TWI440720B - Reagents and a process for preparing fast transforming competent cells - Google Patents

Description

Reagents and methods for preparing rapid transformation competent cells

The present invention relates to an agent and method for preparing competent cells, and more particularly to an agent for providing rapid transformation of competent cells.

Escherichia coli is the most widely used microorganism in modern molecular biotechnology. It is an indispensable host cell especially in the fields of molecular biology research and genetic engineering. Almost all biotechnology-related laboratories use various genetic mutations of Escherichia coli. The strain acts as a host cell for large-scale replication of DNA or protein molecules. The technique of feeding a foreign DNA molecule into an E. coli host cell is called "transformation"; and a host cell which is initially treated to easily absorb foreign DNA molecules is called a "competent cell".

Thus, the importance of manufacturing competent cells and transforming competent cells into modern genetic engineering can be seen. The invention can be traced back to Mandel, M. and Higa A. (J. Mol. Biol. 53: 159). -162) The published CaCl ₂ chemical transformation method, after nearly 30 years of improvement, the operation time of the transformation method still needs 1.5~3.0 hours. The main reason is that the chemical treatment process causes some damage to the cells, and needs to be added. The recovery step of the medium allows the cells damaged by the transformation process to have sufficient time and the nutrient to restore the physiological function to produce the drug resistance ability, and then the step of screening the bacteria can be carried out, otherwise the transformation efficiency will decrease several times. In recent years, there has been a rapid transformation technique called electroporation. Although it is possible to use a transient current to deliver DNA molecules into E. coli host cells, the cells subjected to electroporation must be added to the medium for a recovery step of about 1 hour. A higher transformation efficiency is obtained (Dower et. al., 1988 Nucleic Acids Res. 16: 6127-6145). In 1988, Golub EI (Nucleic Acids Res. 16: 1641) published a one-minute transformation method. Although the recovery step of adding the medium was omitted, the efficiency was only 10 ⁴ ~ 10 ⁵ transform colonies / μg plastid DNA. .

In recent years, Yisheng Biotech Development Co., Ltd. has developed a rapid transformation technology (US Patent No. 6,864,088), which can eliminate the lengthy steps of traditionally recovering E. coli cells after transformation, without reducing the original transformation. effectiveness. In this embodiment, the plastid DNA and the E. coli cells are mixed in a tube and subjected to heat shock treatment, and then the transformed cells are directly applied to the lower temperature screening medium at a lower temperature coating tool without adding The non-screening medium (SOC, LB...) performs the recovery step, thereby shortening the conventionally-converted 1.5-3.0 hour multi-step transformation process to several minutes, and the single tube can be completed without reducing the original transformation efficiency. However, although this method has been improved in various previous transformation methods, it must be supplemented with a temperature-control device for heat shock treatment, and the cells are placed in a tube for heating, and there is a possibility of uneven heating. Since then, the method of rapid transformation of Taiwan Patent Application No. 95116017 has been developed. This method omits the traditional heat shock treatment, which makes the process of transformation more simplified, and replaces the traditional heat shock mode with a warm screening medium. A competent cell and preparation reagent can be widely used in this rapid transformation technology, which will be more helpful for the application and implementation of the transformation technology.

The purpose of the present invention is to provide an agent and a method for preparing a rapid-transformation competent cell, the main feature of which is that the reagent contains an anti-freeze composition containing dimethyl sulfoxide (DMSO), so that the competent cells can be kept at a low temperature without being required. By heat-shock treatment, the transformation efficiency of more than 10 ⁶ colonies per microgram of transformant/μg plasmid can be achieved, which simplifies the steps of heat shock treatment and the recovery of cells after heat shock. The lengthy steps, and will not reduce the original transformation efficiency.

The transforming cells can be prepared from a variety of microorganisms, and most of them are prepared from different strains of Escherichia coli, such as HB101, DH5α, GM2929, XL1-Blue, TG1, BL21, and JM109. Escherichia coli is the most widely used microorganism in modern molecular biotechnology. It is an indispensable host cell especially in the fields of molecular biology research and genetic engineering. Almost all biotechnology-related laboratories use various genetic mutations of Escherichia coli. The strain acts as a host cell for large-scale replication of DNA or protein molecules. The technique of feeding a foreign DNA molecule into an E. coli host cell is called "transformation"; and a host cell which is initially treated to easily absorb foreign DNA molecules is called a "competent cell".

According to the rapid transformation method of Escherichia coli according to U.S. Patent No. 6,864,088, the plastid DNA is mixed with E. coli competent cells, the mixture is treated with heat shock, and the mixture is applied to a low temperature screening medium by a low temperature coating tool. Further, screening culture of the transformed strain is carried out.

In addition to the advantages of the above method, the reagent of the invention enables the competent cells to achieve the purpose of gene transformation without heat shock, simplifies the steps of heat shock treatment, and omits the step of cell recovery after cell transformation. The mixture of plastid DNA and competent cells can be directly applied to the screening medium to achieve the transformation of the competent cells, and the efficiency of the transformation can be further increased by the warm screening medium.

The invention provides a reagent for rapid transformation of competent cells and a preparation method thereof, which comprises the following steps: a. cultivating the strain; b. arranging the reagent, comprising an acid-base buffer [pH 4~9], a monovalent ionic compound, a divalent ionic compound, an antifreeze composition; c. after centrifuging the bacterial liquid, adding a reagent and Mixing; d. mixing the plastid DNA with competent cells suspended in the reagent to form a mixture; e. applying the mixture to the screening medium; and f. incubating the mixture on the screening medium.

This reagent preparation of competent cells capable of rapid transfer shaped, and has a greater than ¹⁰⁶ colonies / microgram Transformation efficiency plasmid DNA (transformants / μg plasmid) of.

The above ionic solution in which the competent cells are suspended comprises calcium (Ca ²⁺ ), magnesium (Mg ²⁺ ), manganese (Mn ²⁺ ), zinc (Zn ²⁺ ), copper (Cu ²⁺ ), chromium (Cd ² ). ⁺ ), a divalent cation of a group consisting of iron (Fe ²⁺ ), strontium (Sr ²⁺ ), and cobalt (Co ²⁺ ), and the ionic solution does not contain calcium (Ca ²⁺ ) alone; In the example, the ion is magnesium (Mg ²⁺ ), wherein the magnesium chloride concentration is 5 mM to 500 mM. The monovalent ion solution may further comprise a compound selected from the group consisting of ammonia (NH ₄ ⁺ ), lithium (Li ⁺ ), sodium (Na ⁺ ), potassium (K ⁺ ), ruthenium (Rb ⁺ ), cesium (Cs ⁺ ), and silver (Ag ⁺ And a covalent cation of a group consisting of cobalt (Co ⁺ ). In a preferred embodiment, the monovalent cation is ruthenium (Rb ⁺ ), wherein the ruthenium chloride concentration is from 5 mM to 500 mM. The acid-base buffer may be Piperazine-1,4-bis (2-ethanesulfonic acid) (Pipes), Tris (hydroxymethyl) buffer [Tris (hydroxymethyl) )-aminomethane (Tris)], Phosphate buffer saline (PBS), 4-hydroxyethyl-piperazine ethanesulfonic acid (HEPES) Or 3-(N-Morpholino)-propanesulfonic acid (MOPES), preferably Pipes, at a concentration of 1 to 100 mM. An antifreeze composition containing dimethyl hydrazine (DMSO), which may be composed of dimethyl hydrazine (DMSO) alone, dimethyl hydrazine (DMSO), polyethene glycol, dimethyl phthalate碸 (DMSO) and glycerol or dimethyl hydrazine (DMSO), glycerol, polyethene glycol three compounds.

The present invention is generally obtained Transformation efficiency of greater than ^106, preferably more than 10 ⁷ Transformation efficiency, most preferably greater than 10 ⁸ Transformation efficiency.

The competent cells prepared by the invention can eliminate the heat shock and the recovery step of adding the culture solution when the shape is transformed, that is, it is not necessary to perform heat shock treatment before being applied to the warm screening medium, and does not need to be applied before the screening medium. Another non-screening medium was added for cultivation for several tens of minutes. The screening medium can be warmed up to 20 ° C ~ 50 ° C in advance, which can further improve the efficiency of the transformation.

E. coli is classified as a gram negative. At present, most of the competent cell preparation and transformation techniques of Gram-negative bacteria or gram positive bacteria are related to the improved technique of Escherichia coli (Method In Enzymology 204: 63-113), so the transformation method of the present invention It can be applied to the transformation technology of most bacteria (Gram-negative or positive bacteria).

The following examples are non-limiting and are merely representative of the various possibilities and features found in the present invention.

Example 1: Preparation of Competent Cells and Method of Preparing Competent Cells

In this example, the DH5α strain was used as a source of competent cells, and the DH5α strain was inoculated from a single colony in 20 ml of LB medium, cultured at 37 ° C for 17 hours, and then diluted 100-fold in LB medium to culture to OD _600. The absorbance is close to 0.2~0.8, preferably 0.4~0.5. The cells are placed in an ice bath at 4 °C, and then centrifuged at 4000 rpm for several minutes to remove the LB supernatant, and the reagent of the present invention is added and uniformly mixed with the cells. Store in a centrifuge tube at -20~-130°C.

The reagent is configured to include an acid-base buffer [pH 5-8], a monovalent ionic compound, a divalent ionic compound, and an antifreeze composition, and the reagent composition is as follows: Add water to 10 mL of the above mixture.

In the present example, the experiment was carried out in the above manner, and the competent cells prepared by the conventional conventional CaCl ₂ method (Mandel, M., and Higa A. 1970, J. Mol. Biol. 53: 159-162) were used as a control group. . This competent cell line is suspended in the reagent of the present invention; whereas the conventional competent cell line is suspended in an aqueous solution containing 100 mM CaCl ₂ . This example has also tested the composition of antifreeze composed of dimethyl sulfoxide alone, polyethylene glycol, dimethyl hydrazine or ethylene glycol and dimethyl hydrazine. The conversion efficiency of the three compounds of ethylene glycol, polyethylene glycol and dimethyl hydrazine is better (data not shown). This example also tested various acid-base buffers other than Pipes but had similar effects on the transformation efficiency (data not shown).

Example 2: Competent cells made with the reagent of the present invention can omit the conventional heat shock treatment steps when transforming

The above two cells of the strain DH5α were transformed into the plastid pBR322. First, 100 μL of the competent cells packed into each tube were taken out from the -80 ° C freezer, and directly thawed in a water bath or tap water at room temperature. Immediately, the plastid pBR322 was added, and the mixture was vigorously shaken for 1 second to be uniformly mixed. The plastid concentration of the competent cells was 10 ^-5 μg/μL, and the concentration of the plastid pBR322 of the conventional competent cells prepared by the CaCl ₂ method was 10 ^-5 μg/μL. Some cells were immediately subjected to heat shock treatment in a 42 ° C water bath for 2 minutes, while the other part was not subjected to heat shock treatment, and then hit with a finger to help mix evenly. Some cells were added to 900 μL of non-screening medium (LB) for 40 minutes at 37 ° C before being applied to the screening medium supplemented with antibiotics, and another part of the cells were directly applied to the screening medium supplemented with antibiotics. The temperature of the screening medium used was 4 ° C or preheated to 37 ° C in advance, and the temperature of the coating tool used was the same as the temperature of the screening medium of the group. The resulting transformation efficiency is as follows:

From the results of the above table, the following three conclusions can be deduced: (1) The recovery step of adding LB can be omitted: the recovery step of adding LB is omitted, and the mixture is directly applied to the medium, and there is no significant difference in the transformation efficiency. The transformation efficiency of competent cells was maintained at 5×10 ⁶ -10 ⁸ colonies/microgram of DNA (transformants/μg plasmid).

(2) The step of heat shock treatment can also be omitted: the heat shock treatment step of the 42 ° C water bath is omitted, and the obtained transformation efficiency is not significantly different. The transformation efficiency of competent cells is still maintained at 10 ⁷ ~ 10 ⁸ colonies / microgram of DNA (transformants / μg plasmid), while the traditional competent cells are still only 10 ⁵ ~ 10 ⁶ colony / microgram of plastid DNA (transformants/μg plasmid).

(3) The competent cells prepared by using the reagent of the present invention have higher transformation efficiency than the conventional method, and the efficiency of screening the culture medium at 37 ° C is higher than that at 4 ° C: the competent cells prepared by using the reagent of the present invention, if the recovery step of adding LB is omitted at the same time And heat shock treatment, the screening medium at 37 ° C instead of 4 ° C screening medium can increase the transformation efficiency by 9 times; but if the traditional CaCl ₂ method is used to prepare the competent cells, the transformation efficiency will not be improved. .

Example 3: Wide applicability of competent cells prepared by the reagent of the present invention to screening culture media of different strains and different temperatures

Competent cells are prepared using different strains of Escherichia coli such as DH5α, JM109, BL21 (DE3) and XL1Blue, and are prepared by using the reagent of the present invention. The test was carried out in the same manner as in Example 2, and the warm screening medium at different temperatures was tested, and the results were as follows:

From the results of the above table, the following two conclusions can be deduced: (1) Competent cells and transformation methods made with the reagent of the present invention have wide applicability to Escherichia coli strains: whether for strains DH5α, JM109, BL21 (DE3) or XL1Blue, the mixture was applied to a screening medium of 25 to 42 ° C, and the resulting transformation efficiency was above 8 × 10 ⁶ colonies / microgram of DNA (transformants / μg plasmid).

(2) Competent cells prepared with the reagent of the present invention have wide applicability to different temperatures of the warm screening medium: the transformation efficiencies are similar when applied at 25 ° C, 37 ° C and 42 ° C.

Example 4: Wide applicability of competent cells made with the reagents of the invention to screening different antibiotics

Different types of antibiotics were added to the warm screening medium, and the 7.4 kb plastid (pBR325-derived plastid containing antibiotic screening markers such as Ap ^r , Tc ^r and Cm ^r ) was tested as follows:

From this result, it can be seen that the obtained transformation efficiency is equivalent, indicating that the competent cells prepared by the present invention have a wide range of antibiotic screening suitability.

Example 5: Competent cells made with the reagent of the present invention for different plastids The wide applicability

The competent cells were tested using plastids of different sizes and the results were as follows:

This result can be seen in plastids of different sizes Transformation of competent cells, resulting in Transformation efficiency was ¹⁰⁶ colonies / microgram plasmid (DNA transformants / μg plasmid) above, this represents a rapid method for different Transformation The size of the plastid and antibiotic concentration has wide applicability.

Claims (20)

An agent for preparing a competent cell, comprising: (1) an acid-base buffer having a pH of 4 to 9; (2) a metal ion composition composed of a monovalent ion compound and a divalent ion compound; and (3) an antifreeze agent A composition comprising dimethyl hydrazine, ethylene glycol and polyethylene glycol. A competent cell reagent set comprising the reagents and competent cells of claim 1 of the patent application. The competent cell reagent set of claim 2, wherein the competent cell line is prepared from Escherichia coli. The reagent of claim 1, wherein the acid-base buffer is piperazine-1,4-bis(2-ethanesulfonic acid (Pipes), trishydroxyl Tris (hydroxymethyl)-aminomethan (Tris), Phosphate buffer saline (PBS), 4-hydroxyethyl hexahydropyridine ethanesulfonic acid [4-(2-Hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES)] or 3-(N-Morpholino-propane-sulfonic acid (MOPES)], pH 5~8, concentration 1~ 100 mM. The reagent of claim 1, wherein the monovalent ionic compound is a compound of cerium, lithium, sodium, potassium or cesium. The reagent of claim 1, wherein the divalent ionic compound is a compound of magnesium, manganese or calcium. The reagent of claim 5, wherein the monovalent ionic compound is chlorine Phlegm. The reagent of claim 6, wherein the divalent ionic compound is magnesium chloride, a divalent metal salt manganese chloride (MnCl ₂ ) or calcium chloride (CaCl ₂ ). The reagent of claim 5, wherein the monovalent ionic compound has a concentration of from 5 to 500 mM. The reagent of claim 6, wherein the divalent ionic compound has a concentration of from 5 to 500 mM. For example, the reagent of the first aspect of the patent application, wherein the content of the ethylene glycol is 0.1 to 10% For example, the reagent of the first aspect of the patent application, wherein the content of the polyethylene glycol is 0.1 to 10%. For example, the reagent of the first aspect of the patent application, wherein the content of the dimethyl sulfoxide is 1 to 20%. A method for preparing a rapid-transformation competent cell, comprising the steps of: a cultivating the strain to an OD ₆₀₀ absorbance value of approximately 0.2 to 0.8; b, removing the supernatant after centrifugation, adding the reagent of the first application of the patent scope; and c sub-packaging Store at -20~-130°C. A method for rapid transformation of a competent cell, comprising the steps of: a thawing a competent cell reagent set of claim 2; b adding plastid DNA to a competent cell reagent set to form a mixture; c smearing the mixture of step b On the screening medium; and d the competent cells are cultured on the screening medium. The method according to Claim 15 patentable scope, which has a DNA Transformation efficiency The number of colonies greater than ¹⁰⁶ / g body mass. The method of claim 15, wherein there is no heat shock treatment before step b. The method of claim 15, wherein the step of recovering the culture solution is not preceded by the step b. The method of claim 15, wherein the screening medium of step c is subjected to a warm heat treatment. For example, the method of claim 19, wherein the temperature of the screening medium is 20 to 50 °C.