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. Furthermore, the incidence among children and teenagers is relatively 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 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, have less side-effects, and are more amenable for improvement.
Biological medicines can be useful treatments for a wide range of diseases, including cancer, diseases of the immune system and so on. However, there are also 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 many patients.
Nevertheless, there are a number of ways to address these problems. For example, increasing production yields in bioreactors by engineering microorganisms, or reducing the time and cost of production in mammalian cells. What’s more, microorganisms have a high cloning capacity, and there are a number of efficient screening methods for microbial libraries which have enabled great progress in biological engineering.
Abstract: Noninvasive gut inflammation detector
We
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
Because of thiosulfate and tetrathionate are indicators of gut inflammation (Levitt et al, 1999; Winter et al, 2010;Jackson et al, 2012; Vitvitsky et al, 2015), this system can be used as sensors of gut inflammation. 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 gut inflammation. Thus, we chose these two molecules as indicators for the detection of gut inflammation using engineered two-component systems.
Schematic diagram of ligand-induced signaling through ThsS/R, and plasmid design of the aTc- and IPTG-inducible sensor components.
Schematic of tetrathionate-induced activation and plasmid design of the aTc- and IPTG-inducible TtrSR components.
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 gut inflammation used sfGFP to show the result (引文), but 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 ___引用2处内容：2012uppsala大学的项目（季），chromoprotein的文章（王宣） to change its color (even in an anaerobic environment). Furthermore, to enable the result to be observed more clearly and easily, we planned to produce a pill in which the modified E. coli is stored, with special walls that would only allow small molecules (like thiosulfate and tetrathionate) to pass through __补充细节，李帛轩. Furthermore, 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. One special reason for choosing it is that this compound can cure or slow down the inflammation to some degree (引文).
The two-component system detector used in our project has the potential to be further modified to produce additional treatments upon detecting inflammation. We also came up with a solution to reduce potential risks in practical treatments. For example, we have discussed creating kill switches, using DNase to destroy the engineered gene to prevent its spread into the normal gut flora.
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. Available at: http://msb.embopress.org/content/13/4/923
Ebba Perman, Josefin Ågren, Delyan Georgiev, Adam Engberg, Fredrik Lindeberg, Konrad Gras, Cecilia Ålander, Adrian Silberman, Dianna Zeleskov, Annie Herbertsson, Joel Striem, Anna Boström, Julia Lundgren, Thomas Andersson, Linnea Westberg, Kira Karlsson, Hanna Eriksson, Dmitry Laso, Björn Greijer (2015) Decyclifier: the one path way to rule them all (Uppsala), iGEM. Available at: https://2015.igem.org/Team:Uppsala/Composite_Part
Liana Verinaud, Stefanie Costa Pinto Lopes, Isabel Cristina Narajo Prado, Fábio Zanucoli, Thiao Alves da Costa, Rosária Di Gangi, Luidy Kazuo Issayama, Ana Carolina Carvalho, Amanda Pires Bonfanti, Guilherme Francio Niederauer, Nelson Duran, Fábio Trindade Marahão Costa, Alexandre Leite Rodrigues Oliveira, Maria Alice da Curz Höfling, Dagmar Ruth Stach Machado, Rodolfo Tomé (2015), Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells, PLOS one Tenth Anniversary. Available at: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125409
