A synthetic oscillatory network of transcriptional regulators

Abstract

Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems. Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function. We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components. Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks.

A generalized model of the repressilator

“…More precisely, the transcription of a certain gene is repressed by the product of the preceding gene. In their mathematical model of the repressilator, Elowitz and Leibler [12] make the additional assumption of identical genes. That is:…”

“…Remark: The mathematical models for the repressilator used in [7,8,9,12] correspond to the limit case γ = 0.…”

“…A milestone in understanding gene regulation by repression has been reached by the experimental preparation of a three membered negative feedback loop, called the "repressilator", on a plasmid by Elowitz and Leibler [12]: Expressed in E.Coli bacteria, the repressilator gave indeed rise to oscillations in living cells. From now on, the availability of an experimental system provides an excellent tool for testing predictions derived from theoretical models.…”

“…In the first system (which is essentially the mathematical model used in [7,8,9,12]) the oscillations for odd numbers of genes are periodic, arising from a stable limit cycle. In the second system (which has not been considered before) there may additionally be aperiodic oscillations resulting from an attracting heteroclinic cycle.…”

A unifying view of 21st century systems biology

“…Two publications demonstrated fundamental properties of positive and negative feedback circuits by virtue of synthetic molecular reconstruction of de novo designed and modeled wiring diagrams [20,21], while a third similar story appeared shortly thereafter [22]. A differential equation-based quantitative model of the budding yeast cell cycle control was released [23].…”