Assisted Living Home Care

Assisted Living Forest

Implementation

Overview：

This year NWU-CHINA-A team is committed to making a non-pathogenic, edible probiotic that can act on intestinal bleeding spots. When the tested person with intestinal micro-bleeding takes the probiotic, it will be activated by heme in the intestinal tract and produce color. We can isolate and cultivate this probiotic from feces, and visualize the micro-bleeding by detecting the expression of pigment protein. At the same time, we have designed a simple filter culture device that can be used at home (non-laboratory environment), which allows users to quickly and efficiently judge whether their intestinal tract has micro-bleeding.

Proposed End Users:

Some intestinal diseases are accompanied by micro-bleeding in the early stage, but the symptoms of bleeding are mild and difficult to detect, and they are often found only when the condition is serious. The probiotics of our project aims to make most or even all patients detect intestinal micro-bleeding at home, so that some intestinal diseases can be found in time at an early stage and treated by professional doctors as soon as possible.

In China, many poor areas have no basic medical facilities, are almost isolated from the outside world, and the traffic is very inconvenient. Many people live in the mountains, and their lives are very primitive and difficult, so it is difficult for people to go to regular hospitals for corresponding check-ups. Moreover, the local residents lack basic medical knowledge and do not pay enough attention to their own health. Often, when diseases are discovered, they have turned into severe cases. Therefore, a convenient and cheap detection device is particularly important.

Based on the consideration of cost and accuracy, this year NWU-CHINA-A team designed a simple isolation and culture device, which can detect intestinal micro-bleeding under non-experimental conditions. Compared with testing in hospitals, the device designed and manufactured by us is cheap, easy to operate, and can be used at home, which saves more time, labor and expenses. Therefore, it is expected to be popularized in poverty-stricken areas and remote mountainous areas, providing more convenience for people in poverty-stricken areas to monitor their health.

Safety and other considerations：

Considering the leakage of the strain, we chose to use the suicide switch that had been verified last year, and inserted two light-induced dimerization domains, nMag and pMag, into different domains of Cas9. They will form a dimer under the activation of blue light, and form a complete Cas9. At this time, Cas9 recovered its nuclease activity and could cut the target gene fragment under the guidance of gRNA.（ Click this link to view related content. Team:NWU-CHINA-A/Implementation - 2021.igem.org ）

 

At the same time, we also considered some other factors. Since the advent of genetically modified products, people's acceptance of them is low. Our probiotic products are considered as genetically modified organisms because of some special changes in the genome. But in recent years, we know that people's resistance to these products has dropped significantly, which provides an opportunity for the successful use of probiotics designed by us.

In addition, we need to measure the feasibility and significance of probiotic diagnosis. Through the follow-up work, we will continue to understand whether our probiotics are safe and harmless, can effectively detect micro-bleeding, and have enough stability, specificity and sensitivity to produce reliable results.

Products for market：

At present, the main diagnostic methods for intestinal micro-hemorrhage are fecal occult blood test and colonoscopy. We compared our project products with the current diagnostic technology, as follows.

Although the fecal occult blood test is low in cost, patients are required to refrain from eating meat, foods containing animal blood and some vegetables which contain iron and vitamin C three days before the test. Not only the error is large, but also there are many influencing factors. False positive results are likely to occur, and the accuracy of detection is low, which is not conducive to follow-up treatment. Moreover, fecal occult blood test generally requires that the content of heme in feces is greater than 6g/ml before it can be detected, and the theoretical minimum heme concentration that our project product can detect is 2μM, which greatly improves the sensitivity of detection;
In China, the cost of a common colonoscopy is between 350-400 RMB (49.18-56.20 $), and that of painless colonoscopy is around 1000 RMB (140.50 $). For people in poor areas, colonoscopy is not only expensive, but also causes psychological pressure on patients, making them feel unwell, and at the same time, it has certain safety risks. Once intestinal perforation occurs, it will cause great damage to the body. At present, the product cost of our project is only about 10 RMB (2.81$), which will be even lower if it is produced in batches. Besides, our probiotics can also regulate the stability of intestinal tract. Therefore, our project products have great advantages in the market.

Future：

Due to COVID-19 epidemic situation in China, our experimental conditions have been limited, and the expected experimental time has been greatly shortened. As a result, the plasmid used in our current experimental stage is still expressed in E.coli BL21 (DE3), which has not yet been expressed in E.coli Nissle 1917. However, at present, we have prepared all the materials needed for transformation to E.coli Nissle 1917, and we will continue our experiment afterwards.

We will improve our project in the following aspects:

1.  Optimize the heme sensing system: We will change promoters with different strengths according to experimental data, and regulate the expression level of pigment protein to achieve the best expression ratio and accuracy.

2.  Try other promoters that can be regulated by heme: for example, HmuO promoter regulated by heme can be used to regulate the expression of pigment protein.

3.  After the transformation into E.coli Nissle 1917 is officially completed, we will conduct in vivo experiments to verify the accuracy of the system and collect data for further correction: our project this year verified and collected data through simulated feces experiments, which makes our results unconvincing, so if conditions permit, we will conduct in vivo experiments in subsequent experiments. At the same time, we will pay attention to all the security and privacy issues involved, and strictly abide by ethics.

4.  Continue to monitor whether our probiotics are safe and harmless, have sufficient stability and high specificity, and can effectively detect micro-bleeding by constantly changing experimental variables.

Quote:

1.  Engineering Bacteria to Monitor the Bleeding of Animals Using Far-Red Fluorescence ， Zi-Zhu Tan, Xiao-Dan Li, Chao-Di Kong, Na Sha, Ya-Nan Hou, and Kai-Hong Zhao ACS Sensors 2021 6 (5), 1770-1778 DOI: 10.1021/acssensors.0c02482

2.  Structures of the Substrate-free and Product-bound Forms of HmuO, a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION， MasakiUnno AlbertArdèvol CarmeRovira MasaoIkeda-Saito ， Journal of Biological Chemistry, Volume 288, Issue 48,2013,Pages 34443-34458,ISSN 0021-9258,

3.  Mimee M, Nadeau P, Hayward A, Carim S, Flanagan S, Jerger L, Collins J, McDonnell S, Swartwout R, Citorik RJ, Bulović V, Langer R, Traverso G, Chandrakasan AP, Lu TK. An ingestible bacterial-electronic system to monitor gastrointestinal health. Science. 2018 May 25;360 (6391):915-918. doi: 10.1126/science.aas9315. PMID: 29798884; PMCID: PMC6430580.