A convenient method for preparing bacterial competent cells and bacterial transformation

Abstract

The present invention discloses a method for conveniently preparing bacterial competent cells and bacterial transformation, the method comprising the following steps: evenly spreading the bacteria onto a culture plate, culturing overnight in a 37°C biological incubator to generate a bacterial lawn; streaking the bacterial lawn and inoculating it onto another culture plate, culturing in a 37°C biological incubator to the logarithmic phase; collecting the logarithmic phase bacteria into a mixed solution containing calcium chloride and glycerol, and incubating on ice to obtain bacterial competent cells. The present invention utilizes solid culture medium for cultivation, a one-step method for rapidly preparing bacterial competent cells without centrifugation, a sufficiently high and stable transformation efficiency, simple operation, and low cost. It is particularly suitable for teaching and scientific research laboratories with relatively limited conditions, as well as for rapid preparation of competent cells and bacterial transformation experiments in primary science, technology, engineering, and mathematics (STEM) education.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology and molecular biology, and in particular to a method for conveniently preparing bacterial competent cells and bacterial transformation. Background Art

[0002] Competent cells are induced through physical and chemical methods to absorb DNA molecules from their surroundings, placing them in a physiological state optimal for uptake and accommodation of foreign DNA. The main principle is to increase the permeability of the cells through treatment. Intuitively, this creates pores on the cell membrane, facilitating the entry of foreign genes or vectors. Due to the fluidity of the cell membrane, these pores are repaired by the cells themselves.

[0003] Escherichia coli is one of the most commonly used bacteria in laboratories. Currently, the most common method for preparing competent E. coli cells is the calcium chloride (CaCl2) method, resulting in cells known as chemically competent cells. While practical, conventional methods for preparing competent E. coli cells are cumbersome, often requiring a long time and advanced equipment to complete in research laboratories. First, the preparation process requires the use of a constant-temperature shaker to culture the bacteria; second, the use of instruments such as a spectrophotometer or microplate reader to monitor bacterial growth during the culture process; and the repeated use of cold CaCl2 solution for treatment and high-speed refrigerated centrifuges for collection of the bacteria place high demands on laboratory facilities. Middle school teaching laboratories, some higher education teaching laboratories, and science, technology, engineering, and mathematics (STEM) institutions often lack these advanced instruments and experimental facilities, hindering the teaching and promotion of this important biotechnology among young students. Although biological companies can provide commercial competent cells with high and stable transformation efficiency, they are expensive and cannot be afforded by teaching laboratories. In addition, competent cells are relatively fragile and have strict environmental requirements. They need to be transported frozen using dry ice, which further increases the price of commercial competent cells and hinders their application. Summary of the Invention

[0004] In order to solve the problem that competent cells are difficult to prepare and transform, the present invention provides a method for conveniently preparing bacterial competent cells, comprising the following steps:

[0005] The bacteria were evenly spread on the culture plate and cultured overnight in a 37°C biological incubator to form a bacterial lawn;

[0006] The bacterial lawn was scratched and inoculated onto another culture plate, and cultured in a biological incubator at 37° C. until the logarithmic phase;

[0007] The logarithmic phase bacteria were collected into a mixture containing calcium chloride and glycerol, and incubated on ice to obtain bacterial competent cells.

[0008] Preferably, the bacteria are evenly spread onto a solid culture plate and cultured in a 37°C biological incubator for 15 hours to form a bacterial lawn; the bacterial lawn is scratched and inoculated onto a new bacterial culture plate, and cultured in a 37°C biological incubator for 3 to 4 hours to the logarithmic phase.

[0009] Preferably, the length of the mushroom lawn is 1 to 2 cm.

[0010] Preferably, the calcium chloride and glycerol mixture is a mixture containing 100 mM calcium chloride and 0-20% (v / v) glycerol.

[0011] Preferably, the logarithmic phase bacteria are collected into a mixture containing calcium chloride and glycerol, and incubated on ice to obtain bacterial competent cells, specifically in the following steps:

[0012] Place the bacterial culture tube on ice and add 1-2 ml of a 0°C mixture of 100 mM calcium chloride and 0-20% (v / v) glycerol;

[0013] Bacteria cultured to the logarithmic phase on the culture plate were collected using a sterile cotton swab soaked in the mixed solution and suspended in the mixed solution in the bacterial culture tube.

[0014] Preferably, the bacteria are Gram-negative bacteria.

[0015] Preferably, the Gram-negative bacteria is Escherichia coli.

[0016] The present invention also provides a method for convenient bacterial transformation using limited conditions, comprising the following steps:

[0017] Add the plasmid to the Epp tube containing the competent bacterial cells prepared by the above method and flick to mix. Incubate on ice for 20-30 minutes.

[0018] Incubate in a 42°C water bath for 45 seconds, then transfer to ice for 1 minute to recover the cells;

[0019] Add 37°C liquid culture medium, transfer to a bacterial culture tube and culture in a 37°C constant temperature incubator.

[0020] Preferably, the plasmid is any plasmid that can transform bacteria.

[0021] Preferably, the amount of the plasmid added is 0.1 to 100 nanograms.

[0022] The important improvement of the present invention is that the traditional liquid culture medium shaking culture method is abandoned and replaced by solid culture medium surface culture. The bacteria on the surface of the culture medium are scraped with a sterile cotton swab and directly collected and resuspended in CaCl2 conversion solution to prepare competent cells. This breaks away from the limitation of the traditional method that must use high-end equipment such as a constant temperature shaker, spectrophotometer, and high-speed refrigerated centrifuge.

[0023] This method, which does not rely on liquid culture shaken culture or high-speed refrigerated centrifugation, provides a one-step, centrifugation-free method for rapid preparation of competent E. coli cells and bacterial transformation. This method is rapid, low-cost, and produces stable competent cells, fully meeting the needs of teaching laboratories and research laboratories with limited funding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flowchart of a method for conveniently preparing competent bacterial cells;

[0025] Figure 2 Flowchart of a method for convenient bacterial transformation;

[0026] Figure 3 The figure shows the transformation effect of plasmid DNA at different dosages. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Example 1

[0029] This embodiment provides a convenient method for preparing bacterial competent cells, comprising the following steps:

[0030] S110. Use a sterile cotton swab to inoculate E. coli from the LB culture plate (moss) onto a new LB culture plate (evenly spread), and culture overnight in a 37°C incubator for 15 hours.

[0031] S120. Use a sterile cotton swab to scrape 1-2 cm of E. coli from the overnight culture plate, inoculate onto a new culture plate, and incubate in a 37°C incubator for 3-4 hours.

[0032] S130. Place a bacterial culture tube on ice, add 2 ml of a mixture of 100 mM calcium chloride and 15% (v / v) glycerol at 0°C, collect bacteria from the culture plate with a sterile cotton swab soaked in the solution and transfer to the bacterial culture tube, and incubate on ice for 30 minutes.

[0033] Gently shake the competent cells in the culture tube to mix them evenly, and dispense 100 μL of the cell suspension into a cold Epp tube. These competent cells are then successfully prepared. The prepared competent cells can be used directly in bacterial transformation experiments or frozen in a -80°C freezer for later use. The transformation efficiency of the prepared competent cells is higher after being frozen in a -80°C freezer.

[0034] Example 2

[0035] This embodiment provides a convenient bacterial transformation method, comprising the following steps:

[0036] S210. Using the competent cells prepared in Example 1, add 2 μl of pBG309 plasmid (0.1-100 ng) to the competent cell suspension in an Epp tube. The plasmid can also be DNA. Gently flick to mix. Then, incubate the Epp tube on ice for 20-30 minutes.

[0037] S220, heat shock: incubate in a 42°C water bath for 45 seconds; immediately after heat shock, transfer the Epp tube to ice for 1 minute to allow cells to recover;

[0038] S230. Add 900 μL of LB liquid culture medium at 37°C to the Epp tube, transfer the bacterial suspension to a bacterial culture tube for recovery, place the bacterial culture tube in a 37°C constant temperature incubator, shake the bacteria manually every 30 minutes, and recover for 2 hours.

[0039] This example uses a spot plate method. 100 μl of the transformed cell suspension is added to 900 μl of LB liquid medium. The bacterial solution is serially diluted 1:10 and then serially diluted to 1:100. The undiluted bacterial solution and the serially diluted bacterial solution are respectively spotted using a 10 μl micropipette on an LB culture plate without chloramphenicol and a culture plate containing chloramphenicol. 10 μl of bacterial solution is aspirated each time. A total of 30 μl of the same concentration gradient is taken and arranged in three rows on the culture plate. After spotting, the culture is cultured and the number of colonies is counted. The transformation efficiency is calculated by comparison. In teaching experiments, the transformation efficiency can be ignored. The transformed bacteria are directly and evenly spread on a selective medium containing antibiotics for culture. Successfully transformed colonies are selected and the culture plate is inverted and placed in a 37°C constant temperature incubator for 18 to 24 hours.

[0040] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for conveniently preparing bacterial competent cells, characterized in that: The steps include: The bacteria are evenly spread on a solid culture plate and cultured in a biological incubator at 37° C. for 15 hours to form a bacterial lawn; wherein the bacteria are Escherichia coli; The bacterial lawn is inoculated onto a new bacterial culture plate and cultured in a biological incubator at 37° C. until the logarithmic phase is reached, which is a culture time of 3 to 4 hours; Place the bacterial culture tube on ice and add 1-2 ml of a 0°C mixture of calcium chloride and glycerol; Bacteria cultured to the logarithmic phase on the culture plate were collected using a sterile cotton swab soaked in the mixed solution and suspended in the mixed solution in a bacterial culture tube. Competent bacterial cells were obtained after incubation on ice.

2. The method for conveniently preparing bacterial competent cells according to claim 1, wherein: The length of the mushroom lawn is 1 to 2 cm.

3. The method for conveniently preparing bacterial competent cells according to claim 1, wherein: The calcium chloride and glycerol mixture is a mixture containing 100 mM calcium chloride and 15% to 20% (v / v) glycerol.

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