LiLac is a biosensor for lactate with a fluorescence-lifetime readout [1]. The fluorescent protein in LiLac is mTurquoise, a low pKa fluorescent protein commonly used in lifetime-contrast sensors. The ligand-binding protein in LiLac is the lactate-binding Cache domain of TlpC. Cache-domain-containing proteins sense extracellular nutrients to guide chemotaxis in bacteria; the N- and C- termini of the nutrient-sensing regions in Cache proteins (like the lactate-binding region of TlpC) are close in space, facilitating modular design. The combination of this modularity and the large diversity of ligands that Cache domains can sense have made this domain family an attractive starting point for building sensors. The mTurquoise is split in the middle of its seventh β-strand (β7) and then inserted the extracellular domain of TlpC at this position [2]. By engineering the connections between split mTurquoise and TlpC using a microfluidics-based screen, we developed LiLac, a lactate biosensor with a robust ~1.2 ns decrease (−35%) in fluorescence lifetime as lactate binds.
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