The role of temperature and bivalent ions in preparing competent Escherichia coli
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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.
KeywordsEscherichia coli Competent cell Bivalent ion Osmotic stress
This work was supported by a Grant (BK20150417) from the Natural Science Foundation of Jiangsu Province of China; a grant (21576110) from the National Natural Science Foundation of China; a grant (HAN2015026) from the Huaian Key Research and Development Project; a grant (JPELBCPI2017003) from Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration Open Project; and Grants (HAGZ201603, HAGZ201604, HAGZ201605, HAGZ201606) from the Huaian Science and Technology Guiding Project.
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Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
- Brooke R, Hopkins J, Laflamme E, Wark P (2009) Effects of temperature-induced changes in membrane composition on transformation efficiency in E. coli DH5α. J Exp Microbiol Immunol 13:71–74Google Scholar
- Chan WT, Verma CS, Lane DP, Gan SK (2013) A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Biosci Rep 33 (6). https://doi.org/10.1042/bsr20130098
- Janjua S, Younis S, Deeba F (2014) High efficiency DNA transformation protocol for Escherichia coli using combination of physico-chemical methods. Int J Agric Biol 16:132–138Google Scholar
- Kurien BT, Scofield RH (1995) Polyethylene glycol-mediated bacterial colony transformation. Biotechniques 18(6):1023–1026Google Scholar
- Liu X, Liu L, Wang Y, Wang X, Ma Y, Li Y (2014) The study on the factors affecting transformation efficiency of E. coli competent cells. Pak J Pharm Sci 27(3 Suppl):679–684Google Scholar
- Sambrook J, Russell DW (2001) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork, p 1Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual, 2nd edn edn. Cold Spring Harbor Laboratory Press, Plainview, pp 49–55 (Chinese) Google Scholar
- Zhiming T, Guangyuan H, Kexiu XL, Mingjie JC, Junli C, Ling C, Qing Y, Dongping PL, Huan Y, Jiantao S, Xuqian W (2005) An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Elec J Biotech 8 (1). https://doi.org/10.4067/s0717-34582005000100014