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3 Biotech

, 8:222 | Cite as

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

  • Jia Zhou
  • Xiangqian Li
  • Jilin Xia
  • Yue Wen
  • Jie Zhou
  • Zhilong Yu
  • Baoxia Tian
Protocols and Methods
  • 101 Downloads

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 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.

Keywords

Escherichia coli Competent cell Bivalent ion Osmotic stress 

Notes

Acknowledgements

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.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

13205_2018_1243_MOESM1_ESM.pdf (818 kb)
Supplementary material 1 (PDF 817 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Life Science and Food EngineeringHuaiyin Institute of TechnologyHuaianPeople’s Republic of China
  2. 2.Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process IntegrationHuaiyin Institute of TechnologyHuaianPeople’s Republic of China

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