Heterologous Reconstruction of a Synthetic Bacterial Oscillator

²Wyss Institute for Biologically Inspired Engineering, Boston, MA USA

Many synthetic biological circuits have been constructed to enhance production of high value compounds via spatial organization or logic gates. However, only a limited number of temporal control systems are currently available and biologically-relevant synthetic timing modules are non-existent.

The circadian clock in cyanobacteria controls global gene expression. The phosphorylation state of KaiC oscillates, facilitated by interactions with KaiA and KaiB. These changes are thought to mediate circadian rhythms in global transcription via output factors SasA, RpaA, RpaB, and CikA; however, the exact role of these output factors is an area of active research.

In order to construct a modular synthetic circadian system and to study the native clock independent of other intrinsic factors, we heterologously expressed the cyanobacterial circadian clock genes and output factors in E. coli. We show that heterologous expression of the cyanobacterial clock proteins in

E. coli results in phosphorylation state changes of KaiC over a 24-hour period. This enabled us to systematically characterize the function of the output factors that transmit circadian information from KaiC to a transcriptional output. Furthermore, using an interaction-dependent transcriptional system with a fluorescent reporter, we show circadian oscillations in E. coli. Taken together, these results indicate that the bacterial circadian oscillator is modular and can be engineered in a heterologous organism in vivo. An engineered circadian clock not only allows us to better understand the physiology of the clock, but also can be incorporated in complex synthetic circuits that may have chronotherapeutic and industrial applications.