Programmable assembly of pressure sensors using pattern-forming bacteria

Abstract

Biological systems can generate microstructured materials that combine organic and inorganic components and possess diverse physical and chemical properties. However, these natural processes in materials fabrication are not readily programmable. Here, we use a synthetic-biology approach to assemble patterned materials. We demonstrate programmable fabrication of three-dimensional (3D) materials by printing engineered self-patterning bacteria on permeable membranes that serve as a structural scaffold. Application of gold nanoparticles to the colonies creates hybrid organic-inorganic dome structures. The dynamics of the dome structures' response to pressure is determined by their geometry (colony size, dome height, and pattern), which is easily modified by varying the properties of the membrane (e.g., pore size and hydrophobicity). We generate resettable pressure sensors that process signals in response to varying pressure intensity and duration.

Figure 1. Programmable material fabrication using engineered pattern-forming bacteria

(a) The circuit consists of T7RNAP that activates its own expression as well as the expression of LuxR and LuxI. LuxI mediates synthesis of AHL, which drives expression of T7 Lysozyme, CsgA containing a 6×-His tag, and an mCherry reporter through activation of LuxR. The circuit is turned ON by exogenous addition of IPTG. (b) Bacteria containing the curli-pattern circuit can form self-organized curli patterns in each colony, which can serve as the scaffold to assemble inorganic materials. (c) The schematic illustrates a “touch pad” that can sense and transduce local pressure variations. The dome shape represents the micro-structured material made from the colony; the orange lines represent conductive wires; and the two blue planes represent supporting surfaces.

Figure 2. Bacterial growth and pattern formation on permeable membranes

(a) The bacterial colonies were grown on permeable membranes. We loaded 0.3% molten agarin 2×YT with IPTG and appropriate antibiotics in a Culture Well multiwell chambered coverslip (Grace Bio-Labs). After the agar solidified, we placed a permeable membrane on top of the culture well and printed bacteria onto the membrane surface. The diagram is not to scale. (b) Experimentally generated dome structures on membranes with different pore sizes. Each column represents the heat map of mCherry fluorescence patterns measured by a confocal microscope after 32 hrs incubation in both vertical (y-axis) and radial (x-axis) directions. The pore size varied from 0.03 to 0.4 μm, as indicated. The contact angles of these membranes varied slightly (from left to right: 64.0°, 59.0°, 58.5°, 57.7°, 55.3°). (c) Simulated dome structures on membranes with varying pore sizes. In our model, we assume that the pore size affects the radius expansion rate v and the nutrient influx rate α₁ (Eqs. (2) and (3)), respectively. Each column represents the heat map of simulated mCherry fluorescence patterns for the varying pore sizes. (d) Experimentally generated dome structures on membranes with different contact angles. Each column represents the heat map of mCherry fluorescence patterns measured by a confocal microscope after 32 hrs incubation in both vertical (y-axis) and radial (x-axis) directions. From left to right, the membrane is PVDF, PC, MCE, NC. The pore size of each membrane is 0.45 μm. Contact angle of each membrane is 134.3°, 63.1°, 38.0°, 1.7°. The most left image is colony directly grow on 0.3% 2×YT (PH=6.5) agar. (e) Simulated dome structures on membranes with different contact angles. In our model, we assume that the contact angle affects the radius expansion rate v according to Eq (2). Each column represents the heat map of mCherry fluorescence using a simulation with different v.

Figure 3. Patterned gold nanoparticles as a resettable pressure sensor

(a) Two opposing colonies were compressed with controlled distance. The distance indicates the displacement of the presser from its starting position. The presser starts to make contact with the device when the displacement is >1 mm. The gray areas in Fig. 3a, c, and e indicate the contact time between pressing device and colony sensor. (b) Different time points are labeled in Fig. 3a. The yellow-blue dome shape represents the colony; yellow dome represents the cells within the colony with gold assembly. The inset image is a higher magnification of the yellow section at nanoscale: gold nanoparticles bind on curli. At time point 3, upon making contact, the two colonies would experience increasing pressure with an increasing displacement distance. (c) Colonies with uniform gold nanoparticles exhibited no differential response to pressure. With uniform expression of induced curli in a colony, gold nanoparticles were uniformly assembled throughout the colony, as illustrated as the yellow solid spherical cap on the right-hand side. Magenta and orange lines indicate currents from colonies grown on membranes with pore sizes of 0.03 μm and 0.1 μm, respectively. The black line indicates response of colonies of pattern-forming bacteria grown on membrane with a pore size of 0.03 μm, without assembling gold nanoparticles (illustrated as the red spherical cap on the right-hand side). The red arrows indicate the electric current pathway. (d) Intensity of electric current as a function of the pressing distance for colonies not containing dome structured gold nanoparticles. The left panel shows responses from a pair of colonies grown on a membrane with a pore size of 0.03 μm. The light magenta line indicates a varying pressing distance from 0 to 1.5 mm; the magenta line indicates a varying pressing distance from 0 to 1.8 mm. The middle panel shows responses from a pair of colonies grown on a membrane with a pore size of 0.1 μm. The light orange line indicates a varying pressing distance from 0 to 1.5 mm; the orange line indicates a varying pressing distance from 0 to 1.8 mm. The right panel shows responses of a pair colonies of pattern-forming bacteria grown on a membrane with a pore size of 0.1 μm, without assembling the gold nanoparticles. The gray line indicates a varying pressing distance from 0 to 1.5 mm; the black line indicates a varying pressing distance from 0 to 1.8 mm. (e) Colonies with the dome structure exhibited differential pressure responses. The red, blue and green solid lines indicate responses from colonies grown on membranes with pore sizes of 0.05 μm, 0.2 μm, and 0.4 μm respectively. The dashed blue line indicates replicate experiment of the solid blue line by using different electrochemical machine of the same model on a different day. The right-hand side illustrates colonies containing dome-structured gold nanoparticles. The red arrows indicate the electrons travel pathway. (f) Intensity of electric current as a function of the pressing distance for colonies containing dome structured gold nanoparticles. The left panel shows responses from a pair of colonies grown on a membrane with a pore size of 0.05 μm. The light red line indicates a varying pressing distance from 0 to 1.5 mm; the red line indicates a varying pressing distance from 0 to 1.8 mm. The middle panel shows responses from a pair of colonies grown on a membrane with a pore size of 0.2 μm. The light blue line indicates a varying pressing distance from 0 to 1.5 mm; the blue line indicates a varying pressing distance from 0 to 1.8 mm. The right panel shows responses of a pair colonies of pattern-forming bacteria grown on a membrane with a pore size of 0.4 μm. The green line indicates a varying pressing distance from 0 to 1.8 mm. Because there is no signal from the colony when the pressing distance is from 0- 1.5 mm, there is no light green line.

Figure 4. Patterned gold nanoparticles respond to pressure derivatives

(a) The pressing distance as a function of time, with the same device configuration as in Fig. 3a. (b) Distributions of gold nanoparticles corresponding to different pressure inputs. 1) the pressure is zero; 2) the pressure and its derivative are both positive; 3) the pressure is positive, but its derivative is zero. The three time points are labeled in Fig. 4A. (c) The pressure sensor responded strongly to changing pressure. The blue and green solid lines indicate current responses from colonies grown on membranes with pore sizes of 0.2 μm and 0.4 μm, respectively.

Figure 5. Robust signal processing by the bacterial pressure sensors

(a) Control of an LED light using a bacterial pressure sensor in response to manual operation. The images indicate LED light intensities when the sensor was pressed to varying degrees or released (also see Supplementary Video 1). All electronic components were from Electroninks Inc. (b) Construction of a noise filter and a signal amplifier using bacterially assembled gold domes. As in Fig. 3E, the input is the pressing distance as a function of time. Two sets of colonies were grown on the membranes with pore size of 0.2 μm and 0.05 μm, respectively. Panel i use the sets of small domes; panel ii use the sets of large domes; panels iii and iv are used the combination of the small and large sets of domes, but with different circuit design. Right of panel i and ii: the electric current readouts of these two sets of colonies being pressed separately. However, after combining the same sets of colonies with more complicated designs of the electronic circuits (panels iii and iv), substantial changes in the current signal were observed. Right panel iii: because the applied voltage polarities were opposite, the current signals from two sets of colonies canceled out. Only the current with higher amplitude was selected, therefore, the electronic circuit combined with the colonies functioned as a noise filter. Right panel iv: because the applied voltage polarities were the same, current signals from two sets of colonies summed up. Hence, the output was amplified in the form of the original input signal, and the electronic circuit functioned as a signal amplifier. The x-axes are of the same scale among i-iv panels. The units of y-axes are all in μA. To obtain reproducible results, all voltage providers used in the electronic circuits are of the same model (Keithley Series 6400 Picoammeters). The vertical alignment between the bottom of the presser and the center of the colony was carefully adjusted manually. The two pressers were controlled with the same mechanical pump to synchronize their operation.