ARTICLES A synchronized quorum of genetic clocks Tal Danino1*, Octavio Mondragon-Palomino1*, �� Lev Tsimring2 & Jeff Hasty1,2,3 The engineering of genetic circuits with predictive functionality in living cells represents a defining focus of the expanding field of synthetic biology. This focus was elegantly set in motion a decade ago with the design and construction of a genetic toggle switch and an oscillator, with subsequent highlights that have included circuits capable of pattern generation, noise shaping, edge detection and event counting. Here we describe an engineered gene network with global intercellular coupling that is capable of generating synchronized oscillations in a growing population of cells. Using microfluidic devices tailored for cellular populations at differing length scales, we investigate the collective synchronization properties along with spatiotemporal waves occurring at millimetre scales. We use computational modelling to describe quantitatively the observed dependence of the period and amplitude of the bulk oscillations on the flow rate. The synchronized genetic clock sets the stage for the use of microbes in the creation of a macroscopic biosensor with an oscillatory output. Furthermore, it provides a specific model system for the generation of a mechanistic description of emergent coordinated behaviour at the colony level. Synchronized clocks are of fundamental importance in the coordina- tion of rhythmic behaviour among individual elements in a com- munity or a large complex system. In physics and engineering, the Huygens paradigm of coupled pendulum clocks1���3 has permeated diverse areas from the development of arrays of lasers4 and super- conducting junctions5 to Global Positioning System (GPS)6 and dis- tributed sensor networks7. In biology, a vast range of intercellular coupling mechanisms lead to synchronized oscillators that govern fundamental physiological processes, such as somitogenesis, cardiac function, respiration, insulin secretion and circadian rhythms8���15. Typically, synchronization helps stabilize a desired behaviour arising from a network of intrinsically noisy and unreliable elements. Sometimes, however, the synchronization of oscillations can lead to severe malfunction of a biological system, as in epileptic seizures16. There is considerable interest in the use of synthetic biology to recreate complex cellular behaviour from the underlying biochemical reactions that govern gene regulation and signalling. Synthetic bio- logy can be broadly parsed into efforts aimed at the large-scale syn- thesis of DNA and the forward engineering of genetic circuits from known biological components. In the area of DNA synthesis, path- ways have been perturbed and replaced17 in an effort to understand the network motifs and transcriptional regulatory mechanisms that control cellular processes and elicit phenotypic responses18. On a larger scale, progress has been made towards the creation of entire genomes, providing new insights into what constitutes the minimal set of genes required for microbial life19. The genetic circuits approach to synthetic biology involves the forward engineering of relatively small gene networks using computa- tional modelling20,21. Here, the original toggle switch22 and oscillator23 have inspired the design and construction of circuits capable of con- trolling cellular population growth24, generating specific patterns25, triggering biofilm development26, shaping intracellular noise27, detecting edges in an image28, and counting discrete cellular events29. In the context of rhythmic behaviour, there have been recent successes in the construction of intracellular oscillators that mimic naturally occurring clocks30���33. As well as their potential as biological sensors, these clock networks have led to insights about the functionality of circadian networks34. A unifying theme for most of the genetic circuit studies is a particularfocus on dynamicalbehaviour. Thus, thecircuits are constructed and monitored in single cells, typically with fluor- escent reporters, and new measurement technologies are often developed in parallel35. Furthermore, because nonlinearities and sto- chasticity arise naturally, tools from the fields of nonlinear dynamics and statistical physics are extremely useful both in the generation of design specifications and for careful comparison between experiment and computational model. Synchronized genetic oscillators The synchronized oscillator design (Fig. 1a) is based on elements of the quorum sensing machineries in Vibrio fischeri and Bacillus Thurigensis. We placed the luxI (from V. fischeri), aiiA (from B. Thurigensis) and yemGFP genes under the control of three identical copiesofthe luxI promoter.The LuxI synthaseenzymaticallyproduces an acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediates intercellular coupling. It binds intracellularly to the constitutively produced LuxR, and the LuxR���AHL complex is a transcriptional activator for the luxI pro- moter36. AiiA negatively regulates the promoter by catalysing the degradationofAHL37.Thisnetworkarchitecture,wherebyanactivator activates its own protease or repressor, is similar to the motif used in other synthetic oscillator designs30���32 and forms the core regulatory module for many circadian clock networks13,38,39. Furthermore, theo- retical work has shown how the introduction of an autoinducer in similar designs can potentially lead to synchronized oscillations over a population of cells40,41. Most quorum sensing systems require a critical cell density for generation of coordinated behaviour42. We modified the local cell density of the synchronized oscillator cells (denoted TDQS1) through the use of microfluidic devices35,43 of differing geometries. The device used for monitoring the bulk oscillations consists of a main nutrient- delivery channel that feeds a rectangular trapping chamber (Fig. 1b). Once seeded, a monolayer of Escherichia coli cells grow in the chamber and cells are eventually pushed into the channel where they then flow to the waste port. This device allows for a constant supply of nutrients 1Department of Bioengineering, 2BioCircuits Institute, University of California, San Diego, La Jolla, California 92093, USA. 3Molecular Biology Section, Division of Biological Science, University of California, Mailcode 0368, La Jolla, California 92093, USA. *These authors contributed equally to this work. Vol 463| 21 January 2010|doi:10.1038/nature08753 326 Macmillan Publishers Limited. All rights reserved ��2010

or inducers and the maintenance of an exponentially growing colony of cells for more than 4 days. We found that chamber sizes of 100 3 (80���100) mm2 were ideal for monitoring the intercellular oscil- lator, as they allowed for sufficient nutrient distribution and optimal cell and AHL densities. In the context of the design parameters, the flow rate can be modulated to change the local concentration of AHL. Furthermore, the device can be modified to permit the observation of spatial waves over longer length scales. After an initial transient period, the TDQS1 cells exhibit stable synchronized oscillations that are easily discernible at the colony level (Fig. 1c, d and Supplementary Movies 1���2). The dynamics of the oscillations can be understood as follows. Because AHL is swept away by the fluid flow and is degraded by AiiA internally, a small colony of individual cells cannot produce enough inducer to activate expres- sion from the luxI promoter. However, once the population reaches a critical density, there is a ���burst��� of transcription of the luxI promo- ters, resulting in increased levels of LuxI, AiiA and green fluorescent protein (GFP). As AiiA accumulates, it begins to degrade AHL, and after a sufficient time, the promoters return to their inactivated state. The production of AiiA is then attenuated, which permits another round of AHL accumulation and another burst of the promoters. To determine how the effective AHL dissipation rate affects the period of the oscillations, we conducted a series of experiments at various channel flow rates. At high flow rate, the oscillations stabilize after an initial transient and exhibit a mean period of 90 6 6 min and mean amplitude of 54 6 6 GFP arbitrary units (Fig. 2a and Sup- plementary Movie 2). At low flow rate, we observed a period of 55 6 6 min and amplitude of 30 6 9 GFP arbitrary units. Notably, the waveforms have differing shape, with the slower oscillator reach- ing a trough near zero after activation, and the faster oscillator decay- ing to levels above the original baseline (Fig. 2b). We swept the flow rate from 180 to 296 mm min21 and observed an increasing oscilla- tory period from 52���90 min (Fig. 2c). Moreover, we found the ampli- tude to be proportional to the period of the oscillations (Fig. 2d), which is consistent with ���degrade-and-fire��� oscillations44 observed in a previously reported intracellular oscillator31. Spatiotemporal dynamics In experiments conducted at low flow rate, we observed the spatial propagation of the fluorescence signal across the 100-mm chamber. To investigate these spatiotemporal dynamics in more detail, we redesigned the microfluidic chip with an extended 2-mm trapping chamber (Supplementary Information). Snapshots of a typical experimental run are presented in Fig. 3a (Supplementary Movies 3 and 4). A few isolated colonies begin to grow and subsequently merge into a large monolayer that fills the chamber (Fig. 3a, 66 min). At 100 min, there is a localized burst of fluorescence that propagates to the left and right in subsequent frames (Fig. 3a, 100���118 min). A second burst occurs near the original location and begins to propa- gate to the left and right as before. To illustrate the spatiotemporal information contained in an entire 460-min image sequence, we plot the fluorescence intensity as a function of time and distance along the chamber (Fig. 3b). Note Figure 1 | Synchronized genetic clocks. a, Network diagram. The luxI promoter drives production of the luxI, aiiA and yemGFP genes in three identical transcriptional modules. LuxI enzymatically produces a small molecule AHL, which can diffuse outside of the cell membrane and into neighbouring cells, activating the luxI promoter. AiiA negatively regulates the circuit by acting as an effective protease for AHL. b, Microfluidic device used for maintaining E. coli at a constant density. The main channel supplies media to cells in the trapping chamber, and the flow rate can be externally controlled to change the effective degradation rate of AHL. c, Bulk fluorescence as a function of time for a typical experiment in the microfludic device. The red circles correspond to the image slices in d. a.u., arbitrary units. d, Fluorescence slices of a typical experimental run demonstrate synchronization of oscillations in a population of E. coli residing in the microfluidic device (Supplementary Movie 1). Inset in the first snapshot is a 3100 magnification of cells. 150 190 235 275 320 40 60 80 100 Period (min) Velocity (��m s���1) 15 30 45 60 40 55 70 85 100 Period (min) Amplitude (a.u.) 0 2.5 5.0 7.5 10.0 0 25 50 75 100 Time (h) GFP (a.u.) 0 1.5 3.0 4.5 6.0 0 25 50 75 100 Time (h) GFP (a.u.) a b c d Figure 2 | Dynamics of the synchronized oscillator under several microfluidic flowconditions. (See also Supplementary Movies 1 and 2.) a, At around 90 min, cells begin to oscillate synchronously after reaching a critical density in the trap. b, The period and amplitude increase for higher flow rates. Magenta curve is at low velocity (240 mm min21), blue is at higher velocity (280 mm min21). c, Period as a function of velocity in the main channel showing tunability of period between 55���90 min. d, Period versus amplitude for all experiments. Magenta circles (c, d) are data from 84 and 90 mm traps, blue crosses are 100 mm traps. Error bars in c and d indicate 61 s.d. for a single channel, averaged over 10���50 peaks each data point represents a different run. NATURE|Vol 463|21 January 2010 ARTICLES 327 Macmillan Publishers Limited. All rights reserved ��2010 Main channel Trapping chamber b c a 0 2 4 6 8 10 0 20 40 60 80 GFP (a.u.) Cell-to-cell coupling AHL LuxR���AHL luxI yemGFP aiiA 417 min 417 min 432 min 432 min 447 min 447 min 507 min 507 min 522 min 522 min 462 min 462 min 477 min 477 min 648 min 648 min 495 min 495 min 537 min 537 min 552 min 552 min 567 min 567 min 627 min 627 min 402 min 402 min �� 20 ��m 5 ��m Time (h) d