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

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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png 3 Biotech Springer Journals

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

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Publisher
Springer Berlin Heidelberg
Copyright
Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature
Subject
Chemistry; Biotechnology; Agriculture; Cancer Research; Bioinformatics; Stem Cells; Biomaterials
ISSN
2190-572X
eISSN
2190-5738
D.O.I.
10.1007/s13205-018-1243-x
Publisher site
See Article on Publisher Site

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.

Journal

3 BiotechSpringer Journals

Published: Apr 17, 2018

References

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