A synchronized quorum of genetic clocks

Abstract

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.

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 neighboring 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 in order 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). (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 100× zoom of cells.

Figure 2

Dynamics of the synchronized oscillator under multiple microfluidic flow conditions (Supplementary Movies 1 and 2). (a) At around 90 minutes, 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(240µm/min), blue is at higher velocity(280µm/min). (c) Period as a function of velocity in the main channel showing tunability of period between 55–90 minutes. (d) Period vs. amplitude for all experiments. Magenta circles (c,d) are data from 84 and 90µm traps, blue crosses are 100µm traps.

Figure 3

Spatiotemporal dynamics of the synchronized oscillators. (a) Snapshots of the GFP fluorescence superimposed over brightfield images of a densely packed monolayer of E. coli cells are shown at different times after loading (Supplementary Movies 3 and 4). Traveling waves emerge spontaneously in the middle of the colony and propagate outwards with the speed of ~8-35µm/min. At later times waves partially lose coherence due to inhomogeneity in cell population and intrinsic instability of wave propagation (see Modeling Box). (b) Corresponding space-time diagram showing the fluorescence of cells along the center of the trap as a function of time. (c) Snapshots of the GFP fluorescence superimposed over the brightfield images of a three-dimensional growing colony of E. coli cells at different times after loading (Supplementary Movie 5). Bursts of fluorescence begin when the growing colony reaches a critical size of about 100µm. These bursts are primarily localized at the periphery of the growing colony. (d) Corresponding space-time diagram showing fluorescence of cells along a horizontal line through the center of the growing colony.

Figure 4

Modeling of synchronized genetic clocks. (a) A typical time series of concentrations of LuxI (cyan circles), AiiA (blue circles), internal AHL (green line), and external AHL (red line). LuxI and AiiA closely track each other, and are anti-phase with the concentrations of external and internal AHL. (b) Period of oscillations as a function of the flow rate µ at cell density d = 0.5 (top panel). Period as a function of the amplitude of oscillations for the same cell density (bottom panel). (c) Period and amplitude as a function of cell density and AHL decay rate µ. Oscillations occur over a finite range of cell densities, and period increases with µ after the bifurcation line is crossed. The results in (c) and (d) compare favorably with the experimental results in Figs. 2c and 2d. (d) Speed of wave front propagation as a function of the diffusion coefficient D₁. The numerical data scale as $V~D_1^{1/2}$ (red line). (e) Space-time diagram of traveling waves propagating through a uniform array of cells corresponding to the experiment depicted in Figs. 3a and 3b. (f) Space-time diagram of bursting oscillations in a growing0020cell population corresponding to the experiments in Figs. 3c and 3d.