A sensing array of radically coupled genetic 'biopixels'

Abstract

Although there has been considerable progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a noisy cellular environment. Such an environment leads to considerable intercellular variability in circuit behaviour, which can hinder functionality at the colony level. Here we engineer the synchronization of thousands of oscillating colony 'biopixels' over centimetre-length scales through the use of synergistic intercellular coupling involving quorum sensing within a colony and gas-phase redox signalling between colonies. We use this platform to construct a liquid crystal display (LCD)-like macroscopic clock that can be used to sense arsenic via modulation of the oscillatory period. Given the repertoire of sensing capabilities of bacteria such as Escherichia coli, the ability to coordinate their behaviour over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field.

Fig. 1

Sensing array of radically coupled genetic biopixels. (a) Network diagram. The luxI promoter drives expression of luxI, aiiA, ndh, and sfGFP in four identical transcription modules. The quorum-sensing genes luxI and aiiA generate synchronized oscillations within a colony via AHL. The ndh gene codes for NDH-2, an enzyme that generates H₂O₂ vapor which is an additional activator of the luxI promoter. H₂O₂ is capable of migrating between colonies and synchronizing them. (b) Conceptual design of the sensing array. AHL diffuses within colonies while H₂O₂ migrates between adjacent colonies through the PDMS. Arsenite-containing media is passed in through the parallel feeding channels. (c) Fluorescent image of an array of 500 E. coli biopixels containing about 2.5 million cells. Inset: brightfield and fluorescent images display a biopixel of 5,000 cells. (d) Heatmap and trajectories depicting time-lapse output of 500 individual biopixels undergoing rapid synchronization. Sampling time is 2 minutes.

Fig. 2

Frequency modulated genetic biosensor. (a) Network diagrams depicting two constructed sensing modules. In thresholding (1), the luxR gene is removed from the oscillator network and supplemented by a new copy driven by an arsenic-responsive promoter. In period modulation (2), a supplemental luxI gene tagged for increased degradation is driven by the arsenic-responsive promoter which affects the period of oscillation. (b) A sample period modulation sensor output following a step increase of 0.8 μM arsenite. Oscillatory period increases from 69 minutes to 79 minutes. (c) (Top) Period versus arsenite concentration for the sensor array. Error bars indicate ± 1 standard deviation averaged over 500 biopixel trajectories. Dotted line represents model-predicted curve. (Bottom) Sensor calibration curve generated from experimental data. Points indicate the maximum arsenite level with 95% certainty for a given measured period as determined statistically from experimental data. (d) Thresholder output following a step increase of 0.25 μM arsenite. A dramatic shift from rest to oscillatory behavior is observed within 20 minutes following the addition of arsenite.

Fig. 3

Computational modeling of radical synchronization and biosensing. (a) Time series of a population of biopixels producing varying amounts of H₂O₂ vapor. Synchronization occurs only for moderate levels while high levels lock ON and low levels oscillate asynchronously. (b) A typical time series for our period modulation sensor undergoing a step increase of arsenite. Oscillations increase in both amplitude and period. (c) A typical time series output for the thresholding sensor. Oscillations arise following the addition of arsenite. (d) Experimental and computational output depicting complex dynamic behaviors between neighboring traps. (Top 2 panels) 1:2 resonance and anti phase synchronization observed when trap size (left, black/blue = 95 μm depth and red/magenta = 85 μm depth) and separation distance (right, same colors) are modified experimentally, (Middle) Scaled-up array experimental data for increased trap separation experiments demonstrating anti phase synchronization, (Bottom) Computational model trajectories depicting 1:2 resonance and anti phase synchronization when trap size (same colors as experimental data) and separation distance are changed.

Fig. 4

Radical synchronization on a macroscopic scale. (a) The scaled-up array is 24 mm × 12 mm and houses over 12,000 biopixels that contain approximately 50 million total cells when filled. (b) Global synchronization is maintained across the array. Heatmap of individual trajectories of all 12,224 oscillating biopixels. (c) Image series depicting global synchronization and oscillation for the macroscopic array. Each image is produced by stitching 72 fields of view imaged at 4X magnification. (d) Schematic diagram illustrating our design for a handheld device utilizing the sensing array. An LED (A) excites the array (B) and emitted light is collected by a photodetector (C), analyzed by an onboard processor (D), and displayed graphically (E).