A universal pipeline for genetically-encoded fluorescent biosensor production

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Advances in high-resolution bio-imaging have revolutionised research in the life sciences and medicine allowing the localisation and movement of proteins within living cells to be visualised and quantified. However, in addition to proteins, there are many other important biomolecules such as metabolites and signal molecules which have not proved so easy to quantify in the same way. The problem is that one has to design a fluorescent sensor protein for each biomolecule. The fluorescence properties of these proteins change when they bind the target molecule, providing a readout of its concentration in the cell. These fluorescent sensors are difficult to make, partly because they rely on identifying a suitable protein that both binds the target molecule and, upon doing so, changes shape substantially - it is this shape change that causes the detectable change in properties of a linked fluorescent protein such as green fluorescent protein (GFP).We have devised an alternative method of constructing fluorescent sensor proteins that does not require the receptor protein to undergo a large shape-change. This massively increases the range of potentially useful receptor proteins and also allows receptor proteins to be identified from antibody libraries. This proposal is to assess the new sensor design by making two test sensors, one for a peptide and one for the hormone estrogen. If successful, our design would represent a universal platform from which biosensors for any molecule could be constructed. We would make this platform available to academic researchers and anticipate that it would dramatically speed up the collecting of critically important data about small molecule dynamics in living organisms.