Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters.

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters.