1140 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt 1200 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg 1260 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc 1320 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga 1380 aaccgacgcc ccagcactcg tccgagggca aaggaataat cagtactgac aataaaaaga 1440 ttcttgtttt caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt 1500 aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga 1560 agttaagtgc gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac 1620 tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa aacggcatgc 1670  

Claims (13)

delete delete delete delete delete delete delete delete delete delete delete delete A minimal medium composition for cleavage yeast culture comprising a hygromycin resistance gene as a marker gene, including monosodium glutamate as a nitrogen source, in addition to hygromycin, and transformed by an expression vector for cleavage yeast having a promoter for an expression vector.

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