Spatiotemporal control of gene expression with pulse-generating networks

Abstract

One of the important challenges in the emerging field of synthetic biology is designing artificial networks that achieve coordinated behavior in cell communities. Here we present a synthetic multicellular bacterial system where receiver cells exhibit transient gene expression in response to a long-lasting signal from neighboring sender cells. The engineered sender cells synthesize an inducer, an acyl-homoserine lactone (AHL), which freely diffuses to spatially proximate receiver cells. The receiver cells contain a pulse-generator circuit that incorporates a feed-forward regulatory motif. The circuit responds to a long-lasting increase in the level of AHL by transiently activating, and then repressing, the expression of a GFP. Based on simulation models, we engineered variants of the pulse-generator circuit that exhibit different quantitative responses such as increased duration and intensity of the pulse. As shown by our models and experiments, the maximum amplitude and timing of the pulse depend not only on the final inducer concentration, but also on its rate of increase. The ability to differentiate between various rates of increase in inducer concentrations affords the system a unique spatiotemporal behavior for cells grown on solid media. Specifically, receiver cells can respond to communication from nearby sender cells while completely ignoring communication from senders cells further away, despite the fact that AHL concentrations eventually reach high levels everywhere. Because of the resemblance to naturally occurring feed-forward motifs, the pulse generator can serve as a model to improve our understanding of such systems.

Fig. 1.

Engineered sender cells are instructed to communicate a signal to the pulse-generating cells, which respond with transient expression of a fluorescent protein. TetR, I, R, CI, and GFP represent the protein products of the tetR, luxI, luxR, cI^*, and gfp^* genes, respectively, where ^* denotes a destabilized version of the protein. (A) Initially, no communication is taking place between the sender and pulse-generating cells. (B) Addition of anhydrotetracycline (aTc) instructs the sender cells to transmit the AHL signal to the pulse-generating cells, which in turn respond by expressing GFP and CI. (C) Continuous transmission of the AHL signal ultimately results in CI concentrations above the threshold required to repress GFP. The fluorescence disappears as GFP decays quickly.

Fig. 2.

Forward-engineering of pulse behavior. (a) Simulated contour map showing how the pulse gain changes with variations in CI RBS efficiency and repressor/operator affinity. (b) Experimental results of the circuit library showing median fluorescence-activated cell sorting fluorescence values of GFP measured every 2.5 to 5 min of different pulse-generator circuits in response to 140 nM AHL (Inset demonstrates the ability to completely regenerate the pulse a second time). (c) The refractory period of a pulse-generator circuit (RBS H, CI O_R1 mut4), as described in the text. The figure indicates average, low, and high fluorescence values for triplicate experiments.

Fig. 3.

The response of the pulse to different AHL concentrations and rates of increase. (a) Pulses resulting from different concentrations of AHL. The concentration of AHL was constant throughout the experiment. (b) Simulations graphing the time-series response to five different rates of AHL increase (all have a final AHL concentration of 50 nM). (c) Experimental results showing the median GFP fluorescence of separate cultures assayed by fluorescence-activated cell sorting with five different rates of AHL increase (all have a final concentration of 47 nM).

Fig. 4.

Spatiotemporal behavior of the circuit. Time-series fluorescence and phase images of pulse-generating cells on an M9 agar slide at two different distances from the senders. Positions 1 and 2 are 2.5 and 3 mm away from the senders, respectively. These images are a cropped portion of the field of view in each position. For the cells shown in the cropped images, positions 1 and 2 cells achieved their highest average fluorescence at 32 and 36 min, respectively.

Fig. 5.

Experimental and simulated spatiotemporal behavior of the pulse generator. Shown are contour maps of average fluorescence and simulated GFP concentrations at different distances from the senders over time. (a) Cells were tracked every 4 min in four different distances away from the senders (2.5, 3, 3.5, and 4 mm) for 130 min. The fluorescence observations (arbitrary units) at each position were fit with a second-order Gaussian curve and then used to compute the map with matlab contour function. Inset shows the time-series response at the first three positions. (b) A simulation of the spatiotemporal behavior of the pulse with cells placed on a 100 × 16-μm grid. A single sender cell was placed in the middle of the grid, and 50 receiver cells were placed linearly away from the sender. The simulation contour map shows the change in GFP levels (in μM) over time in the 20 receiver cells closest to the sender. The bold black lines in both figures connect the points of maximum amplitudes for the particular positions.