About our project

1.Our ultimate goal

The ultimate goal of our project is to solve the problem of chronic functional constipation.

2.Why our project is "gut-Sweeper"?

Constipation is a common clinical condition, and the elderly are at high risk of developing chronic constipation. The main clinical manifestations of constipation are decreased frequency of defecation, decreased volume of defecation, dry feces, and difficulty in defecation.

Constipation not only jeopardizes patients' physical and mental health and reduces life quality, but also generates considerable medical costs, which aggravates the economic burden of both patients and society. At the same time, the majority of constipated patients do not have a good understanding of the problem of constipation. Hong Fei et al ^[1] found in their interviews with constipated patients that despite the fact that constipated patients are deeply troubled and have an urgent therapeutic need, some of them still lack proper knowledge about constipation. Our team can provide constipation-related knowledge along with the program so that they can understand the importance of early prevention and treatment. This interview also found that many chronic constipated patients actively cooperate with diet and exercise, but still with poor results. This is because a significant number of them use medication irregularly, or seek medical help only when constipation is severe, or are frequent to use stimulant laxatives that work quickly, which cannot restore normal bowel physiology, resulting in unsatisfactory medical outcomes.

In recent decades, there has been a growing recognition of the role of gut flora in maintaining human health. In fact, the therapeutic use of bacteria can be seen everywhere in life. Taking the yogurt as an example, the yogurt on the market contains probiotics such as Lactobacillus acidophilus, Bacillus subtilis, Bifidobacterium bifidum, etc. Moreover, through the advertisements of the sellers, more and more people are realizing that bacteria are not all harmful and that the human body is a balanced system of human and microorganisms. Many people even favor purchasing yogurt with particular probiotics. So who would say NO to a cup of yogurt that can easily solve constipation troubles? As probiotics can be administered orally to improve the efficacy and minimize systemic side effects, their therapeutic role in gastrointestinal diseases has gained increasing attention.

Therefore, it is of pivotal importance to develop interventions for constipation, targeting risk factors and at-risk populations. Our team is trying to create an engineered bacteria product that is easy for patients to take in daily life without psychological burden. We plan to add our engineered bacteria to yogurt, so that patients can subconsciously intervene in the occurrence of constipation in their daily diets.

3.Why do we chose E.coli as the biological chassis?

In forward MR (Mendelian randomization, gut microbiome as exposure), we identified SNPs (single nucleotide polymorphisms) with constipation phenotypes associated with the gut microbiome. IVW (inverse-varianceweighted) analysis showed that an increase or decrease in the abundance of microflora in about 20 was associated with the occurrence of constipation. However, the changes in abundance of the above flora are weakly associated with the risk of constipation after FDR (FalseDiscovery Rate) correction.

Among the various host strains available, our choice for employing E. coli as the foundation for genetically engineered bacteria stemmed from its established prowess in serving as a dependable expression vector for DH5α recombinant protein. It should be noted that the Enterobacteriaceae family, including the aforementioned species, has not been associated with the emergence of constipation. Nonetheless, the impact of constipation symptoms on the abundance of E. coli bacteria within the gut remains a pertinent inquiry in the realm of genetically engineered bacteria.

Therefore, in reverse MR (Mendelian randomization, constipation as exposure), we used IVW, MR-Egger, WM and other methods to analyze the causal effects of constipation and Enterobacter abundance. Based on the results of IVW and WM analysis, it was suggested that constipation is associated with increased abundance of Enterobacteriaceae. These results showed that in the case of constipation, the abundance of Enterobacteriaceae in patients would increase, which was conducive to the colonization and function of genetically engineered bacteria based on E. coli.

Click here to see more information about our Mendelian randomization in Model !

Further, due to safety considerations, we determined Bl21 and ECN strains as our chassis, in the early proof of concept and final application, respectively. Both of them are non-pathogenic strains of E. coli and have been widely utilized in researches.

4.How can we insure the engineered bacteria are safe and effective?

The sensory module: Composed of pchA and Cl protein, can sense the concentration of butyrate and express the corresponding amount of Cl protein^{[2, 3]}.

The Cl protein has an inhibitory effect and can inhibit the metabolic module in a healthy human body^[3].

The metabolic module: Composed of TPH and TDC, and was placed downstream of the plam promoter.

TPH and TDC are two enzymes that catalyze the production of 5-HT ^[4], which can bind to 5-HT receptors in the intestinal wall to promote intestinal motility and secretion^[5], thereby improving constipation.

The safety module: Two lines consisting of drug control and temperature control.

Part1 drug control: We utilized bistable switch to achieve drug control when the engineered bacteria are in human body. If there is low dose of tetracycline or its analog atc in the environment, the plac promoter and its downstream tetR will be inhibited and a positive loop is formed to express MazF, thereby inducing bacterial death^[6].

Part2 temperature control: Using toxin-antitoxin system, the engineered bacteria have different survival states in different scenarios.

In transportation scenario, the temperature is below 30°C with rhamnose addition, allowing for simultaneous expression of mazE and mazF, preventing bacterial death.

In in-vivo scenario, the temperature is 37°C, only mazE can be expressed, so the bacteria can survive.

In excretion scenario, the temperature is below 30°C and rhamnose is diluted. Only mazF can be expressed. Therefore the bacteria die. The backbone of our circuit is originated from Wang, et al^[7].

Our inspiration

This year, 5 March was the annual Lei Feng Memorial Day. In order to pay tribute to Lei Feng's spirit of serving the people wholeheartedly, we participated in a volunteer activity in an old people's home. During the activity, we found that many old people suffered from constipation.

After reviewing the literature, we found that 90 million people in China suffer from constipation, and the prevalence increases with age. However, the current medication and surgery for constipation can't solve the problem well, with temporary therapeutic effect only.

Therefore, we came up with the idea of making a product that can have a long-term therapeutic effect to relieve constipation. By designing a specific genetic circuit, we made a modified E. coli Gut-sweeper that combines prevention, treatment and monitoring.

Click here to see more information about our inspiration in Human Practice !

Our project goal

Our project aims to develop brand-new engineered bacteria to solve the constipation problem, making the treatment of constipation more convenient and routine.

1.Conduct a review of current constipation treatment.

2.Review the current selection of chassis bacteria and determine E.coli as the most suitable one in our project.

3.Consult with experts in the field of microorganisms and synthetic biology to evaluate the feasibility of this project and receive suggestions for improvement.

4.Investigate the view of stakeholders about our project and determine the future prospects.

5.Design a genetic circuit for the purpose of allowing engineered modified E. coli to sense different concentrations of butyrate within the environment and to promote downstream gene expression, and experimentally determine the feasibility of the circuit.

6.Design kill switch circuit for engineered bacteria for in vivo urgent containment and environmental protection and develop it in iterations.

7.The role of our engineered bacteria in synthesizing serotonin and treating constipation was simulated through modeling.