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Abstract

This year, we aim to develop a modular system for signaling pathways 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 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). To modify these proteins into signaling proteins, we need both an extracellular sensor domain as well as intracellular domains which can relay the signal. On the extracellular part, we will click aptamers in dual forms, such that two different outer membrane proteins will be brought into close proximity in the presence of their ligands. For the intracellular part, we consider a number of signaling methods which respond to a change in proximity. These methods include the use of TEV Proteases and two component signaling, as well as a read out mechanism to verify that the sensors indeed come into close proximity of each other.

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 continued across the membrane by binding of the two intracellular components.

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 of diagnosing certain pathologies.