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Project

Background

Inflammatory Bowel Disease (IBD) is a term for autoimmune diseases that are mainly divided into two types—Crohn’s disease (CD) and ulcerative colitis (UC). Crohn’s disease can affect any part of the gastrointestinal tract, especially the terminal ileum, while ulcerative colitis only occurs in the colon and rectum. However, both CD and UC can have extra-intestinal manifestations such as eye problems and arthritis. Mesalazine is an efficient treatment for UC, while antibiotics have a better effect on Crohn’s disease. IBD affects a large number of people around the world - over 4 million people in Europe as and the United States. And the incidence among children and teenagers is even higher. Additionally, IBD is more likely to occur in industrializing countries like India and China. A survey conducted by Ray, G shows that IBD is an emerging problem in India and UC is becoming more severe and widespread.

In order to treat the IBD disease, people have tried different methods. In the past, most active pharmaceutical ingredients were small molecules. However, with the development of biological technology, nowadays there are a number of medicines that are composed of biological macromolecules. Such drugs are often more specific in targeting, with less side-effects, and are more amenable for improvement.

There are still some disadvantages of biological medicines. Usually, it is hard to mass-produce them, and they usually cannot be stored for a long time. Thus, although such medicines are efficient, they often cost too much for most patients and inapplicable in real life. But there are ways to deal with it, like increasing the production to a larger quantity to make it worth be provided with special transporting methods.

Back to the research level of detecting gut inflammation. In previous researches, it is very hard to detect molecules that can indicate gut inflammation, like H2S, mainly because the environment is too complicated and it is too difficult to simulate intestinal environment. Thus, previous scientists are facing mainly two problems: first, making bacterial strains to sense easily distinguishable metabolites produced in the gut, and second, developing methods to assay reporters’ gene expressions from those strains in animals with an intact microbiota. Then, we found that thiosulfate and tetrathionate are two ideal targets in studying gut inflammation (Kristina N-M Daeffler, 2017). In order to solve the second issue, we decided to express the detection result by showing a different color. Thus, we are then able to detect and gut inflammation.

Abstract: Noninvasive gut inflammation detector

Because thiosulfate and tetrathionate are indicators of intestinal inflammation (Levitt et al, 1999), this system can be used to detect it noninvasively. Although the detailed process and reasons for their production are still unknown, it has been established that the level of thiosulfate and tetrathionate is directly proportional to the seriousness of intestinal inflammation. Until now, scientists were able to detect thiosulfate and tetrathionate using a detector based on a two-component system, which includes two parts: detector and reporter. The detector was derived from a marine Shewanella species, and previous experiments on the detection of intestinal inflammation used sfGFP to show the result. However, we thought this method can be further improved.

The SHSBNU_China team worked on changing the reporter part to display the results more clearly and visibly, not requiring specially-produced ultraviolet light or prolonged contact with oxygen. In our system, E. coli would produce a chromo-protein to change its color (even in an anaerobic environment). Furthermore, to enable the result to be observed more easily and to ensure its safety, we planned to produce a pill in which the modified E. coli is stored, with special walls that would only allow small molecules to pass through. In addition to the conventional chromo-proteins it can produce, we designed and produced an additional plasmid using violacein as the reporter. Violacein was successfully produced and the corresponding strain showed obvious purple color under anaerobic conditions. The special reason for choosing violacein is that this compound can cure or at least slow down the inflammation to some degree.

The two systems function as follows:

ThsSR system: pSB4K5-thsS+pSB1C3-thsR-sfGFP / BBa_K1033919/ BBa_K1033932/ BBa_K592009/ protoviolaceinic acid

TtrSR system: pSB4K5-ttrS+pSB1C3-ttrR-sfGFP/ BBa_K1033919/ BBa_ K1033932/ BBa_K592009/ protoviolaceinic acid


References

Álvarez, B., & Fernández, L. Á. (2017). Sustainable therapies by engineered bacteria. Microbial Biotechnology.

Levitt MD, Furne J, Springfield J, Suarez F, DeMaster E (1999) Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa. J Clin Invest 104: 1107 – 1114

Kristina N-M Daeffler, Jeffery D. Galley, Ravi U sheth, Laura C Ortiz-Velez, Christopher O Bibb, Noah F shroyer, Robert A britton, & Jefrey J Tabor (2017), Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation, EMBOpress [online]. Available at: http://msb.embopress.org/content/13/4/923 [Accessed Oct 11, 2017]

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