Background and Inspiration
With the development of living standard, pets are playing an increasingly important role in people's lives. According to the statistics, the number of pet dogs has reached 55.03 million in China’s urban area, making pet health an important issue than ever before. From the surveys, we also realized that pet medical is becoming the biggest barrier for animal raising(https://2021.igem.org/Team:HZAU-China/Human_Practices ).
Inflammatory bowel disease (IBD) is an immune-mediated disease at the intersection of interactions between genetics, environment and gut microbiota. The common symptoms of IBD are so deceptive that they are always been recognized as normal diarrhea by non-professionals. Unlike human-beings, pets could not voice their illness correctly and timely, causing delayed diagnosis and increasing the risk of complications. The applicated methods of diagnosis are quite complicated and expensive as well. Thus, it is urgent to develop a convenient home-based method to detect IBD.
When IBD occurs, the concentration of nitrate and thiosulfate in the focus of intestine would increase significantly, making them ideal biomarkers to reflect the overall pathological changes in intestine. Probiotic E.coli Nissle 1917 was selected as our chassis. The engineered bacteria would be capable of sensing concentration changes of the biomarkers and producing a strong and recognizable signal to alarm owners to realize the appearance of disease. Anti-inflammatory substance would be released at the same time to relieve the symptoms. What’s more, we modified our bacteria to produce azurin constantly, which could enhance anti-cancer ability of the product. As for the biosecurity, two suicide switches were designed to ensure death of the engineered bacteria as soon as it get out of the gut.
We learned from senior vet Donghai Zhou that human-being and dogs share similarities in the clinical characteristics of IBD, which means it is possible for us to apply the design on human one day in the future(https://2021.igem.org/Team:HZAU-China/Human_Practices).
The name of our project is P.E.T: Pets' Enteric Test. We aim to develop a method based on synthetic biology to detect IBD (Inflammatory bowel disease) at an early stage. Considering that pets can’t speak for themselves like humans when suffering from IBD, we take pet dogs suffering from IBD as an example to verify the feasibility of our method, and we plan to apply this project as a new method to more mammals in the future. Through the "Detection and Report" module, engineered bacteria can accurately and promptly inform the pet owner of the pet's unhealthy conditions. In addition, the "Anti-inflammatory" module can temporarily help pets alleviate the effects of inflammation, and the "Health-care" module can prevent further inflammation from worsening. Moreover, the "Health-care" module that continues to function can also take a certain health effect in normal times. Considering safety issues, we also add a "Suicide" module to our engineered bacteria. These modules are briefly described in the following paragraphs.
Detection and Report
This module plays the role of feeling abnormal conditions in the intestines and reporting outwards. This module is based on two TCS (NarXL and ThsSR) and an enzyme (ScGS). Among them, NarXL is an endogenous two-component system of Escherichia coli, and ThsSR is a two-component system from marine Shewanella species. They can respectively sense the concentration of nitrate and thiosulfate to affect the downstream loop. ScGS can help E.coli synthesize geosmin, which is an odorous substance with a very low odor threshold for humans.
Studies have found that when IBD occurs, the concentration of nitrate and thiosulfate in the intestine will increase significantly. Therefore, we choose nitrate and thiosulfate as the biomarkers for the occurrence of IBD. When the concentration of nitrate or thiosulfate increases, we think it is a normal fluctuation of the concentration of the substance in the intestine, and no geosmin will be produced to report. Only when the concentrations of both increase at the same time, we think that IBD has already occurred in the intestine. At the same time, the "Report" module will be activated to inform the pet owner through the change of smell and the "Anti-inflammatory" module successively begin to relieve inflammation. After the pet owner discovers the smell, he will take the pet to hospital for further treatment.
When the disease of IBD is detected by our engineered bacteria, the "Anti-inflammatory" module will be activated after the "Report" module. This module can alleviate intestinal inflammation by producing a small peptide LL37-LTA, which can kill some bacteria that can cause inflammation.
This module is always activated. When IBD occurs, the azurin produce by this module can prevent the further deterioration of inflammation, and it can also stabilize the p53 protein, which is a protein expressed by tumor suppressor genes, to produce a certain anti-cancer effect.
In order to prevent the engineered bacteria from leaking into the environment, we add a “Suicide” system for the engineered bacteria. If the engineered bacteria enter the external environment, the “Suicide” system will be activated.
Since our probiotics will produce geosmin to report the occurrence of pet IBD to users, we need to use an odor sensor to receive this signal. In order to prevent the loss of the odor, we designed a hardware “Box”, which can show the concentration of geosmin on its screen after receiving the signal. Moreover, the box can also degrade geosmin.
In order to allow our users to know the long-term health of the pet's intestines, we provide a simple APP to achieve this function. In addition，we link the APP and the hardware through the Bluetooth module, so that users can operate our box more conveniently.