Project
Background
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-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 solutions 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.
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 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. 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 .
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
