Abstract:

Bioluminescence is a naturally occurring process that has been observed across a diverse range of organisms. This light-emitting reaction consists of the oxidation of a small molecule luciferin by a luciferase enzyme. For decades, the chemistry behind bioluminescence has been harnessed for investigating biological processes in living systems. In particular, bioluminescence enables real-time, non-invasive imaging with high spatial and temporal resolution. However, naturally existing luciferin-luciferase pairs are limited in the scope of their applications, thus necessitating further modification in the laboratory. Current protocols for developing new pairs, though, are laborious and low-throughput. The advancement of bioluminescence as a practical biotechnological tool has thus lagged behind other imaging modalities. In this work, I present several contributions toward expanding luciferase engineering methods. I describe multiple efforts towards advancing the speed and ease of discovering new bioluminescent tools. I developed a continuous directed evolution platform for generating luciferase libraries. I further build on this work by coupling continuous evolution to an optogenetic circuit. I designed a new system for enabling an alternative readout, fluorescence, for reporting on bioluminescent signal. This brighter readout increases screening throughput of luciferase-based tools by several orders of magnitude. Collectively, this work highlights advances towards expanding the bioluminescent toolkit of bright, sensitive probes for use in vitro and in vivo.