The Repressilator

To build their clock they began with three different repressors and their corresponding promoters. The repressor proteins were TetR, LacI, and λ cI, and their cognate promoters were, respectfully, pTet, pLac, and λP_R. So, pTet is repressed by TetR, pLac (from the lac operon) is repressed by LacI, and λP_R is repressed by cI. All three promoters are on by default; it is only when their repressor is present that they are shut off.

They arranged these components in a circular negative feedback loop on the repressilator plasmid (see diagram at right). They also build a second plasmid containing GFP regulated by pTet (also shown at right). Let me walk you through a cycle of this clever system. First, say that the cell contains TetR cI. Then the only active promoter will be pLac, which causes the production of only TetR. Since no cI is being produced, its concentration will eventually drop enough that the λP_R promoter is activated (clockwise from pLac). This promoter ramps up production of LacI, which shuts down pLac and the production of TetR. Now, as the levels of TetR go down, pTet is activated, which begins to produce cI. This cI shuts down λP_R and LacI production. As the level of LacI falls, pLac is activated once again and the cycle continues.

To summarize, the repressilator creates oscillations in the levels of the three repressors, but those levels are too small to be directly detectable. So Elowitz and Leibler relied on their reporter plasmid containing GFP regulated by pTet. At points in the cycle where TetR concentrations are very low, GFP on the reporter plasmid is expressed, and the E. coli cells begin to glow green. When pLac is active, TetR is produced and represses the pTet that controls the production of GFP. Since GFP has a particular half-life (it does not remain in the cell forever but degrades over time), the intensity of fluorescence will fall.

There are several interesting side notes about this elegant yet simple experiment. First, the period of the GFP oscillations was roughly 150 minutes, which is about three times as long as the length of one E. coli generation under the conditions of the experiment. However, the two offspring cells (E. coli reproduces by splitting in two) lost their synchronization after a little over one generation time. So it can be said that Elowitz and Leibler observed a lot of noise in their system. Random variation in the levels of repressors and mRNA, as well as other factors, caused the repressilator to behave somewhat unpredictably.

All in all, these authors were able to produce an oscillating biological clock using the tools of synthetic biology. The clock wasn't perfect as it lost synchronization with each new generation, and it doesn't allow for an entire culture of bacteria to blink in sync with one another. However it is remarkable that this novel system worked as well as it did. And did you notice how abstraction came in to the picture? The promoters and repressor genes are parts, each promoter-repressor pair is a device, and the entire clock/reporter can be thought of as a system. By incorporating more specific control elements, a redesign of the repressilator might reach (or more optimistically, surpass) the fidelity of the biological clocks that natural selection has produced.

Image Credit: Personal image; Timreid (via Wikimedia)

References and Further Reading:

Elowitz, M. B. & Leibler, S. A Synthetic Oscillatory Network of Transcriptional Regulators. Nature 403, 335-338 (2000).