Preparation of Competent E. coli Cells and Plasmid Transformation
News 16 7 月, 2025
Competent Escherichia coli (E. coli) cells are host cells made receptive to foreign DNA by calcium chloride (CaCl₂) treatment, which temporarily increases cell membrane permeability. This process enables efficient plasmid transformation.
1. What Are Competent Cells?
- Competence is a physiological state that allows cells to uptake and incorporate foreign DNA.
- Competent cells are prepared via physical or chemical treatments to reach peak DNA receptivity.
- Log-phase (actively dividing) cells are ideal for preparing competent cells.
- Plasmid transformation is the process by which plasmid DNA or recombinant plasmids are introduced into bacterial cells.
2. Principle of Competent Cell Preparation
- Gene expression requires that recombinant plasmids enter bacterial cells via transformation.
- CaCl₂ method: When E. coli is incubated in cold, hypotonic CaCl₂ solution, cells swell, and Ca²⁺ ions alter membrane structure, creating a state of competence.
- Transformation mechanism: Ca²⁺ facilitates DNA adherence to the membrane. A brief heat shock at 42°C disrupts the membrane, allowing DNA to enter.
- Once inside, the foreign DNA is replicated and expressed. Positive colonies can be selected on antibiotic-containing media.
3. Step-by-Step: CaCl₂ Competent Cell Preparation
- Strain Activation: Recover frozen strains (e.g., DH5α, Top10, DE3, BL21) on LB agar without antibiotics and incubate at 37°C.
- Culture: Pick a single colony into 10 mL LB broth and grow for ~12 hours.
- Inoculation: Dilute into 20 mL fresh LB broth (2% inoculum), grow 2–3 hours until OD600 = 0.4–0.5.
- Chill and Centrifuge: Ice-bath for 10 min, centrifuge at 4°C, 3000 rpm, 10 min.
- Resuspension: Wash cells with 10 mL cold 0.05M CaCl₂, incubate on ice, centrifuge again.
- Final Suspension: Resuspend in 6 mL cold CaCl₂ (with 15% glycerol for storage), aliquot 50 µL per tube, freeze at –80°C.
4. Plasmid Chemical Transformation Procedure
- Thaw competent cells on ice.
- Prepare plasmid by diluting 1 ng in 10 µL buffer or water.
- Mix 1 µL of plasmid with 50 µL competent cells, incubate on ice for 30 min.
- Heat shock at 42°C for 60–90 s, then return to ice for 2–3 min.
- Recovery: Add 300 µL prewarmed LB (no antibiotics), shake at 150 rpm for 45 min at 37°C.
- Plate 100 µL on antibiotic LB agar, incubate at 37°C overnight.
- Calculate transformation efficiency using colony count and DNA input:
- Total colonies = (colonies × dilution factor × total volume) ÷ plated volume
- Efficiency = Total colonies ÷ DNA mass in µg
- Max efficiency: up to 1×10¹⁰ colonies/µg plasmid
5. Best Practices & Notes
- Maintain strict sterility throughout to prevent contamination.
- Temperature-sensitive steps (e.g., antibiotic handling) require careful monitoring.
- Control cell density and plasmid DNA quality for high efficiency.
- Record transformation data for evaluating success.