The role of temperature and bivalent ions in preparing competent Escherichia coli

Several factors including the culture temperature, bivalent ion, and osmotic stress were gradually optimized for preparing efficient Escherichia coli competent cells. The effect of culture temperature on the transformation efficiency (TrE) of E. coli DH5α was tested with 100 mM CaCl₂. The lower culture temperature at 18 °C resulted in higher TrE of 2.5 × 10⁶ cfu/μg, which was about 3.5 times of that obtained at 37 °C. Bivalent ions including Ca²⁺, Mn²⁺, Mg²⁺, and Ni²⁺ were tested independently or combinatorially at a total concentration of 100 mM. Ni²⁺ showed a significantly inhibition on the TrE, and various concentration combinations of Ca²⁺, Mg²⁺, and Mn²⁺ were tested. The TrE was improved up to 1.8 ± 0.4 × 10⁸ cfu/μg, when a combination of 25 mM Ca²⁺, 50 mM Mg²⁺, and 25 mM Mn²⁺ was applied. Further supplement of 0.8% (w/v) PEG₆₀₀₀ lead to a slight decrease in the TrE, whereas supplement of 25 mM sucrose contributed to another increase in the TrE by 17% up to 2.1 ± 0.3 × 10⁸ cfu/μg. These results indicated that the culture temperature and bivalent ion were important factors affecting the TrE of E. coli. A chemical method for preparing efficient competent cells of E. coli was provided.