Link:  Pharm/Biotech Resources

Title:  Fast method of transforming competent cells

United States Patent:  6,864,088

Issued:  March 8, 2005

Inventors:  Chen; Tzu-Chih (Taipei, TW); Hua; Wei-Ni (Taipei, TW)

Assignee:  Yeastern Biotech Co., Ltd. (Taipei, TW)

Appl. No.:  157421

Filed:  May 29, 2002

Abstract

A fast method of transforming competent cells is described. The competent cells are thawed at room temperature or in a water bath. Plasmid DNAs and competent cells are mixed together, then the mixture is subject to heat shock treatment. After plating the mixture on a low-temperature selective medium by a low-temperature plating tool, the competent cells are cultured on the selective medium.

Description of the Invention

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Taiwan application serial no. 90,127,915, filed Nov. 9, 2001, the full disclosure of which is incorporated herein by reference.

BACKGROUND

1. Field of Invention

The present invention relates to a gene transformation method. More particularly, the present invention relates to a fast method of transforming competent cells.

2. Description of Related Art

The host cells after preliminary treatment to be more permeable to DNA molecules are called competent cells, and the technology of delivering DNA molecules in the surrounding medium into host cells is called transformation. Hence, producing competent cells and transforming competent cells is very important in view of recent developments of genetic engineering.

The technology described above can be retraced to Mandel, M. and Higa, A. (J. Mol. Biol. 53:159-162), who published a chemical transformation method using CaCl₂. After 30-year-improvement, the time needed for transformation is still 1.5-3.0 hours because the host cells are injured by the chemical treatment. The injured host cells require a recovering step. In the recovering step, the host cells are cultured in a nutrient medium to allow the injured host cells to recover their physiological function and drug resistance. Then the host cells are plated on a selective medium to screen the successfully transformed host cells. Otherwise, the transformation efficiency would decrease by several times. Recently, a fast transformation method called electroporation has been developed. Although the electroporations can deliver DNA molecules into the E. coli host cells by transient current, the host cells after the transient current treatment still need an hour of recovery to obtain a higher transformation efficiency (Dower et. al., 1988 Nucleic Acids Res. 16: 6127-6145). In 1988, Golub E. I. (Nucleic Acids Res. 16: 1641) also published a method of one-minute transformation. Although a recovering step is performed, the transformation efficiency is only 10⁴ -10⁵ colonies/.mu.g plasmid DNA.

SUMMARY OF THE INVENTION

It is therefore an objective of the present invention to provide a fast method of transforming competent cells to save operation time.

In a preferred embodiment of the present invention, the fast transformation method comprises the following steps. The competent cells in a container are thawed at room temperature or in a water bath. Plasmid DNAs and competent cells are mixed together in the container, and then the mixture is subjected to heat shock treatment for about 0-180 seconds. After plating the mixture on a selective medium of about 0^oC.-30^oC. by a plating tool of about -90^oC.-30^oC., the mixture is cultured on the selective medium to obtain competent cells.

In another preferred embodiment, a step of incubating the competent cells in an ice bath can be inserted between the mixing step and the heating step for about 0-90 minutes. Another step of incubating the competent cells in an ice bath can be inserted between the heating step and the plating step for about 0-60 minutes.

As described above, the recovering step of the conventional transformation method is omitted, and the transformed cells are directly plated on a low-temperature selective medium by a low-temperature plating tool. Therefore, in contrast with the conventional required recovery time of 1.5-3.0 hours, the method according to the present invention needs only several minutes, and possibly as little as several seconds.

In addition to the short operation time, another advantage of the fast transformation method is that one container can be used from the thawing step to the plating step. Therefore, an automatic transformation apparatus can be used to conduct the fast transformation method of competent cells as described above.

In the fast transformation method conducted by an automatic transformation apparatus, competent cells are first thawed in a container held in a first tank at room temperature. Then, plasmid DNAs are pipetted into the container to mix with the competent cells in the container to form a mixture by a micropipette. Next, the container is transferred by a robot arm to a second tank heated by a temperature control unit for about 0-180 seconds, whereby the mixture in the container is subjected to heat shock treatment. A plate filled with a selective medium at about 0-30^oC. and a plating tool of about -90^oC. to 30^oC. on the selective medium are provided by a plate-loading unit. The plating tool is moved on the selective medium to spread the mixture thereon by the plating unit. Finally, the mixture is cultured on the selective medium in an incubating unit to obtain transformed cells.

It is to be understood that both the foregoing general description and the following detailed description are by examples, and are intended to provide further explanation of the invention as claimed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The recovering step of a conventional transformation method is the most time-consuming and troublesome step. In the recovering step, new tubes with SOC or LB culture solutions are needed, and then the tubes with culture solution and transformed cells need to be incubated at 37^oC. with shaking for at least 1-2 hours. If the recovering step can be omitted or the time needed for the recovering step can be reduced, the transformation method can be largely simplified.

In a conventional transformation method, a recovering step of culturing the transformed cells, which have absorbed plasmid DNAs capable of resisting antibiotics, in a SOC or LB culture solution at 37^oC. with shaking for 1.0-2.0 hours is usually performed before a plating and a selective step. The purpose of the recovering step is to allow the transformed cells time to express the antibiotic resistance of the absorbed plasmid DNAs. Hence the transformed cell can survive in a selective medium with antibiotics in the selective step. If the time needed for the recovering step is reduced, the injury to the host cells during the steps of thawing, incubating in a ice bath, and heat shock also have to be reduced to make the transformed cells develop antibiotic resistance more rapidly.

In a preferred embodiment of the present invention, the fast transformation method comprises the following steps. The competent cells in a tube are thawed at room temperature or in a water bath. Plasmid DNAs and competent cells are mixed together in the tube, and then the mixture is subjected to heat shock treatment. After plating the mixture on a low-temperature selective medium by a low-temperature plating tool, the competent cells are cultured on the selective medium.

In this preferred embodiment of the present invention, the transformed cells are directly plated on the selective medium at a low temperature, and the conventional recovering step, which is usually performed before the plating and selective step, is omitted. Therefore, the drawbacks of the conventional transformation method, such as multiple steps (multiple tubes) and time-consumption (about 1.5-3.0 hours) are overcome by the advantages of the present invention, such as a short time (several seconds to several minutes) and use of a single tube.

Generally, competent cells are stored at -70^oC. Before mixing plasmid DNAs and competent cells, the competent cells first have to be thawed. A method of thawing the competent cells is incubating them for about 5-3 minutes in an ice bath for slow thawing. A method of fast thawing the competent cell is to incubate them in a water bath for about 5 seconds to 5 minutes.

The competent cells can be prepared form, for example, various Escherichia coli (E. coli) strains such as HB101, DH5.alpha., GM2929, XL1-Blue, TG1, BL21, and JM109 etc. E. coli is the most widely applied microorganism. Especially in the field of molecular biology and genetic engineering, E. coli variants are obligate host cells used in labs for mass-producing different kinds of DNAs or proteins.

Then, plasmid DNAs and the competent cells are mixed together. No special limiting conditions are needed for the competent cells. The competent cells can be purchased from any commercial available sources or made according to any known technology. The plasmid DNAs can be obtained from any natural sources or after recombination by genetic engineering. The competent cells and the plasmid DNAs can be mixed either by gently inverting the tube with the fingers or by violently shaking the tube with a shaker.

After uniformly mixing, the mixture of the plasmid DNAs and the competent cells are incubated in an about 36-48^oC. water bath for heat shock treatment. The time needed for the heat shock treatment is preferably for about 0-180 seconds and more preferably for about 10-90 seconds.

Next, the mixture is plated on a low-temperature selective medium by a low-temperature plating tool. The plating tool can be a conventional one such as glass beads or a glass loop. The temperature of the plating tool is preferably about -90^oC.-30^oC. and more preferably about -20^oC.-8^oC. The temperature of the selective medium is preferably about 0^oC.-30^oC. and more preferably about 0^oC.-4^oC.

Finally, the competent cells are cultured on the selective medium. An antibiotic is added to the selective medium. The amount of the antibiotic added can be varied as needed or according to the known arts. For example, the concentration of an antibiotic such as ampicillin can be about 25-100 .mu.g/mL.

According to another preferred embodiment of the present invention, a step of incubating in an ice bath can be inserted between the mixing step and the heat shock step. That is, after mixing the plasmid DNAs and the competent cells, the mixture is incubated in an ice bath then subjected to the heat shock treatment. The time for the mixture incubated in the ice bath is preferably about 0-90 minutes and more preferably about 0-30 minutes. Another step of incubating in an ice bath can be inserted between the heat shock treatment and the plating step. The time for incubating the mixture in the ice bath is preferably about 0-60 minutes and more preferably about 0-30 minutes.

Without further elaboration, it is believed that the above description has adequately enabled the present invention. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications cited herein are hereby incorporated by references on their entire.

Preparation of Competent Cells

The competent cells of the present invention were produced by the following four chemical methods. The four chemical methods are CaCl₂ (Mandel, M., and Higa A. 1970, J. Mol. Biol. 53: 159-162), TB (Inoue H, 1990, Gene 96: 23-28), TSS (Chung C. T., Proc. Natl. Acad. Sci. USA., 86:2172-2175), and TFB (Hanahan D., 1983, J. Mol. Bio. 166: 557-580). The strain of E. coli used was DH5.alpha., which is commonly used in the research of molecular biology. The genetic information of DH5.alpha. strain is F^- (.PHI.80d lacZ.DELTA.M15) .DELTA. (lacZYA-argF) U169 supE44 hsdR17(r_.kappa. -m_.kappa. +) recA1 gyrA96 endA1 thi-1 relA1 deoR .lambda.^-.

In the transformation procedures, a clone of the DH5.alpha. strain was inoculated in a LB culture solution and cultured at about 37^oC. for about 16 hours. The cells of the DH5.alpha. strain were then diluted by the LB culture solution to a concentration of about 1/100 of the original concentration. The diluted DH5.alpha. strain cells solution was divided into four bottles and was continuously cultured in the LB culture solution until the absorbance was about 0.2-0.6. Then, the competent cells were respectively produced by the four chemical methods described above (Methods in Enzymology, 1991, 204:63-113) and stored at about -70^oC.

Claim 1 of 16 Claims

What is claimed is:

1. A fast method of transforming competent cells, comprising:

thawing competent cells, prepared from Escherichia Coli, in a container at room temperature or in a water bath;

mixing a plasmid DNA and the competent cells in the container to form a mixture;

plating the mixture on a selective medium of about 0^oC. to less than room temperature by a plating tool of about -90 ^oC. to less than room temperature without heating the mixture and recovering the competent cells in a culture medium before the plating step; and

culturing the mixture on the selective medium to obtain transformed cells.

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