What is our objective?

Our objective is to develop an intelligent cholesterol regulatory system based on synthetic biology methodologies, aiming to establish a revolutionary hypercholesterolemia prevention program. Through the application of synthetic biology techniques, we are dedicated to creating a personalized, efficient, and sustainable health management solution, enhancing the overall quality of life for individuals.

Function Parts

Cholesterol regulation genes

Cholesterol degradation genes refer to specific genes in an organism's genetic makeup that encode enzymes and proteins responsible for breaking down（or indirectly impacting） cholesterol molecules which play a crucial role in regulating cholesterol levels in the body. Cholesterol degradation is a complex process involving multiple enzymes that convert cholesterol into various products, which can be eliminated from the body or used in the synthesis of essential molecules. Through the insights of cholesterol regulationtion genes, we may better intervene in various conditions related to abnormal high level of cholesterol in blood, such as hypercholesterolemia

IsmA

The IsmA gene is involved in the first and last steps of cholesterol conversion by encoding a hydroxysteroid dehydrogenase (HSD). Eubacterium coprostanoligenes (ATCC 51222) is the only publicly available coprosterol-producing strain. The gene ECOP170 of this strain is homologous to the IsmA gene.

BSH

The BSH gene produces bile saline hydrolase, which lowers cholesterol levels (indirectly) in two main ways:
1. Unconjugated bile acids produced by enzymatic catalysis have higher hydrophobicity and lower solubility, and their flow to the intestine is reduced
2. Promote the excretion of bile acids in the stool.
These two processes result in a decrease in the circulating pool of bile acids in the gut, which promotes the conversion of cholesterol to bile acids and, in turn, lowers cholesterol levels.

Promoter

Oleic acid inducer

After some thought and discussion, we decided to link the initiation of a functional gene module that carries out cholesterol regulation to a high-fat dietary process that may increase the risk of developing hypercholesterolemia. In doing so, we identified a very suitable class of promoters: the oleic acid inducers. Oleic acid, as a frequent substance in high-fat diets, can serve us well as an indicator of whether or not people are on a high-fat diet, meaning that it serves as an excellent signal to tell functional genes whether or not they should start working. In addition, in the investigation of this promoter, we also gained the surprise that by modifying this promoter, we can realize the intelligence and customization of the whole cholesterol regulation system. By modifying the FadO operator sequence in the promoter sequence, we can change the threshold of oleic acid concentration that can cause the expression of the promoter. Through the verification of experiments and modeling plates, we can obtain the appropriate induction threshold interval, by matching the content of oleic acid in high fat diet defined according to different human constitution. We can design cholesterol-degrading strains based on the principle of oleic acid inducer to adapt to different human needs in the enteral nutrition environment.

Permutation

In order to find the optimal solution to the cholesterol down-regulation answer, in addition to testing the effect of individual genes on cholesterol levels, we also arranged the three genes in combinations and tested their effects on cholesterol down-regulation individually, and unsurprisingly, in the process, we got a different answer.

Killing switch

Considering the safety of the project, we cooperated with Beijing University of Chemical Technology (BUCT) to design and test a killing switch, which can effectively activate the suicide module in the presence of oxygen to inhibit the growth of engineered bacteria. Based on the appealing design, we are able to prevent the leakage of engineered bacteria in vitro and in this way.

Capsule

For a second layer of assurance, we made microcapsules from sodium alginate and chitosan, also known as ACA. More details can visit hardware.

Result

We have successfully constructed engineering strains with complex cholesterol regulation and oleic acid inducer reporter strains, and functionally validated our target genes and engineering strains. The experimental results indicate that our engineered strain has a successful effect and great potential for cholesterol regulation. At the same time, the experiment also proved the success of the oleic acid inducer, which can respond to gradient oleic acid and has the potential for dynamic regulation. For detailed information on this section, please refer to the Proof of Concept section.
At the same time, facing the future development of our project, in order to achieve better safety and delivery results, we have provided capsule design for our engineering strain and collaborated with Beijing University of Chemical Technology to develop an oxygen suicide switch. In order to ultimately produce a dynamic cholesterol regulation system for our project, we introduced the design concept of changing the oleic acid induction threshold regulation system by modifying the FadO operator of the oleic acid inducer. Based on our project, we have also conducted in-depth research and extensive publicity in the social sector. Through our research, we aim to improve our project by taking into account the widest range and achieving better inclusiveness, bringing more positive impacts to the world.