Difference between revisions of "Part:BBa K4988002"

Latest revision as of 04:38, 10 October 2023

We identified and cloned the galU gene, which encodes UDP glucose pyrophosphorylase (GalU) in Strept

Considering that cholesterol in the blood may not be completely removed by the Isma gene, we decided to design in another bacterium, EPS, which interferes with the absorption of cholesterol in the intestines by binding to cholesterol and finally eliminating it from the body（1）.In this way we can effectively remove the residual cholesterol.

Usage and Biology

Figure 1 Design of the galU.

We use the oleic acid promoter to express the galU gene, and the constructed plasmid is then introduced into E. coli Rosetta.

Characterization

Figure 2 Gel electrophoresis of galU.

Figure 3 The effect of GalU gene.

GalU was cloned into pET28a vector and transformed into E.coli Rosetta. Inoculated into LB medium, cultured OD600=0.4 at 37℃ and 180 rpm, and induced 16h with 0.5 mM IPTG. Adjust OD600 to 1. Take 10 mL of bacterial solution and use anthrone sulfuric acid method (see: EPS measurement method) to detect EPS yield. The EPS yield of blank strain was 46 mM, and the EPS yield of engineering strain under 0.5 mM IPTG induction was 105mM. The results of this study clearly indicate that EPS production in E. coli can be enhanced by overexpression of galU in figure A. Each bacterial culture was cultured in LB medium and then diluted to OD600 = 0.1. At 0 h, 8 mg/mL ethanol cholesterol solution was added into LB medium to make the final concentration reach 80 μg/mL. The cholesterol concentration in each group's medium was detected with a quantitative cholesterol kit (mlbio, ml094955). OD600 and cholesterol concentrations were detected again after 12 h of culture. Δcholesterol/ΔOD600, which represents the absorption of cholesterol by bacteria during growth, was calculatedin figure B.Figure C,D,E and F are the effect of increased EPS content on adsorption of cholesterol

Potential application directions

Experiments shows that EPS production in E. coli can be enhanced by overexpression of galU. As a result, galU gene is going to be useful in producing low cholesterol foods and helps decrease the cholesterol intake of potential high cholesterol patients and helps decrease the risk of diseases such as cardiovascular disease caused by high cholesterol. galU gene can be used as a clinical treatment for high cholesterol in the future and also provide new ideas and solutions.

Sequence and Features