4-bit adhesion logic enables universal multicellular interface patterning – Nature

Plasmids and strains

Plasmids were transformed into chemically competent cells following standard protocol¹⁰. Plasmids were sourced from our earlier work¹⁰ and are deposited in Addgene (Supplementary Table S1). To better match the metabolic load between cells, all strains used here expressed at most a single adhesin. However, it is possible to use strains expressing several adhesins. For example, instead of mixing two cell types expressing Ag2 and Ag3, one could use one strain expressing both adhesins¹⁰. Doing so produces the expected logical interactions that dictate interface formation but with greater variance in growth rates.

Cell culture

The MG1655 E. coli strain obtained from E. Coli Genetic Stock Center (CGSC #6300) was used for all experiments in this study. For overnight growth, cells were shaken at 37 °C and 300 rpm with antibiotics in Luria broth (LB) media. No ATc was added for overnight growth. Cell stocks were stored in glycerol at −80 °C. For experiments, single colonies were inoculated from agar plates containing antibiotics streaked out from frozen stocks.

Soft agar

To prepare soft agar gels, 20 g l⁻¹ of LB broth powder (Affymetrix #75852) and 0.225% w/v Bacto Agar (BD 214050) was added to distilled water. This mixture was autoclaved and allowed to cool to 50 °C before adding antibiotics or ATc inducer as needed. This mixture was then pipetted into Petri dishes, at 10 ml for 10-cm dishes, 5 ml for 60-mm dishes, 2 ml for 35-mm dishes and six-well plates, and 25 ml for 15-cm dishes. The plates were covered and allowed to cool at room temperature for at least 2 h and used on the day of preparation. For confocal imaging, the soft agar percentage was increased to 0.25% w/v for greater imaging stability.

To seed cells for experiments, 1 μl of overnight culture was pipetted onto the surface of the gel without puncturing the surface using low-adhesion tips (VWR 89174-520). For interface-formation experiments, a multichannel pipette was used to ensure consistent 9-mm spacing between the colonies. For experiments needing greater patterning complexity, stencils were cut out of 4.5-mm acrylic sheets (TAP Plastics) using a laser cutter (Dremel LC40). The stencils were designed so that the pipette tips were just above the surface of the agar by using shims and varying the radii of laser-cut holes.

Soft agar plates were placed in a 37 °C incubator right side up, with the lid on and no circulating air. Cells were typically grown for 18 h on soft agar before imaging. Note that E. coli transitions to a swarming phenotype in swarming agar, resulting in greater expansion compared with the agar concentrations most commonly used in labs for various purposes. When specified in the text, cells were incubated at room temperature for 48 h, with all other conditions kept constant.

Small peptide inhibitors

Small peptide inhibitor EPEA and the scrambled PEAE were synthesized by GenScript¹⁰. Frozen aliquots were stored at −80 °C. To create gels with a specific concentration of inhibitor, aliquots of peptide were first thawed and diluted in distilled water to a 10× working concentration. Soft agar gel solutions were prepared at 1.1× concentration to account for the liquid volume in the 10× peptide solution and kept warm in a 50 °C bath before pouring.

Titrating cell seeding concentration

For experiments needing variable cell concentrations such as the delayed growth experiment, cells were grown in a shaking incubator to stationary phase overnight and diluted in LB media to the desired concentration immediately before seeding.

Mixed cell populations

For experiments that required seeding mixed populations of different types, cells were first separately grown in a shaking incubator to stationary phase. Immediately before seeding, cells were pipetted from the stationary cultures and mixed in polymerase chain reaction tubes.

Imaging

Fluorescence stereomicroscopy

Fluorescence stereomicroscopy images were obtained using a Leica M205 fluorescent microscope, with a Planapo 2.0× objective (10450030, Leica Biosystems) and DSR (10447412, Leica Biosystems), YFP (10447410, Leica Biosystems) and CFP (10447409, Leica Biosystems) filter sets. Oblique illumination was used for non-fluorescent channels.

Confocal images

Confocal images were captured using an inverted Zeiss LSM700 using 405-nm, 488-nm and 555-nm laser lines and a 63× 1.4-NA (0.19 mm FWD) Plan-Apochromat objective (44 07 62, Carl Zeiss AG). Soft agar plates were allowed to equilibrate to room temperature for at least an hour before imaging. Immediately before imaging, a no. 1.5 glass coverslip was carefully placed on the surface of the agar. The soft agar plates were then mounted upside down using a custom laser-cut stage. Images in a tiled set were taken within a few minutes, owing to viscoelastic creep over time. Confocal stacks were acquired at 2-μm slices up to a depth of approximately 20 μm, at which point soft agar undergoes displacement. Furthermore, low-viscosity immersion oil (Resolve M2000, Epredia) was used to minimize the effects of drag on agar as the stage moved around.

Epifluorescent wide-field time lapses

Epifluorescent wide-field time lapses were captured using an inverted Zeiss LSM700, with a 20× Plan-Apochromat objective (440640-9903, Carl Zeiss AG), a 1.4-MP CCD monochrome camera (Zeiss AxioCam MRm, Carl Zeiss AG) and a light-emitting diode (LED) lamp (X-Cite XYLIS, Excelitas Technologies Corp.). Plates were mounted as described above and frames were captured every 2 min.

Macroscopic images

Macroscopic images were captured using a DSLR camera (D5600, Nikon). Samples were illuminated using oblique illumination from LED ring lights. To correct for the uneven brightness inherent in oblique illumination, a Gaussian blur filter was applied to a copy of the image and this image was then subtracted from the original image using ImageJ⁴². Subsequently, image contrast was adjusted using ImageJ. These corrections were applied in an unbiased manner solely for the purpose of enhancing visually clarity, and no quantifications were made on these images. Specifically, the following panels in this paper were processed this way: Figs. 1b,c,d, 2a,e,f,g,h, 3e and 4a,b,e,f,g,h.

Time-lapse macroscopic images

Time-lapse macroscopic images were captured using a Raspberry Pi Camera V2 controlled by a Raspberry Pi Zero. The sample was placed in a small humidified chamber and an LED ring light was used for intermittent illumination. A custom Python script was written to control the Raspberry Pi Camera V2 and ring light activation. Frames were captured every 5 min.

Measuring interface profiles

To measure the fluorescence profile along an interface, the fluorescence intensity was measured in raw images along a 6-mm line aligned with the axis connecting the initial seeding points. The fluorescence intensity was binned across a width approximately 1.5 mm thick. These profiles were fit using equation (1).

Measuring interface angles

To measure the interface angles between seeds, the interface was first manually traced using ImageJ. The angle between each leg of the interface and the line connecting the two starting seeding points was calculated using linear regression in Python.

Image processing for confocal images

Confocal images were cropped to remove smearing caused by the viscoelastic response of the soft agar to stage motion during imaging. The cropped region was always the top 128 × 2,048 pixels, in an image of 2,048 × 2,048 pixels.

For pair-correlation analysis, confocal images were segmented using DeepCell⁴³. Cell positions were defined by the centroids of segmentation results. To calculate the mixed-species pair-correlation function, a custom Python script was used. Briefly, for each cell, the script counted the number of cells in a ring r + dr away. This count was normalized to the area of the ring, as well as the overall cell density. Area corrections were made for rings cut off by the image edges.

Fitting interface profiles and calculating interface widths

The transition profiles measured using fluorescent microscopy (such as in the graphs at the bottom of Fig. 2a,c, which were measured from the bottom half of the composite images on top) were fitted using the following heuristic equation:

Here n is the Hill coefficient, k_a is the x-position at half the maximum value along the transition and z weights the contribution of the Hill function. The variable a weights the contribution of the Gaussian distribution and b is the standard deviation of the distribution. y is a vertical offset for the overall curve.

The rationale for fitting the profiles to this equation is as follows: equation (1) is a sum of a Hill function and a Gaussian. The Hill function is primarily responsible for fitting the transition region, whereas the Gaussian compensates for any slope in the plateau region of the measured curve.

The width of the transition w is then defined as the distance between the two points of maximum curvature κ, calculated using:

Free-standing patterned sheets

To produce a free-standing patterned material, patterned agar was covered with 10 ml 1× PBS before a scalpel was used to cut out a rectangular section. For smaller sections, gentle agitation of the PBS by shaking allowed the cut-out patterned sheet to lift off from the Petri dish. For larger sheets, a thin object such as a scalpel or coverslip was slid under the agar to separate it from the Petri dish. Alternatively, a glass slide or coverslip was placed in the bottom of the Petri dish before pouring the agar. In this case, covering with PBS and using a scalpel to cut along the edge of the glass was sufficient for the patterned sheet to lift off without any further agitation or manipulation.

Sheets up to a few centimetres in length could be transferred between dishes by pouring the PBS, and sheets up to a centimetre in length could be lifted out of the PBS on the edge of a coverslip, scalpel or tweezer.

Patterned wetting

For wetting experiments, strains were seeded and grown on soft agar at room temperature for 48 h to produce strong interfaces. Then, 1× PBS was poured at one end of the plate and allowed to flow. The shape of the PBS front demonstrates the effect of bacterial patterning on surface wetting. For droplet capture in square patterns, 1× PBS was gradually added to the square using a pipette.