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Description



Overview



This year, we aim to develop a modular sensor system based on the clickable outer membrane proteins (COMPs), developed by last year’s iGEM team. These COMPs contain a non-natural amino acid with an azide functional group. The team has shown that DNA functionalized with a DBCO-group could be clicked on these proteins using the SPAAC click chemistry reaction. For more information on last year’s project, we refer to their iGEM wiki page (see iGEM TU Eindhoven 2014). Instead of using COMPx, we will be working with the OmpX variant.
To modify OmpX into signaling proteins, we need both an extracellular sensory domain and an intracellular domain that can relay the signal. The extracellular part will be a pair of dual aptamers, each clicked on a different membrane protein. This results in two outer membrane proteins to be brought to close proximity if the aptamer’s ligand is present. For the intracellular part, we consider a number of signaling methods that respond to a change in proximity. As proof of concept we use a split luciferase and BRET (Bioluminescence resonance energy transfer). The split luciferase can only emit light when its two parts are connected. BRET is another promising possibilty when we use NanoLuc and mNeonGreen, since these two proteins have no affinity towards each other. Too much intracellular interaction could influence the proximity of the proteins even with the absence of the ligand. After the testing phase an intracellular TEV protease system could be used to release a transcription factor in order to activate protein transcription.

Figure 1: Schematic overview of the device: the left part depicts the situation where no ligand is bound, the middle shows thrombin bound by the aptamers and the right parts shows that the signal is transducted across the membrane by binding of the two intracellular components.


Future Applications



The device we aim to develop can have a wide range of applications, due to its inherent modularity. A particular application which we will consider during our iGEM project is the use of the system within the gastrointestinal tract. It is known that disturbances within the immune system in the intestines are associated with many different pathologies, such as Crohn’s disease and intestinal cancer. Moreover, aptamers for the signaling molecules of the immune system, cytokines, have been examined within the scientific community. In the future, we hope the system could thus be used to detect immunological disturbances within the intestines and serve as a means to diagnosing certain pathologies in early stages of the disease. The application in the gastrointestinal tract isn't limited to human disease only; Q fever, a local problem, could benefit from earlier detection with our system as well. Nowadays, Q fever is a huge problem in the area around Eindhoven and other parts of the Netherlands. Spreading of this disease from farm to farm or even from goats to humans can be prevented when detection is possible in an earlier stage. Furthermore, the system can be used to create an interactive pesticide sensor system or microfluidic device that allows testing of multiple biomarkers at once. When used on the field, the system could release pesticides only when it senses particular fungi. This can reduce the use of pesticides drastically, which will be beneficial for both nature and the economy.