Simple method for producing competent cells and transformed cells

Abstract

To provide methods for producing competent cells of unicellular microorganisms that can be produced simply while having practical transformation efficiency comparable to that of the conventional technique and also provide methods for producing transformed unicellular microorganisms that can perform the production of competent cells and transformation by a one-tube method.SOLUTION: The present invention provides a culture medium comprising a minimal essential medium, polyethylene glycol, and a bivalent cation, and methods for producing competent cells of unicellular microorganisms using the same, and methods for producing transformed unicellular microorganisms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a simple method for producing competent cells and a method for producing transformed cells. More specifically, this invention relates to a simple method for producing competent cells of single-celled microorganisms and a method for producing transformed single-celled microorganisms. [Background technology]

[0002] The development of genetic engineering technology, which introduces genes from one organism into the cells of another, has brought about significant progress in cell biology. In particular, transformation methods that use vectors such as plasmids to introduce target genes into bacterial cells such as E. coli have been widely used since their discovery in the 1970s for applications such as DNA cloning and gene or protein expression analysis.

[0003] Because cells are protected by a cell membrane, it is usually not possible to easily introduce extracellular molecules, especially macromolecules, into the cell. In order to introduce foreign DNA, such as vectors, into bacterial cells by passing through the cell membrane, it is necessary to manufacture competent cells with increased cell membrane permeability by some means.

[0004] In 1972, Cohen et al. discovered that transformation with plasmid DNA was possible by recovering E. coli in the logarithmic growth phase, treating it with calcium ions, and then briefly heat-treating it (Non-Patent Literature 1). In 1983, Hanahan published a method for producing competent cells using a buffer containing calcium ions, other divalent cations, and DMSO, and E. coli that had been previously grown in a normal medium to the logarithmic growth phase (Non-Patent Literature 2). In 1989, Chung et al. published a method for producing competent cells using a buffer containing polyethylene glycol (PEG), magnesium ions, dimethyl sulfoxide (DMSO), and the like, and E. coli that had been previously grown in a normal medium to the logarithmic growth phase (Non-Patent Literature 3).

[0005] In addition, in 1988, Dower et al. published an electroporation method in which plasmid DNA is introduced by harvesting E. coli that has been grown in conventional medium up to the logarithmic growth phase, repeatedly washing it with a low ionic strength solution, and then applying a high voltage to the bacterial cells suspended in 10% glycerol (Non-Patent Literature 4).

[0006] In a comparative analysis of the transformation efficiency of competent cells produced by the methods described in Non-Patent Documents 1-3 when transformation was performed without electroporation, the transformation efficiency of competent cells produced by the method described in Non-Patent Document 1 was 10 5 -10 7 The transformation efficiency of competent cells produced by the method described in Non-Patent Document 2, using CFU / μg DNA, is 10 4 -10 8 The transformation efficiency of competent cells produced using the method described in Non-Patent Document 3 with CFU / μg DNA is 1 × 10⁻⁶. 3 -10 5 It has been shown that the concentration was CFU / μg DNA (Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cohen et al., PNAS, 69(8), 2110-2114, 1972 [Non-Patent Document 2] Hanahan, J. Mol. Biol., 166(4), 557-580, 1983 [Non-Patent Document 3] Chung et al., PNAS, 86(7), 2172-2175, 1989 [Non-Patent Document 4] Dower et al., Nucl. Acid. Res., 16(13), 1988 [Non-Patent Document 5] Chan et al. Biosci Rep, 33(6), e00086, 2013 [Overview of the project] [Problems that the invention aims to solve]

[0008] Conventional methods for producing competent cells and transformation methods using competent cells require several steps: preparing a culture medium by culturing E. coli overnight to obtain cells that have grown to the logarithmic growth phase; treating the cells separated by centrifugation with a buffer for competent cell production; and transferring the cells separated by further centrifugation to a buffer for storage or transformation. This process requires considerable time and effort for cultivation, frequent centrifugation, and cell washing. Furthermore, while these conventional methods can be used to produce large quantities of competent cells from one or a few strains, they pose a significant problem in that they require considerable time and effort to produce small quantities of competent cells from multiple strains.

[0009] The present invention aims to provide a method for producing competent cells of single-celled microorganisms that can be easily manufactured while having transformation efficiency equivalent to that of the prior art. Furthermore, the present invention aims to provide a method for producing transformed single-celled microorganisms in which the production of competent cells and transformation can be carried out in a single tube. [Means for solving the problem]

[0010] As a result of diligent research to solve the above problems, the inventors of the present invention have found that E. coli can be grown in a culture medium prepared by adding polyethylene glycol and divalent cations to the basal culture medium of E. coli. Furthermore, plasmid DNA can be introduced by directly adding plasmid DNA to the grown E. coli, achieving the same results as the conventional technology. 5 We have completed this invention by discovering that we can produce competent cells that can stably achieve transformation efficiencies of CFU / μg DNA or higher.

[0011] The present invention is not limited thereto, but includes the following embodiments. [1] A culture medium for producing competent cells of single-celled microorganisms, containing a basal medium, polyethylene glycol, and divalent cations. [2] The culture medium according to [1], wherein the single-celled microorganism is Escherichia coli. [3] The culture medium according to [1] or [2], wherein the divalent cation is derived from MgSO4 or from MgSO4 and CaCl2. [4] The method according to any one of [1] to [3], wherein polyethylene glycol is contained at a concentration of 5 to 10% by weight. [5] The culture medium according to any one of [1] to [4], wherein polyethylene glycol is PEG8000. [6] The culture medium according to any one of [3] to [5], wherein MgSO4 is contained at a concentration of 25 to 100 mM. [7] The culture medium according to any one of [3] to [6], wherein CaCl2 is contained at a concentration of 10 to 50 mM. [8] A kit for transformation of single-celled microorganisms, containing the culture medium according to any one of [1] to [7]. [9] (Method 1) Culturing single-celled microorganisms in a culture medium containing a basal medium, polyethylene glycol, and divalent cations to prepare a culture; and (Method 2) Obtaining the single-celled microorganisms contained in the culture as competent cells A method for producing competent cells of single-celled microorganisms, comprising the above steps.

[10] The method according to [9], which does not include a centrifugation step.

[11] The method according to [9] or

[10] , wherein the single-celled microorganism is Escherichia coli.

[12] The method according to any one of [9] to

[11] , wherein the divalent cation is derived from MgSO4 or from MgSO4 and CaCl2.

[13] The method according to any one of [9] to

[12] , wherein the culture medium contains polyethylene glycol at a concentration of 5 to 10% by weight.

[14] The method according to any one of [9] to

[13] , wherein polyethylene glycol is PEG8000.

[15] The method according to any one of the items [9] to

[14] , wherein the culture is performed at 25°C to 37°C for 12 to 24 hours.

[16] The method according to any one of the items in [9] to

[14] , wherein the culture is performed at 37°C for 1 to 3 hours.

[17] The method according to any one of

[12] to

[16] , wherein the culture medium contains MgSO4 at a concentration of 25 to 100 mM.

[18] The method according to any one of

[12] to

[17] , wherein the culture medium contains CaCl2 at a concentration of 10 to 50 mM.

[19] (1) A step of preparing a culture by culturing single-celled microorganisms in a culture medium containing a base medium, polyethylene glycol, and divalent cations; (2') Adding a transformation vector to the culture and incubating at 4°C or below. A method for producing transformed single-celled microorganisms, including [the specified component].

[20] The method according to

[19] , which does not involve a centrifugal separation step. [twenty one] The method according to

[19] or

[20] , wherein the single-celled microorganism is Escherichia coli. [twenty two] The method according to any one of

[19] to

[21] , wherein the incubation is performed on ice in step (2'). [Effects of the Invention]

[0012] The present invention provides a method for producing competent cells of single-celled microorganisms that can be easily manufactured while having transformation efficiency suitable for practical use. Furthermore, the present invention provides a method for producing transformed single-celled microorganisms in which the production of competent cells and transformation can be performed in a single tube. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows an overview of the manufacturing and transformation of competent cells according to the present invention. [Figure 2] Figure 2 shows the composition of TFM in relation to polyethylene glycol (PEG8000) concentration. [Figure 3] Figure 3 shows the composition of TFM with respect to MgSO4 concentration. [Figure 4] Figure 4 shows the composition of TFM in relation to CaCl2 concentration. [Figure 5] Figure 5 shows the results of the study on incubation times at temperatures ranging from 25°C to 37°C. [Figure 6] Figure 6 shows the results of an investigation into incubation times at 37°C for a short period of time. [Figure 7] Figure 7 shows the results of examining the duration of standing on ice after DNA addition. [Figure 8] Figure 8 shows the simultaneous transformation of multiple E. coli strains. [Modes for carrying out the invention]

[0014] <Single-celled microorganisms> The single-celled microorganisms used in this invention can be any that are used for genetic recombination by transformation, and yeasts, bacteria, etc., can be suitably used. Suitable yeasts include those of the genera Saccharomyces, Schizosaccharomyces, Candida, and Aspergillus. Suitable bacteria include, for example, Escherichia coli (E. coli), Acinetobacter, Pseudomonas, Salmonella, Vibrio, Campylobacter, Legionella, Yersinia, Shigella, Haemophilus influenzae (H. influenzae), Staphylococcus, Lactococcus and other lactic acid bacteria, actinomycetes such as Streptomyces, and Bacillus subtilis.

[0015] <Culture medium for producing competent cells> This invention relates to a culture medium for producing competent cells of single-celled microorganisms. The culture medium of this invention contains a basal medium, polyethylene glycol, and divalent cations, thereby enabling the cultivation of single-celled microorganisms inoculated into the culture medium and the simultaneous production of competent cells.

[0016] The culture medium of the present invention may contain, in addition to the base medium, polyethylene glycol, and divalent cations, any additional components such as serum, growth factors, pH indicators, antibiotics, and coagulants such as agar and gelatin. The culture medium of the present invention preferably does not contain dimethyl sulfoxide (DMSO).

[0017] The basal medium included in the culture medium of the present invention refers to a medium that supports the growth of single-celled microorganisms, containing a sufficient source of amino acids, vitamins, minerals, and carbohydrates necessary for the growth of single-celled microorganisms. The basal medium in the present invention is not particularly limited as long as it does not inhibit the growth of single-celled microorganisms, and a basal medium prepared from a known composition may be used, or a commercially available basal medium may be used. Examples of basal media for E. coli include LB medium, TB medium, YT medium, 2×YT medium, and mixed media thereof.

[0018] The polyethylene glycol contained in the culture medium of the present invention can be a commercially available polyethylene glycol or its derivative having a number average molecular weight of 200 to 20,000, for example, PEG8000 with a number average molecular weight of 8,000 can be used.

[0019] The concentration of polyethylene glycol contained in the culture medium of the present invention can be appropriately determined depending on the type of single-celled microorganism used, but for example, it can be set to a concentration of 0.01 to 30% by weight, 1 to 20% by weight, or 5 to 10% by weight.

[0020] As the divalent cations contained in the culture medium of the present invention, ions of Group 2 elements (alkaline earth metals) can be used, for example, those derived from MgSO4 or those derived from MgSO4 and CaCl2 can be used.

[0021] The culture medium of the present invention may contain MgSO4 at concentrations of 10-200 mM, 15-150 mM, or 25-100 mM. The culture medium of the present invention may contain, in addition to the above-mentioned MgSO4, 1 to 200 mM, 5 to 150 mM, or 10 to 50 mM of CaCl2.

[0022] <Transformation Kit> The culture medium of the present invention allows for the simultaneous cultivation of single-celled microorganisms inoculated into the culture medium and the production of competent cells. Furthermore, by directly adding an arbitrary transformation vector to the culture containing the produced competent cells, the production and transformation of competent cells can be carried out in a single tube.

[0023] Therefore, in another aspect, the present invention relates to a kit for transforming single-celled microorganisms, comprising the culture medium of the present invention described above. The transformation kit of the present invention may include, in addition to the culture medium of the present invention, additional components such as an optional transformation vector, instructions for the transformation procedure, and / or a package containing such instructions.

[0024] <Method for producing competent cells from single-celled microorganisms> In another aspect, this invention relates to a method for producing competent cells of single-celled microorganisms. Specifically, this invention relates to a method for producing competent cells of single-celled microorganisms. (1) A step of preparing a culture by culturing single-celled microorganisms in a culture medium containing a base medium, polyethylene glycol, and divalent cations; and (2) A step to obtain competent cells from single-celled microorganisms contained in the culture. Method for producing competent cells of single-celled microorganisms, including Regarding.

[0025] The present invention provides a method for producing competent cells of single-celled microorganisms, comprising the steps of (1) preparing a culture by culturing single-celled microorganisms in a culture medium containing a basal medium, polyethylene glycol, and divalent cations. The culture medium used in step (1) is as described above. The single-celled microorganisms to be cultured can be a portion of those cultured separately in liquid, picked from colonies, or thawed frozen stocks such as glycerol stocks, as appropriate. Those picked from colonies or thawed frozen stocks such as glycerol stocks may be directly inoculated into the culture medium without going through a separate extended culture step.

[0026] According to the method for producing competent cells of single-celled microorganisms of the present invention, culture and production of competent cells can be performed simultaneously in step (1). There are no particular restrictions on the amount of culture medium used in step (1), but it is preferable to use a liquid culture medium having a volume in the microliter to milliliter range. According to the method for producing competent cells of single-celled microorganisms of the present invention, competent cells can be easily produced even in small quantities, and therefore, competent cells can be produced simultaneously using a large number of bacterial samples. For example, competent cells can be produced simultaneously from 5 or more, 10 or more, 100 or more, or 1000 or more bacterial samples.

[0027] The culture conditions in step (1) are not particularly limited as long as single-celled microorganisms can grow, but it is preferable to carry out the process at 25°C to 37°C for 12 to 24 hours, or at 37°C for 1 to 3 hours. The present invention provides a method for producing competent cells of single-celled microorganisms, comprising step (1) followed by step (2) of obtaining competent cells from the single-celled microorganisms contained in the culture prepared in step (1). The single-celled microorganisms contained in the culture prepared in step (1) can be obtained as competent cells as is. Therefore, the culture prepared in step (1) can be used directly in the transformation step without any additional processing such as centrifugation or filtration. Alternatively, the culture prepared in step (1) may be centrifuged or filtered to obtain concentrated competent cells.

[0028] The method for producing competent cells of single-celled microorganisms according to the present invention may not include a centrifugation step. The method for producing competent cells of a single-celled microorganism according to the present invention may consist of steps (1) and (2).

[0029] <Method for producing transformed single-celled microorganisms> The culture medium of the present invention allows for the simultaneous cultivation of single-celled microorganisms inoculated into the culture medium and the production of competent cells. Furthermore, by directly adding an arbitrary transformation vector to the culture containing the produced competent cells, the production and transformation of competent cells can be carried out in a single tube.

[0030] In another aspect, this invention relates to a method for producing transformed single-celled microorganisms. Specifically, the present invention is (1) A step of preparing a culture by culturing single-celled microorganisms in a culture medium containing a base medium, polyethylene glycol, and divalent cations; (2') Adding a transformation vector to the culture and incubating at 4°C or below. Method for producing transformed single-celled microorganisms, including Regarding.

[0031] The present invention provides a method for producing transformed single-celled microorganisms, which includes step (1) preparing a culture by culturing single-celled microorganisms in a culture medium containing a basal medium, polyethylene glycol, and divalent cations. Step (1) is the same as step (1) in the method for producing competent cells of single-celled microorganisms described above.

[0032] The present invention provides a method for producing transformed single-celled microorganisms, comprising step (1) followed by step (2'), in which a transformation vector is added to the culture and incubated at 4°C or below. The single-celled microorganisms contained in the culture prepared in step (1) can be obtained as competent cells. Therefore, the culture prepared in step (1) can be used directly in the transformation step (2') without any additional processing such as centrifugation or filtration.

[0033] There are no restrictions on the transformation vector used in step (2'), but a DNA vector is preferred. Examples of DNA vectors include plasmid vectors, cosmid vectors, fosmid vectors, DNA viral vectors such as lambda phage vectors, and artificial chromosome vectors such as BAC and PAC.

[0034] In step (2'), the transformation vector is added to the culture and incubated at 4°C or below. Incubation may be performed on ice. The incubation time can be adjusted as appropriate, for example, from 1 minute to 1 hour, 5 minutes to 30 minutes, or 10 minutes to 20 minutes. If incubation is performed on ice, it can be from 5 minutes to 15 minutes, or from 5 minutes to 10 minutes.

[0035] The method for producing transformed single-celled microorganisms of the present invention may not include a centrifugation step. The present invention's method for producing transformed single-celled microorganisms may consist of steps (1) and (2').

[0036] Transformed single-celled microorganisms produced by the method for producing transformed single-celled microorganisms of the present invention can be used directly for culture and analysis without any further additional steps. [Examples]

[0037] The details of the present invention will be specifically described below with reference to experimental examples, but the present invention is not limited thereto. Furthermore, unless otherwise specified in this specification, numerical ranges are described as including their endpoints.

[0038] <1. Manufacturing and transformation of competent cells> Competent cells were prepared and transformed by the following method unless otherwise specified. E. coli colonies or glycerol stocks were suspended in 50 μL of Transformation Medium (TFM, the culture medium of the present invention) in 1.5 mL or 0.2 mL microtubes. TFM was prepared by the following method: 2xYT medium (16 g Bacto Tryptone (BD Biosciences), 10 g Bact Yeast Extract (BD Biosciences), 5 g NaCl in 1 L H2O) was used as the basal medium, to which PEG 8000 (Sigma-Aldrich), MgSO4, and CaCl2 were added in concentrations of 7.5%, 50 mM, and 10 mM, respectively. Competent cells were prepared by incubating E. coli in TFM overnight at 30°C. Transformation was performed by placing the competent cells on ice, adding plasmid DNA, and incubating on ice for 10 minutes. Transformed E. coli were spread on LB agar containing a suitable antibiotic (Figure 1).

[0039] <2. Examination of TFM composition> Transformation was carried out using TFM in which only the concentration of PEG8000 was changed to 0, 2.5, 5, 7.5, 10 wt%. The Escherichia coli strain BW25113 (F- DE(araD-araB)567 lacZ4787(del)::rrnB-3 LAM- rph-1 DE(rhaD-rhaB)568 hsdR514) was used. Also, the plasmid to be introduced was pUC19-cat with chloramphenicol resistance (Nozaki and Niki, J. Bacteriol., 201(5):e00660-18, 2019, doi: 10.1128 / JB.00660-18.). Colonies of the BW25113 strain were suspended in 50 μL of TFM with the concentration of PEG8000 changed in a 0.2 mL microtube, the lid was closed, and the mixture was statically cultured at 30 °C for 20 hours. The tube was transferred onto ice, 2 ng of pUC19-cat was added, and the mixture was placed on ice for 20 minutes. Then, the whole volume was spread onto an LB agar medium containing 15 μg / mL of chloramphenicol and cultured overnight at 37 °C, and the transformation efficiency was measured from the number of colonies that appeared (Figure 2). The averages of the experiments performed three times under each condition were shown. Error bars indicate the standard deviation.

[0040] When polyethylene glycol is added to be 5-10 wt%, it was found that the transformation efficiency of about 10 5 CFU / μg DNA can be stably achieved. Similarly, transformation was carried out and the transformation efficiency was measured using TFM in which only the concentration of MgSO4 was changed to 0, 25, 50, 75, 100 mM (containing 7.5 wt% of PEG8000 and 10 mM of CaCl2), and TFM in which only the concentration of CaCl2 was changed to 0, 5, 10, 25, 50 mM (containing 7.5 wt% of PEG8000 and 50 mM of MgSO4) (Figure 3, Figure 4).

[0041] From the results in Figure 3, it was found that when MgSO4 is added at a concentration of 25-100 mM, the transformation efficiency of about 10 5 ~10 6 CFU / μg DNA can be stably achieved. The results in Figure 4 show that even TFM without CaCl2 is stable at 10 5 Competent cells capable of achieving transformation efficiencies of approximately CFU / μg DNA can be manufactured, and even when CaCl2 is added at concentrations of 5-50 mM, they remain stable at 10 5 It was found that competent cells capable of achieving transformation efficiencies of approximately CFU / μg DNA can be manufactured.

[0042] <3. Examination of culture temperature and optimal culture conditions> Colonies of BW25113 were suspended in 50 μL of TFM in 0.2 mL tubes. The tubes were placed at 25°C, 30°C, and 37°C and incubated statically for 4, 8, 12, 16, and 24 hours, respectively. The tubes were then transferred to ice, 1 ng of pUC19-cat was added, and the tubes were left on ice for 10 minutes. Subsequently, the entire volume was spread onto LB agar medium containing 15 μg / mL chloramphenicol and incubated overnight at 37°C. Transformation efficiency was measured from the number of colonies that appeared (Figure 5). The average of three experiments performed under each condition is shown. Error bars indicate the standard deviation.

[0043] 10 5 Competent cells capable of achieving transformation efficiencies of CFU / μg DNA or higher can be produced, and when cultured at 30°C for 12 hours or more, 10 6 We were able to produce competent cells that could achieve transformation efficiencies exceeding CFU / μg DNA.

[0044] <Investigation of short-term culture at 4.37°C> Colonies of BW25113 were suspended in 50 μL of TFM in 0.2 mL tubes. After static incubation at 37°C for 0, 1, 2, and 3 hours, the cultures were transferred to ice, 1 ng of pUC19-cat was added, and the cultures were left to stand on ice for 10 minutes. Subsequently, the entire volume was spread onto LB agar medium containing 15 μg / mL chloramphenicol and incubated overnight at 37°C. Transformation efficiency was measured from the number of colonies that appeared (Figure 6). The average of three experiments performed under each condition is shown. Error bars indicate the standard deviation.

[0045] Just mixing the colony with TFM will get you 10 5 We were able to produce competent cells that achieved a transformation efficiency of approximately CFU / μg DNA, and we found that culturing them at 37°C for 1 to 3 hours could produce even more competent cells with improved transformation efficiency.

[0046] <5. Examination of the duration of standing on ice after DNA addition> Colonies of BW25113 were suspended in 50 μL of TFM in 0.2 mL tubes. After standing incubation at 30°C for 20 hours, the cells were transferred to ice, 1 ng of pUC19-cat was added, and the cells were left standing on ice for 0, 5, 10, or 20 minutes. The entire volume was then spread onto LB agar medium containing 15 μg / mL of chloramphenicol and incubated overnight at 37°C. Transformation efficiency was measured from the number of colonies that appeared (Figure 7). The average of three experiments performed under each condition is shown. Error bars indicate the standard deviation.

[0047] Even just adding DNA to competent cells can cost 10 5 While we were able to achieve a transformation efficiency of approximately CFU / μg DNA, we found that the transformation efficiency could be further increased by allowing the mixture to stand on ice for 5 to 20 minutes, preferably 5 to 10 minutes.

[0048] <6. Simultaneous transformation of multiple E. coli strains> 8 types of E. coli strains, MG1655 strain, SN1187 strain (MG1655 ΔhsdR ΔendA ΔrecA), MC1061 strain (hsdR, mcrB, araD139, Δ(araABC-leu)7679, ΔlacX74, galU, galK, rpsL, thi), BW25113 strain, JM109 strain (recA1, endA1, gyrA96, thi, hsdR17(rK- mK+), e14- (mcrA-), supE44, relA1, Δ (lac-proAB) / F'[traD36, proAB+, lac Iq, lacZΔM15]), DH5α strain (F-, Φ80d lacZΔM15, Simultaneous transformation of Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rK- mK+), phoA, supE44, λ-, thi-1, gyrA96, relA1), BL21(DE3) strain (F-, ompT, hsdSB(rB- mB-), gal(λcI 857, ind1, Sam7, nin5, lacUV5-T7gene1), dcm(DE3)), and ClearColi BL21(DE3) strain (Lucigen) was attempted. Each strain was inoculated from glycerol stocks into 50 μL of TFM in 0.2 mL tubes. After 20 hours of static incubation at 30°C, the cultures were transferred to ice, 1 ng of pUC19-cat was added, and the cultures were left to stand on ice for 10 minutes. Subsequently, the entire volume was spread onto LB agar medium containing 15 μg / mL chloramphenicol and incubated overnight at 37°C. Transformation efficiency was measured from the number of colonies that appeared (Figure 8). The graph shows the average of three experiments. Error bars indicate the standard deviation. Plasmid DNA was prepared from two colonies obtained from each strain using Qiagen's Qiaprep mini spin miniprep kit. Plasmid DNA was confirmed by 0.7% agarose gel electrophoresis. It was confirmed that pUC19-cat had been introduced into all strains. [Industrial applicability]

[0049] According to the present invention, it is possible to perform a culture method that is almost equivalent to the conventional method (10) without requiring the preparation of E. coli in the logarithmic growth phase, frequent centrifugation, cell washing, and other complicated procedures, as well as expensive shaking culture equipment. 5 This method allows for the simple production of competent cells with transformation efficiencies of CFU / μg DNA or higher. Transformation can be easily performed by simply adding the desired DNA to the produced competent cells and placing them on ice for a short time, making it possible to produce and transform competent cells in a single tube. Furthermore, because the reaction can be carried out with a small reaction system, numerous samples can be processed simultaneously in a space-saving manner. Therefore, it is possible to easily produce and transform competent cells of multiple bacterial strains simultaneously, which opens up possibilities for applications in high-throughput analysis.

Claims

1. A culture medium for use in a method for producing competent cells of single-celled microorganisms, The culture medium comprises a basal medium, polyethylene glycol, and divalent cations. The method described above is (1) A step of preparing a culture by culturing single-celled microorganisms in the culture medium; and (2) A step to obtain competent cells from single-celled microorganisms contained in the culture. Includes, The aforementioned single-celled microorganism is Escherichia coli. The divalent cation is derived from MgSO₄ or from MgSO₄ and CaCl₂. The culture is carried out at 25°C to 37°C for 12 to 24 hours, or the culture is carried out at 37°C for 1 to 3 hours. The culture medium.

2. The culture medium according to claim 1, wherein the method does not include a centrifugal separation step.

3. A method for producing competent cells of a single-celled microorganism, (1) A step of preparing a culture by culturing single-celled microorganisms in a culture medium containing a base medium, polyethylene glycol, and divalent cations; and (2) A step to obtain competent cells from single-celled microorganisms contained in the culture. Includes, The aforementioned single-celled microorganism is Escherichia coli. The divalent cation is derived from MgSO₄ or from MgSO₄ and CaCl₂. The culture is carried out at 25°C to 37°C for 12 to 24 hours, or the culture is carried out at 37°C for 1 to 3 hours. The aforementioned manufacturing method.

4. The method according to claim 3, which does not include a centrifugal separation step.

5. A method for producing a transformed single-celled microorganism, (1) A step of preparing a culture by culturing single-celled microorganisms in a culture medium containing a base medium, polyethylene glycol, and divalent cations; (2') A step of adding a transformation vector to the culture and incubating it at 4°C or below. Includes, The aforementioned single-celled microorganism is Escherichia coli. The divalent cation is derived from MgSO₄ or from MgSO₄ and CaCl₂. The culture is carried out at 25°C to 37°C for 12 to 24 hours, or the culture is carried out at 37°C for 1 to 3 hours. The aforementioned manufacturing method.

6. The method according to claim 5, which does not include a centrifugal separation step.

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