Open AccessEffects of Sr2 + on the preparation of Escherchia coli DH5α competent cells and plasmid transformation
Author(s) -
Yonggang Wang,
Xinjian Wang,
Linmiao Yu,
Yuan Tian,
Shaowei Li,
Feifan Leng,
Jianzhong Ma,
Jixiang Chen
Publication year - 2020
Publication title -
peerj
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.927
H-Index - 70
ISSN - 2167-8359
DOI - 10.7717/peerj.9480
Subject(s) - plasmid , puc19 , transformation (genetics) , escherichia coli , transformation efficiency , zeta potential , scanning electron microscope , chemistry , microbiology and biotechnology , analytical chemistry (journal) , materials science , biophysics , biology , chromatography , gene , biochemistry , nanotechnology , agrobacterium , composite material , nanoparticle
Bacterial gene transformation used with Escherichia coli as a desired microorganism is one of the important techniques in genetic engineering. In this study, the preparation of E. coli DH5α competent cells treated with SrCl 2 and transformation by heat-shock with pUC19 plasmid was optimized by Response Surface Methodology (RSM). Other five E. coli strains including BL21 (DE3), HB-101, JM109, TOP10 and TG1, three different sizes plasmids (pUC19, pET32a, pPIC9k) were used to verify the protocol, respectively. The transformation mechanism was explored by scanning electron microscope combined with energy dispersive spectrometer (SEM-EDS), atomic absorption spectroscopy (AAS) and Fourier-transform infrared spectroscopy (FT-IR). An equation of regression model was obtained, and the ideal parameters were Sr 2 + ions of 90 mM, heat-shock time of 90 s and 9 ng of plasmid. Under this conditions, the transformation efficiency could almost reach to 10 6 CFU/µg DNA. A small change of the cell surface structure has been observed between E. coli DH5α strain and competent cells by abovementioned spectrum technologies, which implied that a strict regulation mechanism involved in the formation of competent cells and transformation of plasmids. An equation of regression model for the competent cells preparation and plasmid transformation could be applied in gene cloning technology