Figure 1   Experimental design schematic diagram
The main objective of our project is to construct a gene circuit that enables our strain to effectively degrade cholesterol in food. 

The T7 promoter expresses a large amount of transport enzyme that expresses cholesterol transport proteins, which will transport cholesterol into the bacterial cell. Then, IsmA and ACS will degrade it to achieve a reduction in cholesterol absorption.

Figure 2  Schematic diagram of gene components

Figure 3  Determination of cholesterol transporters（MFS）
For the experimental group and control group strains, 8 mg/mL ethanol cholesterol stock solution was added to LB medium at 0 h to achieve a final concentration of 80 μg/mL. The cholesterol concentration in each group's medium was detected using a cholesterol quantitation kit. After 12 h of cultivation, OD600 and cholesterol concentration were measured again. To standardize the data, we calculated Δcholesterol/ΔOD600, which represents the cholesterol uptake by bacteria during growth. All values were determined based on the average of all duplicate measurements. Figure A shows the relative content of MFS cholesterol absorption in the control group and experimental group. Figures B and C show the cholesterol uptake at 0 h and 12 h. At 12 h, the cholesterol concentration in the control group was approximately 72.16 µg/mL, while in the experimental group it decreased to about 55.83 µg/mL. In comparison, the cholesterol concentrations in the control group and experimental group decreased by approximately 7.84 µg/mL and 24.17 µg/mL, respectively. Figure D compares the growth of strains in the control group and experimental group, and Figure E compares the cholesterol content in the experimental group and control group.

Figure 4  The expression of ismA .
The ismA gene was cloned into the pET28a plasmid and validated before being transformed into Escherichia coli Rosetta.     A 10 mL bacterial culture was taken, centrifuged to remove the supernatant, and the cell pellet was resuspended .   The cells were then subjected to ice-cold sonication to collect the crude enzyme solution, and the protein concentration was determined using the Bradford assay.    The crude enzyme solution was mixed with 5 mg/mL cholesterol and incubated for 12 hours.    Subsequently, the cholesterol concentration was determined by measuring the fluorescence emission using the Amplex™Red Cholesterol Assay Kit.    The results, as shown in the figure, indicate that the engineered strain significantly degraded cholesterol compared to the wild-type.

The ACS was also  cloned into the pET28a plasmid and validated before being transformed into Escherichia coli Rosetta. However, the experimental results were not satisfactory, and ACS did not play a significant role.

Cholesterol  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.In this way we can effectively remove the residual cholesterol。

Figure5   Schematic diagram of gene components 
We use  promoter to express the galU gene, and the constructed plasmid is then introduced into E. coli Rosetta.

Figure6   Images of experimental results
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 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.

We interviewed a nutrition expert and learned that oleic acid has the effect of reducing cholesterol. Inspired by this, we hope to combine our project with oleic acid, so we designed an oleic acid promoter. We hope that the addition of oleic acid can activate the expression of downstream genes, while the intake of oleic acid can also degrade cholesterol. Moreover, during the fermentation stage in the factory, because there is no oleic acid in the factory, it will not be produced and the downstream genes will not be expressed, reducing the pressure on the bacteria. Therefore, we chose to use the oleic acid promoter as our promoter.

Figure7  Schematic diagram of gene components. 
The LB medium was added with different concentrations of oleic acid and inoculated with E.coli Rosetta. The OD600 value of E.coli Rosetta was measured by spectrophotometer. The results showed that 5 mM oleic acid had little effect on bacterial growth, while 10 mM oleic acid inhibited bacterial growth.

Figure8  Effect of oleic acid on bacterial growth
The LB medium was added with different concentrations of oleic acid and inoculated with E.coli Rosetta/pOleic ACID-MRFP. Adjust the initial OD600 to 0.1. After 12 hours, the OD600 value and mRFP fluorescence intensity were measured using an microplate reader. The results showed that under the induction of oleic acid at 0mM and 5 mM, oleic acid promoter mediated the engineering bacteria to produce about 4.5 Fold change under the induction of 5mM oleic acid comparing to the result produced by 0mM oleic acid.

Figure9   Response of oleic acid promoter.
The ismA gene was cloned into the downstream of pOleic acid and transformed into Escherichia coli Rosetta after verification.The recombinant strain Rosetta was then placed in LB medium containing different concentrations of oleic acid. After 12 hours, the crude enzyme was extracted according to the above method. 5 mg/mL crude enzyme solution was incubated with 200 μM cholesterol for 12 h. The Amplex™Red cholesterol test kit (Sigma, A12216) was then used to determine cholesterol concentrations based on fluorescence emission. Under the induction of 5mM of oleic acid , the cholesterol concentration goes through a dramatic decrease due to the expression of IsmA gene, comparing to the concentration when 0mM oleic acid is present. The result shows oleic acid can stimulate the oleic acid promoter to express IsmA gene which can then lower cholesterol concentration. 

Figure10    The expression of ismA is controlled by oleic acid promoter

Figure 11   Gel electrophoresis of MFS,galU and ismA.

We have constructed a complete gene circuit that aligns with our expectations and is also practically meaningful.For patients with high cholesterol, it is recommended to cook using oils rich in oleic acid, such as sunflower oil, and simultaneously consume our engineered probiotics during meals. In the intestines, the oleic acid promoter senses the presence of oleic acid and initiates the expression of downstream genes. Cholesterol transport proteins will transport cholesterol into bacterial cells, where it will be degraded by IsmA and ACS to reduce cholesterol absorption.