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Description

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). 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, which can only emit light when the two parts are connected. We are also looking into the use of BRET between NanoLuc and mNeonGreen, since these 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.