Construction of a minicircle DNA vector carrying enhanced green fluorescence protein (egfp) gene

Abstract

Two-factor and three-level fractional factorial design was employed for evaluation of the effect of Glycine and Triton X-100 on the secretion and expression of ZZ–EGFP fusion proteins. Varying contents of glycine (0%, 1%, 2%) and Triton X-100 (0%, 1%, 2%) were added into shaking flasks, respectively, and supplied with appropriate volume of ampicillin (total 9 combinations; group at concentration zero serving as control) to promote more ZZ–EGFP diffuse into liquid culture medium. Fluorescent intensity in the culture supernatant was detected. A standard curve could be generated on the basis of fluorescent intensity and protein concentration. The expression level of ZZ–EGFP fusion proteins was estimated by checking the protein standard curve concentration fluorescene intensity. Results show that when the culture medium contains 2% Glycine and 1% Triton X-100, the expression level of ZZ–EGFP was able to be greatly increased. Further experiments revealed that absorbance value (A600) in the experiment group, whose culture medium contains 2% Glycine and 2% Triton X-100, is significantly lower than other groups in the present experiment. These results indicate that the culture medium containing appropriate quantity of Glycine and Triton X-100 is favourable to the secretion and expression level of ZZ–EGFP in gene-engineering bacteria Escherichia coli HB101.