Plasmid Construction — Preparation of Competent Cells

In plasmid construction experiments, a type of Escherichia coli called competent cells is used during the transformation stage. The main role of competent cells is to take up exogenous fragments and amplify them. Competent cells refer to the physiological state of the recipient cells in which they are most receptive to foreign DNA fragments and can be transformed. There are two types of bacterial competence: natural competence and artificial competence. The E. coli used in plasmid construction belongs to artificially induced competence.

Common methods for preparing competent cells include electroporation, calcium chloride method, Hanahan method, and Inoue method.

Here, we will briefly introduce the preparation of competent cells using the calcium chloride method as an example.

Preparation:

·         Prepare LB liquid medium, 0.1M CaCl2, and 80% glycerol, then sterilize them by autoclaving.

·         Prepare LB solid and liquid media, sterilize them by autoclaving, and when the temperature drops to 50°C, pour the plates.

Streak Culture:

·         Retrieve a tube of E. coli DH5α strain from the -80°C freezer and allow it to thaw naturally on ice.

·         Take a small amount of the bacterial solution and streak it onto an LB agar plate without antibiotics.

·         Invert the plate and incubate it in a 37°C incubator. After 12-16 hours, check for colony formation.

Picking Single Colonies:

Select a single colony from the streaked plate and transfer it into a 5mL LB medium tube without antibiotics. Label the tube and place it in a shaking incubator for culture. The incubation conditions are: 37°C, 250 rpm, for 6-8 hours, allowing the bacteria to grow and proliferate in large quantities.

Preparing Bacteria in the Log Phase:

Take 400mL of bacterial culture (inoculated at a 1:500 ratio) and transfer it into a 200mL LB medium without antibiotics in a conical flask. Place the flask in a bacterial shaker for incubation under the following conditions: 37°C, 250 rpm, for 2-3 hours. During the incubation, use a spectrophotometer to monitor the OD value of the bacteria. Once the OD600 value reaches 0.6-0.8 (which indicates that the bacteria are in the log phase), stop the shaking culture.

Ice Bath Cooling:

Transfer the bacterial culture from the conical flask into pre-chilled sterile 50mL centrifuge tubes and place them on ice for 10 minutes.

Bacterial Resuspension and CaCl2 Incubation:

Add 10mL of pre-chilled 0.1M CaCl2 to each tube to resuspend the bacterial pellet. Centrifuge at 4°C, 4000 rpm for 10 minutes to pellet the bacteria.

Next, add another 10mL of pre-chilled 0.1M CaCl2 to resuspend the bacterial pellet again. Place the tubes on ice for 10 minutes, then centrifuge at 4°C, 4000 rpm for 10 minutes to pellet the bacteria once more.

Competent Cell Aliquoting:

Add 2mL of 0.1M CaCl2 and 0.5mL of 80% glycerol solution to each tube to resuspend the bacterial pellet. Then, aliquot the competent cells into pre-chilled, sterile 1.5mL EP tubes, with 50mL per tube. Once aliquoted, immediately place the competent cells in a -80°C freezer for long-term storage.

Transformation Efficiency Testing:

Transform the plasmid into freshly prepared competent cells, plate the transformed cells on agar plates, and incubate at 37°C. After incubation, count the number of positive colony formations to assess the transformation efficiency.