4-bit adhesion logic enables universal multicellular interface patterning

Abstract

Multicellular systems, from bacterial biofilms to human organs, form interfaces (or boundaries) between different cell collectives to spatially organize versatile functions^1,2. The evolution of sufficiently descriptive genetic toolkits probably triggered the explosion of complex multicellular life and patterning^3,4. Synthetic biology aims to engineer multicellular systems for practical applications and to serve as a build-to-understand methodology for natural systems^5-8. However, our ability to engineer multicellular interface patterns^2,9 is still very limited, as synthetic cell-cell adhesion toolkits and suitable patterning algorithms are underdeveloped^5,7,10-13. Here we introduce a synthetic cell-cell adhesin logic with swarming bacteria and establish the precise engineering, predictive modelling and algorithmic programming of multicellular interface patterns. We demonstrate interface generation through a swarming adhesion mechanism, quantitative control over interface geometry and adhesion-mediated analogues of developmental organizers and morphogen fields. Using tiling and four-colour-mapping concepts, we identify algorithms for creating universal target patterns. This synthetic 4-bit adhesion logic advances practical applications such as human-readable molecular diagnostics, spatial fluid control on biological surfaces and programmable self-growing materials^5-8,14. Notably, a minimal set of just four adhesins represents 4 bits of information that suffice to program universal tessellation patterns, implying a low critical threshold for the evolution and engineering of complex multicellular systems^3,5.

Fig. 1. Swarming E. coli expressing heterophilic synthetic cell–cell adhesins form programmable interfaces, which enables complex tessellation and tiling patterns at the tissue-level scale^,.

a, Schematic of the experimental procedure: overnight cultures of swarming E. coli seeded on soft agar form macroscopically visible interfaces when expressing complementary pairs from a library of heterophilic synthetic adhesins (Nb2/Ag2 and Nb3/Ag3 corresponding to yellow/blue and green/red, respectively). b, A visible interface is formed between two colonies with complementary adhesins (Supplementary Video S1). Scale bar, 5 mm. c, Six alternating colonies form a hexagonally symmetric interface pattern. Scale bar, 9 mm. d, Observations (a–c) suggest the potential to engineer more complex 2D target patterns P (top), posing the inverse design problem of finding valid seeding patterns S of cells (bottom) that have the necessary spatial organization and express suitable adhesins. Seeding positions are colour-coded by strain as in a, with mixed-colour half-circles representing homogeneous mixtures of strains. The approach to find the seeding pattern is described in the main text (see also Supplementary Text S2). e,f, Simulations (e) based on the periodically repeating seeding pattern in d quantitatively predict the experimentally observed interface patterns (f) (Supplementary Videos S2 and S3), realizing an Escher-like transmutation. Scale bars, 5 mm. Throughout this work, non-linear background subtraction was consistently applied to all non-fluorescent macroscopic images to correct for inhomogeneous illumination and improve visual contrast; this had no impact on image analysis or interpretation (see Methods).

Fig. 2. Key interface properties can be rationally controlled in quantitative agreement with a biophysical continuum model.

a, Composite images of oblique transmitted illumination microscopy and fluorescent microscopy (top) and normalized quantification of fluorescence (bottom) show sharp and gradual interfacial transitions with adhesion (left) and without adhesion (right), respectively (for fit, see equation (1)). Note that colours of fluorophores (RFP, YFP) should not be confused with colour labels (blue, yellow) for two adhesins. Scale bar, 2 mm. b, The continuum model incorporates cell density, adhesion (leading to aggregation and immobilization), (active) diffusion and logistic population growth (equation S1). c, Model simulations recapitulate experiments and interface profiles from a (Supplementary Video S4). Scale bar, 2 mm. d, Interface widths can be experimentally tuned by adjusting adhesion avidity (K) using an adhesin inhibitor (EPEA). e, Symmetric interfaces form when complementary strains have identical growth and motility properties. Scale bars, 5 mm. f, Interface angle and position can be tuned by delaying swarming initiation for one of the two colonies (experimentally, by using lower seeding density). Scale bars, 5 mm. g, Interface curvature and position can be tuned by using strains with different expansion rates (Supplementary Video S5). Scale bars, 5 mm. h, Non-point-source seeding generates interfaces consistent with the linear superposition of many point sources; for example, a line and a point generate the mathematically expected parabolic interface. Scale bars, 5 mm. i, Model and experiment agree for interface angle over a wide range of seeding densities (compare to f; see also Supplementary Fig. S2).

Fig. 3. At the microscopic intercellular level, interfaces are formed by means of a ‘swarming adhesion’ mechanism, which enables a combinatorial 4-bit cell–cell adhesion logic to control interface formation.

a, Confocal images show that adhesive cells form a porous interface with punctate cellular aggregates (left) compared with substantially smaller clusters without adhesins (middle). Multicomponent pair correlation (right) between different cell types in adhesive (black) and non-adhesive (grey) conditions. Solid lines and shaded area are mean and 1 standard deviation, n=9 experiments, dashed lines are a visual guide for g(r) = 1 and radius = 1 μm. Scale bar, 15 μm. b, Three-colour confocal image for interface region includes an extra non-adhesive strain (CFP), which is excluded from the adhesive cell clusters (RFP and YFP). c, Epifluorescent images of a three-strain experiment, with non-adhesive (CFP) and adhesive (RFP, YFP) strains (left). Fluorescent profiles of mixed culture experiment (right); colours correspond to the expressed fluorophore, black curve is fit to model as in Fig. 2a. Scale bar, 3 mm. d, 4-bit cell–cell adhesion logic: composable set based on two adhesin pairs depicting the nine elements of practical interest, that is, the ‘null’ element without adhesins, four strains with a single adhesin (‘singlets’) and four strains with two adhesins (‘doublets’). Colour-coding corresponds to adhesin identity (singlets as in Fig. 1a); not to be confused with fluorescent colours. (Note that in Fig. 1d, adhesin mixes were represented by two half-circles instead.) e, Pairwise combinations of all doublet elements follow expected interface formation logic. Scale bar, 9 mm.

Fig. 4. General interface patterning can be achieved with just four adhesins, providing an engineering platform for applications such as smart biomaterials and human-readable molecular diagnostic devices.

a, Generating regular triangular, square and hexagonal tilings requires one to two adhesin pairs. Scale bars, 3 mm (left) and 9 mm (middle and right). b, Generating Voronoi tessellation with four doublet elements is guaranteed by the four-colour theorem (Supplementary Video S8). Scale bar, 9 mm. c, Schematic of how a non-Voronoi tessellation can be realized by solving for the associated Voronoi pattern and hiding the added interfaces. d, Definition of closed, open, visible and hidden interfaces. General open interface patterns can be realized using only the four doublet elements and the null element. e, A three-step algorithm for sequentially identifying seed configurations on regular triangular lattices enables arbitrary interface patterning using only two adhesin pairs (Supplementary Video S9). Scale bar, 9 mm. f, Complex curved interface patterns generated with differential expansion rates and seeding densities (green indicates the Nb3-1 strain). Scale bar, 9 mm. g, Thin sheets with interface patterns can be cut out and handled, shown here floating in a PBS bath. Scale bar, 1 cm. h, Surface wettability of biomaterials can be patterned owing to differential hydrophilicity of interface patterns. PBS added to an interface pattern shows that the liquid surface closely tracks the patterned interface (Supplementary Fig. S29). Scale bar, 1 cm. i, Liquid droplets can be captured in regularly tiled squares (Supplementary Fig. S29). Scale bar, 5 mm. j, Demonstration of an environmentally dependent patterning and molecular diagnostic application in which a human-readable indicator (‘\’ or ‘X’) is formed, depending on whether a molecular inhibitor against the Nb2 adhesin (EPEA, Fig. 2d) is present or not. Scale bar, 2 mm. k, Implementation of the common 16-segment digital display, programmed to write ‘U of A’, demonstrates complex combinatorial patterning with human-readable output (Supplementary Video S8). Colour codes defined in Fig. 3d; for continuum simulations of b and j, see Supplementary Fig. S27. Scale bar, 9 mm.