Genetic circuits for directed evolution of bioluminescent proteins

Abstract

Bioluminescent technologies are amongst the most commonly used tools for quantifying and visualising biological processes, and novel functions are being engineered for their application inside and outside the lab. Current methods to engineer bioluminescence-based tools rely on random or semi-random mutagenesis approaches which require intensive genotype screening, and are typically performed on expensive robotic workstations. In this project, we aimed to develop an in vivo platform to automate the screening of bioluminescent protein libraries using engineered bacteria. We repurposed a light-sensing circuit that allows individual bacteria to transduce the bioluminescence emission of an intracellular luciferase variant into expression of antibiotic resistance genes. This synthetic ability should allow bacteria expressing brightest luciferase variants to outcompete the rest of the population and become automatically selected by simply growing them under the appropriate antibiotic pressure. The current version of the genetic circuit confers modest but detectable growth and survival advantages to glowing cells; however, it requires further optimisation for robust performance.