Effects of Sr2 + on the preparation of Escherchia coli DH5α competent cells and plasmid transformation

Abstract

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 SrCl2 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 Sr2 + 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 106 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

Keywords

• Escherchia coli
• Response surface methodology
• Competence
• Transformation mechanism