Protocols for in vitro reconstitution of the cyanobacterial circadian clock

Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24-h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.