Synthetic biology is an interdisciplinary field that combines biology, genomics, engineering, informatics, and other fields. It integrates methods of observation and analysis from life sciences with the design thinking of engineering, enabling humans to engineer, modify, and even synthesize biological systems with specific functions. In recent years, synthetic biology and its applications have had a profound impact on the development of fields such as chemical engineering, food, consumer goods, energy, healthcare, and agriculture, creating significant social and economic value.

The main objective of our project is to construct a gene circuit that enables our strain to effectively degrade cholesterol in food. We encountered some issues during the design and testing phase, but we made adjustments and designed the final version of our gene circuit, testing its effectiveness. The gene circuit largely aligns with our expectations and goals.

Design

Figure 1   Experimental design schematic diagram
Our initial gene circuit includes genes encoding cholesterol transport proteins and two genes that can degrade cholesterol (ACS and IsmA).

Build

First, 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

Test

Figure 3  Determination of cholesterol transporters（MFS）
While maintaining identical experimental conditions, we establish trials to measure cholesterol concentrations in untreated plasmid E. coli Rosetta and plasmid E. coli Rosetta treated with protein pT7-MFS after twelve hours of cultivation conducted in LB medium. As illustrated by Fig. B and Fig. C, with identical initial cholesterol concentrations of 80 µg/mL, the untreated plasmid displayed an ultimate cholesterol concentration of roughly 72.16 µg/mL, while the cholesterol concentration plasmid treated with protein pT7-MFS dropped to approximately 55.83 µg/mL. Comparatively, the cholesterol concentrations reduced were approximately 7.84 µg/mL and 24.17 µg/mL, respectively for untreated and protein-treated plasmid samples, as shown in Fig. E. In this case, precise calculations reveal that the concentration of cholesterol is reduced by roughly 9.8% in untreated plasmid E. coli Rosetta, while in the protein-treated plasmid E. coli Rosetta, the concentration is reduced by approximately 30.21%.

Figure 4  The expression of ismA .
The ismA gene was cloned into the pET28a plasmid and validated before being transformed into Escherichia coli Rosetta. The recombinant Rosetta bacteria were then cultured in LB medium containing 0.5 mM IPTG and incubated at 16°C for 12 hours. A 10 mL bacterial culture was taken, centrifuged to remove the supernatant, and the cell pellet was resuspended in 20 mM Tris-HCl (pH 7.0). 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.

Learn

Preliminary results have been obtained, but there is still room for improvement.We plan to collaborate with other teams to introduce EPS to enhance the adsorption of cholesterol.

Design

Considering that cholesterol  may not be completely removed by the Isma gene, we decided to produce  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.

Build

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.

Test

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 (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.

Learn

After introducing the gene galU, the effect is significantly improved. However, in order to initiate gene expression more efficiently, we would like to design a switch to make gene expression controllable.

Design

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 promoter 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.

Build

Figure7  Schematic diagram of gene components 

Test

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 omM 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 omM 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

Learn

The gene design is largely consistent with our expectations and goals, and it has shown good results.

After three rounds of iterative design, we have constructed a complete gene circuit that aligns with our expectations and is also practically meaningful. In the factory, our engineered probiotics will be added into high cholesterol food such as egg and cheese. Besides, oils that are rich in oleic acid, such as sunflower oil will also be added during the process to help stimulate oleic acid promoter. The oleic acid promoter can sense 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.