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

Abstract

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 100mM 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 100mM. 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 25mM Ca2+, 50mM Mg2+, and 25mM Mn2+ was applied. Further supplement of 0.8% (w/v) PEG6000 lead to a slight decrease in the TrE, whereas supplement of 25mM 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.