Jan 232009
ResearchBlogging.orgNumerous and diverse biological processes depend on the functioning of an internal clock. Biological timers determine your heart rate, the frequency of cell division, and the way you feel at 3 AM, among other things. Similarly, mechanical and electronic clocks serve essential functions in many kinds of man-made devices. As we begin to develop synthetic organisms for medical and industrial purposes, it will be useful for us to be able to construct timers within these micro-organisms to control their activity. In two recent papers, scientists have created molecular systems in mammalian and bacterial cells with tunable oscillation periods.

Although the methods used to construct these oscillators and the kinds of cells they were made in differ significantly, the two systems had one key similarity. Both oscillators used both a positive and a negative feedback loop. In principle, it should be possible to construct an oscillating system using only a negative feedback loop. For instance, you could have a system in which a transcriptional activator enhances the expression of a functional protein as well as that of a transcriptional repressor. As the concentration of the repressor increases, that of the activator falls, causing levels of the functional protein and the repressor to fall, allowing the concentration of the activator to rise again. By tuning the lag in this system one could in theory produce an oscillator with a range of possible frequencies. Yet many systems seem to have evolved with a positive feedback loop as well (in which the activator enhances its own expression).

This curious feature was the subject of a series of simulations reported by Tsai et al. (1) last July in Science. Their studies indicated that a system using only a negative feedback loop would produce a periodic oscillation just as expected. However, they also found that systems relying only on negative feedback were limited in that it was difficult to adjust the frequency of the oscillation without also altering its amplitude. Introducing a positive feedback loop stabilized the system so that the oscillator could be tuned to a wider range of frequencies without altering peak amplitude.

The benefits of this approach were demonstrated in data reported by Stricker et al. last November in Nature (2). They constructed a circuit that expressed green fluorescent protein in an oscillatory manner in response to stimulation by arabinose and isopropyl β-D-thiogalactopyranoside (IPTG). They created a circuit (see their figure, right) in which every component ran off a hybrid promoter that could be activated by AraC (which binds arabinose) and inhibited by LacI (which binds IPTG). Arabinose binds to and activates AraC, while IPTG binds to and inactivates the LacI repressor, with the result that all three genes are transcribed, and the cells fluoresce due to the presence of GFP. As the concentration of LacI increases, the activating power of the IPTG decreases, leading to an eventual repression of transcription and the end of fluorescence. As the LacI proteins get degraded the IPTG concentration again becomes sufficient to activate transcription, leading to a new fluorescent phase.

Stricker et al. found that they could alter the frequency and amplitude of this oscillation by altering the growth conditions of the bacteria (temperature and nutrient availability) as well as by adjusting the concentrations of the activating reagents arabinose and IPTG. By attempting to match computer models of their oscillator to the data they collected, they found that the time needed for translation, folding, and multimerization played a critical role in establishing the existence and period of the oscillation. Stricker et al. constructed an additional circuit using only negative feedback from LacI, proving that this was possible, but they found that in this case the period was not very sensitive to IPTG concentration and the oscillations were not as regular.

A similar system was constructed in Chinese hamster ovary cells by Tigges et al., who described their results recently in Nature (3). The circuit they constructed used tetracycline (TC) and pristinamycin I (PI) as activating molecules. The tetracycline-dependent transactivator (tTA) served as the positive feedback lood, activating transcription of itself, GFP, and the pristinamycin-dependent transactivator (PIT). In this system, increased levels of PIT cause the production of antisense RNA to tTA, causing its mRNA to be destroyed prior to protein production. This, in turn, diminishes production of all proteins until the reduced levels of PIT allow tTA to again activate transcription. They found that they could control the period of oscillation by altering the gene dosage (i.e. the quantity of DNA used to transfect the cells).

The oscillating systems constructed in these papers serve more as test cases and examinations of principles than as functional pieces of synthetic systems. You will not be using an E. coli alarm clock any time soon. However, it has always been true that you learn more from trying to build something than from trying to tear it apart. These attempts to construct artificial periodic oscillators have provided interesting insights into those that have evolved naturally. The knowledge gained from these experiments will help us to understand oscillatory systems like the circadian rhythm and cardiac pacemaker, in addition to illuminating design principles for synthetic biology.

(1)T. Y.-C. Tsai, Y. S. Choi, W. Ma, J. R. Pomerening, C. Tang, J. E. Ferrell (2008). Robust, Tunable Biological Oscillations from Interlinked Positive and Negative Feedback Loops Science, 321 (5885), 126-129 DOI: 10.1126/science.1156951

(2)Jesse Stricker, Scott Cookson, Matthew R. Bennett, William H. Mather, Lev S. Tsimring, Jeff Hasty (2008). A fast, robust and tunable synthetic gene oscillator Nature, 456 (7221), 516-519 DOI: 10.1038/nature07389

(3)Marcel Tigges, Tatiana T. Marquez-Lago, Jörg Stelling, Martin Fussenegger (2009). A tunable synthetic mammalian oscillator Nature, 457 (7227), 309-312 DOI: 10.1038/nature07616

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