Engineering a Circadian Clock in Escherichia coli

View all posters

Anna Chen, Daniel C. Ducat, Jeffrey C. Way, Pamela A. Silver

Harvard University, United States

Synthetic biological circuits and engineered pathways can now incorporate many modules, such as logic gates and scaffolds for spatial organization. However, temporal control and time-sensing modules are still relatively under-developed. The circadian clock in the cyanobacterium, Synechococcus elongatus PCC 7942, maintains a robust day-night cycle by controlling the expression of over 50% of its genes. Consisting of just three core proteins, KaiA, KaiB, and KaiC, the circadian clock oscillates over 24 hours without a transcriptional-translational circuit. Instead, a posttranslational readout, selective phosphorylation of KaiC during the night, conveys information from the core oscillator to downstream components. We engineered the kai circadian clock heterologously in Escherichia coli. After synchronization of cultures, KaiC phosphorylation state was shown to oscillate via western blot, demonstrating functionality of the core oscillator in vivo. In order to construct a downstream transcriptional output at the single cell level, mCherry was put under the control of candidate circadian regulated promoters. Three proteins known to operate downstream of the Kai clock, SasA, RpaA and RpaB were also expressed. An orthogonal strategy utilizing a bacterial two-hybrid system, taking advantage of the known circadian-dependent binding of SasA and KaiC, is also being tested. Synchronization of the clock was achieved by either changing the ATP/ADP ratio through a minimal media starvation shock or by pulse overexpression of KaiA which promotes KaiC phosphorylation. Current progress is being made on connecting the clock with a light inducible promoter, allowing for easy synchronization. An engineered circadian clock not only allows us to better understand the physiology of the native clock in cyanobacteria, but also can be incorporated in complex, time dependent synthetic biology circuits that may have medical and industrial applications.