(61f) Engineering Genetically-Encodable Oxygen-Independent Fluorescent Reporters Using Lov Proteins

The Green Fluorescent Protein (GFP), DsRed, and their derivative fluorescent proteins have revolutionized our biochemical understanding of cellular processes over the last several decades. These fluorophores are used as platforms to build genetically-encodable sensors for a wide range of phenomena and analytes, including pH, ATP, NADH, protease activity, and gene expression. However, these fluorescent proteins require an oxygen-independent post-translational modification to develop fluorescence, rendering this family of probes ineffective in anaerobic organisms. To illuminate this anaerobic “shadow,” we have engineered light-, oxygen-, and voltage-sensing (LOV) proteins to act as anaerobically compatible fluorescent reporters. LOV proteins bind the ubiquitously available flavin as a chromophore, eliminating the need for exogenous cofactors, including oxygen. However, LOV proteins suffer poor brightness and spectral diversity, and do not have a reliable design paradigm for facile sensor construction.

Toward making LOV proteins highly useful tools for synthetic biology, we have greatly reduced the size of iLOV, eliminating 10 residues making it one of the smallest fluorescent proteins available, while retaining 86.5 ± 2.2% of wildtype iLOV fluorescence intensity. We demonstrate its ability to track localization of proteins through several cellular compartments. Further, we have created a circularly permuted version of iLOV to facilitate construction of molecular sensors.

We also present the development of a LOV-based sensor for protease activity, as well as one sensing adenosine triphosphate (ATP), demonstrating its utility to construct highly adaptable sensors. These engineering efforts are strongly facilitated by adapting sensors based on GFP, indicating a “plug-and-play” paradigm of sensor engineering.