Building a Synthetic Transcriptional Oscillator

Abstract

Reaction circuits mimicking genetic oscillators can be realized with synthetic, switchable DNA genes (so-called genelets), and two enzymes only, an RNA polymerase and a ribonuclease. The oscillatory behavior of the genelets is driven by the periodic production and degradation of RNA effector molecules. Here, we describe the preparation, assembly, and testing of a synthetic, transcriptional two-node negative-feedback oscillator, whose dynamics can be followed in real-time by fluorescence read-out.

Autonomous dynamic control of DNA nanostructure self-assembly

“…The oscillator was tuned to operate in a regime of high RNase H concentration, because RNase H is needed to promote nanotube regrowth by degrading RNA invader bound to the tiles. Obtaining large amplitude oscillations in this regime requires an increase in RNAP concentration [22]. Amplitude is defined as the peak-well difference in T21 fluorescence, corresponding to the on-off state of switch S21; this amplitude is directly proportional to the on-off ratio of the insulator as well, which is has the same activation/inhibition domains of T21.…”

“…Amplitude and frequency in this molecular oscillator cannot be independently tuned [22]: large amplitude correlates with a large (slow) oscillation period. In addition, there is significant variability in the achievable oscillator behavior depending on the lot of RNAP and RNase H. As a result of these two challenges, our experiments focus on only two operating points: in the first we obtained a single, slow oscillation with large amplitude followed by a slow drift to a steady state; in the second, we tuned the circuit to exhibit two faster oscillations.…”

From electric circuits to chemical networks

“…Inspecting (9), the chemical species v ± out and i ± are involved in autocatalytic cycles. (9,10). A ball-headed arc from x to y denotes a reaction x → x + y, and a double-tailed arc from x and y denotes a reaction x + y → ∅.…”

“…From a systems biology point of view, we take it as given that an abstract CRN can be turned into a collection of molecules. In fact, multiple target architectures are possible, from short oligonucleotides in solution [8] to gene networks [10]. The ability to generate molecular configurations from a vast existing library of (electric, or other) circuits is appealing for systematizing the generations of synthetic organisms.…”

Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

“…In vitro regulatory models can be stripped of cellular machinery for protein translation and may use nucleic acid-based programs to design biochemical networks. In this section we will discuss simplified in vitro synthetic regulatory models using the synthetic transcription-based genelet system as an example [37].…”