Method for preparing novel competent cell

Abstract

The present invention addresses the problem of providing a method for preparing a competent cell derived from Thermus thermophilus, which can be stored for a long period. The problem is solved by providing a method for preparing a competent cell, the method including a step for cryopreserving Thermus thermophilus after culturing.

Description

Novel method for preparing competent cells

[0001] The present invention relates to a method for preparing competent cells using Thermus thermophilus. The present invention also relates to a method for amplifying a plasmid using the competent cells.

[0002] Thermus thermophilus, a model thermophilic microorganism, is known for its high ability to take up foreign DNA, and this property has led to the establishment of a genetic manipulation system for it. For example, various genetic manipulation methods are applicable, such as the development of a shuttle vector for E. coli based on the commonly used plasmid pTT8 present in the HB8 strain of Thermus thermophilus, and gene insertion and deletion in the chromosome by utilizing the high recombination efficiency with homologous sequences.

[0003] In transformations performed in such genetic engineering experiments, fresh cells of Thermus thermophilus have always been prepared for experiments (Non-Patent Document 1). However, such conventional competent cell techniques using Thermus thermophilus require significantly more time and labor than the frozen competent cell method currently mainstream for Escherichia coli and the like, which allows for long-term storage. Specifically, conventional competent cell techniques using Thermus thermophilus require the preparation of competent cells every time a transformation experiment is performed, which requires a great deal of labor and not only makes the cells unavailable for immediate use when desired, but also results in extremely inefficient results.

[0004] Furthermore, Patent Document 1 discloses a highly active P450scc enzyme protein and describes Thermus bacteria as an example of a host into which a vector containing DNA encoding the protein is introduced, but does not disclose any specific experimental examples of transformation using Thermus bacteria as competent cells.

[0005] Furthermore, Patent Document 2 discloses a method for causing a thermophilic bacterium with an optimum growth temperature of 50°C or higher to produce an enzyme derived from a psychrophilic bacterium or a mesophilic bacterium with an optimum growth temperature of 50°C or lower that is 10°C or higher lower than the optimum growth temperature of the thermophilic bacterium. Patent Document 2 also discloses in its Examples an example of transformation using a thermophilic bacterium of the genus Geobacillus. However, it does not disclose any examples of transformation using bacteria of the genus Thermus.

[0006] International Publication No. WO 2010 / 079594 International Publication No. WO 2017 / 169751

[0007] Y Koyama et al., Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp., J Bacteriol., 1986, Apr;166(1):338-40. doi: 10.1128 / jb.166.1.338-340.1986.

[0008] In view of the above, an object of the present invention is to provide a method for preparing competent cells derived from Thermus thermophilus that can be stored for a long period of time.

[0009] As a result of extensive research aimed at solving the above problems, the present inventors discovered the following new findings, and based on these findings, they have completed the present invention. The present inventors have succeeded in producing competent cells that can be stored for long periods of time using a simple procedure while maintaining the transformability of the Thermus thermophilus HB27 strain. The present inventors have succeeded in producing competent cells that are stable for storage in a -20°C freezer, without using liquid nitrogen or an ultra-low temperature storage cabinet at -80°C, which are commonly used for storing competent cells. While conventional competent cells have generally been produced through the steps of pre-culture, main culture, cell collection, resuspension, aliquoting, and cryopreservation, the present invention has succeeded in simplifying these steps. Specifically, the present inventors have succeeded in producing competent cells simply by cryopreserving Thermus thermophilus after culture. The resulting competent cells had transformation efficiency equivalent to that of conventional methods in which the cells are prepared just before use, and were stable for long periods of time.

[0010] That is, one aspect of the present invention relates to the following: [1] A method for preparing competent cells, comprising a step of cryopreserving Thermus thermophilus after culture. [2] A method for preparing competent cells, comprising a step of culturing Thermus thermophilus, and a step of cryopreserving the culture solution containing Thermus thermophilus obtained in the step. [3] A method for preparing competent cells, comprising a step of culturing Thermus thermophilus, a step of dispensing the culture solution containing Thermus thermophilus obtained in the step, and a step of cryopreserving the culture solution containing Thermus thermophilus dispensed in the step. [4] The method of any of [1] to [3], further comprising a step of adding a cryopreservation agent before the cryopreservation step. [5] The method of any of [1] to [3], wherein the cryopreservation is performed at a temperature higher than -80°C. [6] The method of any of [1] to [3], wherein the Thermus thermophilus is the HB27 strain or the HB8 strain. [7] The method according to [4], wherein the cryopreservative is at least one selected from the group consisting of glycerol, dimethyl sulfoxide (DMSO), polyvinylpyrrolidine, and polyethylene glycol. [8] A method for amplifying a plasmid using competent cells prepared by the method according to any one of [1] to [3]. [9] The method according to [8], comprising the steps of thawing competent cells prepared by the method according to any one of [1] to [3] and transforming the thawed competent cells.

[0011] According to the present invention, competent cells derived from Thermus thermophilus can be stored for a long period of time. Therefore, there is no need to prepare them every time a transformation experiment is performed, which saves labor and allows experiments to be performed efficiently. Furthermore, the competent cells of the present invention have the advantage that they can be stored at relatively high temperatures and can be used immediately when desired.

[0012] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0013] (1. Definitions) As used herein, "Thermus thermophilus" refers to a type of Gram-negative aerobic bacterium classified in the genus Thermus. Its optimum growth temperature is 75°C, and it is classified as a thermophile. Furthermore, as used herein, "thermophiles" refer to microorganisms whose optimum growth temperature is 50°C or higher.

[0014] As used herein, the term "competent cells" refers to cells or microorganisms for transformation. Competent cells have increased membrane permeability and can incorporate foreign DNA and other substances into their interiors.

[0015] As used herein, "cryopreservation" refers to freezing a culture medium and storing it at a low temperature. In particular, cryopreservation refers to freezing a culture medium containing a specific microorganism (e.g., Thermus thermophilus) and storing it at a low temperature (e.g., -20°C).

[0016] As used herein, the term "transformation" refers to the introduction of foreign DNA or the like into a cell or the like to change its genetic properties.

[0017] (2. Method for Preparing Competent Cells) In one embodiment of the present invention, there is provided a method for preparing competent cells, which comprises a step of cryopreserving Thermus thermophilus after culture.

[0018] Furthermore, one embodiment of the present invention provides a method for preparing competent cells, which comprises the steps of culturing Thermus thermophilus and cryopreserving the culture solution containing Thermus thermophilus obtained in the above step.

[0019] Furthermore, in one embodiment of the present invention, there is provided a method for preparing competent cells, comprising the steps of culturing Thermus thermophilus, dispensing the culture solution containing Thermus thermophilus obtained in the above step, and freezing and storing the culture solution containing Thermus thermophilus dispensed in the above step.

[0020] The present inventors focused on the competent cell method and conducted studies aimed at dramatically improving the efficiency of genetic engineering experiments using Thermus thermophilus. Specifically, they attempted to produce competent cells that could be stored for long periods of time using a simple procedure while maintaining the transformability of the commonly used Thermus thermophilus strain HB27. Furthermore, regarding storage, they investigated whether it was possible to produce competent cells that could be stored in a more conventional -20°C freezer, rather than using liquid nitrogen or an ultra-low temperature storage cabinet at -80°C, which are commonly used for storing competent cells. Using competent cells produced based on this concept, they confirmed their transformability and storage stability using various separately developed vectors. They successfully developed competent cells that could be used for at least three months without a significant decrease in transformation efficiency. Furthermore, the transformation efficiency was found to be equivalent to that of conventional methods where competent cells are prepared just before use. It is surprising that such highly functional competent cells could be obtained using such a simple method.

[0021] INDUSTRIAL APPLICABILITY The present invention significantly improves the efficiency of genetic recombination experiments using Thermus thermophilus, and is therefore extremely useful in research fields that use Thermus thermophilus.

[0022] <Culturing (Main Culturing) Step> In one embodiment of the present invention, the method for preparing competent cells preferably includes a step of culturing Thermus thermophilus. In the culturing step, Thermus thermophilus is cultured to mass-produce Thermus thermophilus.

[0023] The Thermus thermophilus to be cultured is not particularly limited, but from the viewpoint of general use, the HB27 strain or the HB8 strain is preferred.

[0024] The medium used for the culture is not particularly limited as long as it is one that is commonly used for culturing microorganisms, such as LB medium (e.g., Lennox (Nacalai)), M9 medium, TB medium, SOB medium, SOC medium, 2X YT medium, and NZCYM liquid medium.

[0025] In one embodiment of the present invention, the medium preferably contains components such as MgSO4, CaCl2, etc. By including these components, the transformation ability of the competent cells of the present invention is increased.

[0026] The culture temperature is not particularly limited as long as it allows Thermus thermophilus to grow, but is, for example, 50 to 90° C., preferably 60 to 85° C., more preferably 65 to 80° C., and even more preferably 70 to 75° C. Since the optimal growth temperature for Thermus thermophilus is 75° C., temperatures around this temperature are more suitable as culture temperatures.

[0027] The culture time is not particularly limited as long as the desired amount of Thermus thermophilus can be obtained, but is, for example, 2 to 8 hours, preferably 3 to 6 hours, and more preferably 4 to 5 hours.

[0028] The vessel used for the culture is not particularly limited, and any vessel commonly used in the art can be used. For example, the culture can be performed using a flask (e.g., an Erlenmeyer flask), a culture tube, etc.

[0029] In one embodiment of the present invention, from the viewpoint of transformation efficiency, it is preferable to proceed to the next step using a culture medium in which Thermus thermophilus is in the growth phase from the logarithmic growth phase to the stationary phase.

[0030] <Freezing and preserving step> The method for preparing competent cells of the present invention includes a step of freezing and preserving the cultured Thermus thermophilus. Since the present invention allows for freezing and preserving the cells at a relatively high temperature, no large-scale equipment is required and the thawing time can be significantly reduced when the cells are used. Therefore, the present invention allows for efficient transformation.

[0031] The cryopreservation temperature is not particularly limited as long as it can maintain the frozen state. When the concentration of the cryopreservative (e.g., glycerol) is a commonly used concentration, the cryopreservation temperature can be, for example, -20°C. The cryopreservation temperature may be higher than the -80°C commonly used in conventional techniques. From the viewpoint of shortening the thawing time and providing for immediate use, the cryopreservation temperature is, for example, -40 to -20°C, and preferably -30 to -20°C.

[0032] The freezing method may be natural freezing or flash freezing, but natural freezing is preferred from the viewpoint of convenience, since it does not require the preparation of liquid nitrogen. Natural freezing is performed, for example, by placing the container containing the culture medium in a storage freezer. Flash freezing is performed, for example, by immersing the container containing the culture medium in liquid nitrogen.

[0033] <Step of adding a cryopreservation agent> In one embodiment of the present invention, the method for preparing competent cells preferably further comprises a step of adding a cryopreservation agent before the cryopreservation step. The cryopreservation agent is used to prevent the competent cells to be stored from being damaged by freezing at low temperatures or warming to room temperature.

[0034] The cryopreservative is not particularly limited, but examples thereof include glycerol, dimethyl sulfoxide (DMSO), polyvinylpyrrolidine, polyethylene glycol, etc. Glycerol is preferably used because it is the most common and inexpensive.

[0035] The amount (concentration) of the cryopreservative to be added is not particularly limited, but for example, the final concentration is 10 to 20% v / v, preferably 12 to 18% v / v.

[0036] The addition of the cryopreservation agent may be carried out at any stage before the cryopreservation step, for example, after the culture step. Furthermore, if a dispensing step described below is carried out, the addition may be carried out before or after the dispensing step. Furthermore, if a resuspension step described below is carried out, the addition may be carried out before or after the resuspension step, or simultaneously with the resuspension step.

[0037] The method for adding the cryopreservative is not particularly limited as long as it is a method in which the cryopreservative and the culture medium are mixed so that the cryopreservative is present in the culture medium, and it is possible to add the cryopreservative to not only the culture medium but also a solution containing the cryopreservative or the cryopreservative alone. From the viewpoint of simplicity, it is preferable to add the cryopreservative to the culture medium.

[0038] <Dispensing Step> In one embodiment of the present invention, the method for preparing competent cells preferably includes a dispensing step in which a medium containing Thermus thermophilus grown by culture is dispensed into amounts suitable for storage and use.

[0039] The amount of the dispensed solution is not particularly limited, but is, for example, 50 μL to 500 μL, preferably 100 μL to 450 μL, and more preferably 150 μL to 400 μL.

[0040] The container for dispensing is not particularly limited, and for example, a microtube (for example, a 1.5 mL tube) can be used.

[0041] <Pre-culture step> In one embodiment of the present invention, the method for preparing competent cells may further include a pre-culture step prior to the culture (main culture) step. In the pre-culture step, Thermus thermophilus is grown to a certain amount in a relatively small amount of medium.

[0042] The pre-culture time is not particularly limited as long as it is a time that allows the desired amount of Thermus thermophilus to be obtained, but is, for example, 6 to 24 hours, preferably 8 to 20 hours, and more preferably 12 to 16 hours.

[0043] The vessel used for pre-culture is not particularly limited, and any vessel commonly used in the art can be used. For example, pre-culture is performed using a culture tube or the like.

[0044] The "ingredients of the medium" and "culture temperature" in the pre-culture step are the same as those described in the <Culturing (main culture) step>.

[0045] <Collecting Step> In one embodiment of the present invention, a step of recovering Thermus thermophilus may be included after the culturing step. The collecting step allows the Thermus thermophilus to be concentrated and other components to be removed.

[0046] The method for collecting the bacteria is not particularly limited, but examples thereof include centrifugation, filtration, etc. Preferably, the bacteria are collected by centrifugation.

[0047] <Resuspension Step> In one embodiment of the present invention, a step of resuspending the Thermus thermophilus collected in the cell collection step may be included. In the resuspension step, the collected Thermus thermophilus is suspended in a fresh medium.

[0048] The dilution ratio at the time of resuspension is not particularly limited, but is, for example, 0.5 to 15 times, preferably 0.7 to 12 times.

[0049] The resuspension method is not particularly limited, and can be carried out, for example, by adding the medium described in the section <Culturing (main culturing) step> and mixing by pipetting, tapping, or the like.

[0050] In the method for preparing competent cells of the present invention, competent cells can be prepared without including a cell collection step and a resuspension step after the culturing step. However, in one embodiment of the present invention, a cell collection step and a resuspension step may be included after the culturing step.

[0051] (3. Method for amplifying a plasmid) In one embodiment of the present invention, there is provided a method for amplifying a plasmid using the competent cells prepared as described above. As described above, the competent cells of the present invention can be stored for a long period of time, so that preparation of the competent cells does not require much time and plasmid amplification can be carried out efficiently.

[0052] In addition, in one embodiment of the present invention, there is provided a method for amplifying a plasmid, which comprises the steps of thawing the competent cells prepared as described above and transforming the thawed competent cells.

[0053] The plasmid to be amplified is not particularly limited as long as it can be introduced into the Thermus thermophilus-derived competent cell of the present invention and can be amplified therein. Such plasmids may include not only previously known plasmids but also plasmids that will be newly discovered in the future. Examples of previously known plasmids include pTT8-type plasmids and pTMY-type plasmids. Examples of pTT8-type plasmids include pTT8, pHB5002d, pHB5008c, pHB5018e, and pAA2-2d. Examples of pTMY-type plasmids include pTMY, pTthSNM1-1c, pTthSNM1-7d, pTthSNM4-1c, pTthSNM6-6d, pTthSNM7-6d, pAA1-1c, and pAA3-7d.

[0054] The method for thawing competent cells is not particularly limited as long as it does not weaken or destroy the function of the competent cells. Competent cells can be thawed, for example, by removing them from a freezer in which they have been stored and thawing them at room temperature or by leaving them to stand on ice for a certain period of time.

[0055] The transformation method is not particularly limited as long as it is a method commonly used in the technical field. Examples of transformation methods include those described in Non-Patent Document 1 above. In one embodiment of the present invention, transformation is performed by the method described in the Examples.

[0056] The temperature for transformation is not particularly limited, but is, for example, 50 to 90°C, preferably 60 to 80°C.

[0057] The transformation time is not particularly limited, but is, for example, 1 to 4 hours, preferably 1.5 to 3.5 hours.

[0058] The time from thawing to completion of transformation is, for example, within 10 hours, preferably within 8 hours, more preferably within 6 hours, even more preferably within 4 hours, and particularly preferably within 3 hours. As mentioned above, in conventional methods, it took a long time from the time when it was intended to perform a transformation experiment to the completion of transformation (for example, a total of 18 to 22 hours, consisting of 12 to 16 hours of pre-culture, 4 hours of main culture, and 2 hours of transformation). However, in the present invention, the time from the time when it was intended to perform a transformation experiment (corresponding to the start of thawing in the present invention) to the completion of transformation can be significantly shortened.

[0059] (4. Other) In one embodiment of the present invention, the following are provided. [1-1] A method for preparing competent cells, comprising the step of cryopreserving Thermus thermophilus after culture. [1-2] A method for preparing competent cells, comprising the steps of culturing Thermus thermophilus, and cryopreserving the culture solution containing Thermus thermophilus obtained in the step. [1-3] A method for preparing competent cells, comprising the steps of culturing Thermus thermophilus, dispensing the culture solution containing Thermus thermophilus obtained in the step, and cryopreserving the culture solution containing Thermus thermophilus dispensed in the step. [1-2'] A method for preparing competent cells, comprising the steps of culturing Thermus thermophilus, adding a cryopreservation agent to the culture solution containing Thermus thermophilus obtained in the step, and cryopreserving the culture solution containing Thermus thermophilus to which the cryopreservation agent has been added in the step. [1-3'] A method for preparing competent cells, comprising the steps of: culturing Thermus thermophilus; adding a cryopreservation agent to the culture solution containing Thermus thermophilus obtained in the step; dispensing the culture solution containing Thermus thermophilus to which the cryopreservation agent has been added in the step; and freezing and storing the culture solution containing Thermus thermophilus dispensed in the step. [1-3''] A method for preparing competent cells, comprising the steps of culturing Thermus thermophilus; dispensing the culture solution containing Thermus thermophilus obtained in the step; adding a cryopreservation agent to the culture solution containing Thermus thermophilus dispensed in the step; and freezing and storing the culture solution containing Thermus thermophilus to which the cryopreservation agent has been added in the step. [1-4] The method according to any one of [1-1] to [1-3], [1-2'], [1-3'] and [1-3''], wherein the cryopreservation is carried out at a temperature higher than -80°C. [1-5] The method according to any one of [1-1] to [1-3], [1-2'], [1-3'] and [1-3''], wherein the Thermus thermophilus is the HB27 strain or the HB8 strain.[1-6] The method according to [1-4], wherein the cryopreservative is at least one selected from the group consisting of glycerol, dimethyl sulfoxide (DMSO), polyvinylpyrrolidine, and polyethylene glycol. [1-7] A method for amplifying a plasmid, using competent cells prepared by a method according to any one of [1-1] to [1-3], [1-2'], [1-3'], and [1-3'']. [1-8] The method according to [1-7], comprising the steps of thawing competent cells prepared by a method according to any one of [1-1] to [1-3], [1-2'], [1-3'], and [1-3''], and transforming the thawed competent cells.

[0060] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0061] (1. Preparation of Competent Cells) Competent cells were prepared using the following strains, media, and methods.

[0062] <Strain> Thermus thermophilus HB27 strain (ATCC BAA-163)

[0063] <Culture medium>

[0064]

[0065] <Additives> MgSO4 (Wako Pure Chemical Industries, Ltd.) CaCl2 (Wako Pure Chemical Industries, Ltd.) Glycerol (Wako Pure Chemical Industries, Ltd.)

[0066] <Method> Competent cells were prepared using the following procedure. 1. Thermus thermophilus HB27 strain was pre-cultured at 70°C for 16 hours. 2. After pre-culture, the Thermus thermophilus HB27 strain was main-cultured at 70°C for 8 hours. 3. Thermus thermophilus HB27 strain was harvested by centrifugation. 4. The harvested Thermus thermophilus HB27 strain was resuspended in a medium containing glycerol (final concentration 16% v / v). 5. The resuspension was dispensed into 1.5 mL tubes in 200 μL aliquots. 6. Store in a freezer (-20°C).

[0067] (2. Transformation) The competent cells prepared above were removed from the freezer, and a DNA solution containing the plasmid was added. The cells were then incubated at 70°C for 2-3 hours. The plasmid used was pIOK9Hg, prepared by the following method.

[0068] (Construction of pIOK9Hg) First, the putative replication gene and the sequence between the preceding and following ORFs of each T. thermophilus plasmid, pTthSNM3-3d (GenBank ID: AP025612), as well as two downstream protein genes of unknown function, were amplified by PCR. Next, the PCR-amplified DNA fragment was inserted in the + orientation into the E. coli vector pHSG298Hg (SEQ ID NO: 1) using Gibson Assembly to construct pIOK9Hg. pHSG298Hg is a pUC-type plasmid that uses a ColE1-type replication mechanism and contains a thermostable hygromycin resistance gene as a selection marker.

[0069] The base sequence of pHSG298Hg (SEQ ID NO: 1) is shown below.

[0070]

[0071] <Vector Preparation> Using vector pHSG298Hg, in which the kanamycin resistance gene of the commercially available vector pHSG298 (manufactured by Takara Bio) was replaced with a heat-stable hygromycin resistance gene, an on was inserted downstream of the Hg resistance gene. A linear full-length plasmid of pHSG298Hg was prepared by inverse PCR using primers of SEQ ID NOs: 2 and 3. KOD one (manufactured by Takara Bio) was used for vector PCR.

[0072] After the reaction, the reaction solution was cooled to room temperature, and 0.5 μl (equivalent to 10 U) of Dpn I (NEB) was added, followed by reaction at 37° C. for 2 hours.

[0073] <PCR materials> DNA template 0.2 μl Fw primer 0.5 μl Rv primer 0.5 μl KOD One 8 μl dH2O 10.3 μl 20 μl

[0074] <PCR conditions> 98℃ 2 minutes, 98℃ 10 seconds, 57℃ 5 seconds, 68℃ 20 seconds *30 cycles, 68℃ 10 seconds

[0075] <Primer>

[0076]

[0077] (Preparation of Ori Fragment) Ori fragment was prepared under the following conditions.

[0078] <PCR materials> DNA template 0.2 μl Fw primer 0.5 μl Rv primer 0.5 μl KOD FENeo 0.4 μl S*Buffer for FXNeo 10 μl 2 mM dNTPS 4 μl dH2O 4.4 μl 20 μl

[0079] <PCR conditions> 98℃ 2 minutes, 98℃ 10 seconds, 57℃ 30 seconds, 68℃ 90 seconds *30 cycles, 68℃ 10 seconds

[0080] <Primer>

[0081] <Gibson Assembly>

[0082]

[0083] A reagent was prepared with the above composition and incubated at 50°C for 1 hour.

[0084] The vector and Ori fragment were separated and purified by agarose gel electrophoresis. A portion of the 30 μl eluate was subjected to Gibson assembly as described above. After incubation at 50°C for 1 hour, a portion of the reaction mixture (0.5 μl) was added to 100 μl of JM109 competent cells for transformation. Plasmids were prepared from colonies that appeared on LB / Hg medium, and sequences were confirmed by the Sanger method.

[0085] The transformation efficiency (CFU / μg) was calculated using the following method: 1. 10 ng of DNA (plasmid) was added to competent cells and transformed. 2. The number of colonies was counted and the number of colonies per 1 μg of DNA (CFU / μg) was calculated.

[0086] (3. Examination of conditions) Next, the conditions for producing competent cells and transformation were examined. The following were used as indicators for examining the conditions. - Growth phase Log phase (OD600 = 0.3-0.5) and stationary phase (OD600 = 1.4-1.6) - Dilution ratio at resuspension Log phase: 1x, 5x, 10x Stationary phase: 1 / 10, 1x, 10x - Mg 2+ , Ca 2+ If added, the concentration is Mg 2+ 0.4 mM Ca 2+ Add to the medium to make it 0.35 mM. - Whether or not to change the medium (whether or not to resuspend) If resuspending, replace with fresh medium.

[0087]

[0088] From the results of the comparative experiments, the following protocol is presumed to be an optimized protocol for producing competent cells, although it goes without saying that the present invention is not limited to the following protocol.

[0089] <Preparation of competent cells> 1. Pre-culture Thermus thermophilus HB27. 2. After pre-culture, Thermus thermophilus HB27 is cultured in a medium containing Mg 2+ , Ca 2+ 3. Add 10 mL of 80% glycerol to the culture solution during the main culture (this will evaporate during the culture, reducing the medium volume to approximately 40 mL). 4. Dispense 200 μL into 1.5 mL tubes (do not flash freeze). 5. Store in a freezer (-20°C).

[0090] <Transformation> 1. Remove the competent cells from the freezer. 2. Add the DNA solution to the competent cells. 3. Incubate at 70°C for 2-3 hours.

[0091] The present invention enables the long-term storage of competent cells derived from Thermus thermophilus, and is therefore extremely useful in transformation experiments. This application is based on Japanese Patent Application No. 2024-048785 (filing date: March 25, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for preparing competent cells, which comprises a step of cryopreserving Thermus thermophilus after culturing.

2. A method for preparing competent cells, comprising the steps of: culturing Thermus thermophilus; and freezing and storing the culture solution containing Thermus thermophilus obtained in the above step.

3. A method for preparing competent cells, comprising: a step of culturing Thermus thermophilus; a step of dispensing the culture solution containing Thermus thermophilus obtained in the step; and a step of freezing and storing the culture solution containing Thermus thermophilus dispensed in the step.

4. The method according to any one of claims 1 to 3, further comprising the step of adding a cryopreservation agent prior to the cryopreservation step.

5. The method according to any one of claims 1 to 3, wherein the cryopreservation is carried out at a temperature higher than -80°C.

6. The method according to any one of claims 1 to 3, wherein the Thermus thermophilus is strain HB27 or strain HB8.

7. The method of claim 4, wherein the cryopreservation agent is at least one of the group consisting of glycerol, dimethyl sulfoxide (DMSO), polyvinylpyrrolidine, and polyethylene glycol.

8. A method for amplifying a plasmid, using competent cells prepared by the method according to any one of claims 1 to 3.

9. The method according to claim 8, comprising the steps of thawing the competent cells prepared by the method according to any one of claims 1 to 3, and transforming the thawed competent cells.

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