Programmable microbial ink for 3D printing of living materials produced from genetically engineered protein nanofibers

Abstract

Living cells have the capability to synthesize molecular components and precisely assemble them from the nanoscale to build macroscopic living functional architectures under ambient conditions. The emerging field of living materials has leveraged microbial engineering to produce materials for various applications but building 3D structures in arbitrary patterns and shapes has been a major challenge. Here we set out to develop a bioink, termed as "microbial ink" that is produced entirely from genetically engineered microbial cells, programmed to perform a bottom-up, hierarchical self-assembly of protein monomers into nanofibers, and further into nanofiber networks that comprise extrudable hydrogels. We further demonstrate the 3D printing of functional living materials by embedding programmed Escherichia coli (E. coli) cells and nanofibers into microbial ink, which can sequester toxic moieties, release biologics, and regulate its own cell growth through the chemical induction of rationally designed genetic circuits. In this work, we present the advanced capabilities of nanobiotechnology and living materials technology to 3D-print functional living architectures.

Fig. 1. Schematics of the design strategy, production, and functional applications of microbial ink.

a E. coli was genetically engineered to produce microbial ink by fusing α (knob) and γ (hole) protein domains, derived from fibrin to the main structural component of curli nanofibers, CsgA. Upon secretion, the CsgA-α and CsgA-γ monomers self-assemble into nanofibers crosslinked by the knob-hole binding interaction. b The knob and hole domains are derived from fibrin, where they play a key role in supramolecular polymerization during blood clot formation. c The protocol to produce microbial ink from the engineered protein nanofibers involves standard bacterial culture, limited processing steps, and no addition of exogenous polymers. Microbial ink was 3D printed to obtain functional living materials.

Fig. 2. Optical and electron microscopy images of functional curli nanofibers and the corresponding hydrogels.

a Transmission electron microscope (TEM) images of self-assembled nanofibers of CsgA-α, CsgA-γ and CsgA-αγ (co-culture of CsgA-α and CsgA-γ) after recombinant expression. Representative images from three independent samples were reported. b Optical images of CsgA-α, CsgA-γ and microbial ink CsgA-αγ hydrogels with the corresponding field-emission scanning electron microscope (FESEM) images show the presence of aligned microscopic fiber bundles. Representative images from three independent samples were reported.

Fig. 3. Rheological properties and 3D printing of CsgA-α, CsgA-γ and microbial ink CsgA-αγ.

The storage modulus (G’) and loss modulus (G”) under frequency sweep (a) and oscillatory sweep (b). c Viscosity as a function of shear rate, d Shear modulus (n = 3) **p = 0.0014, ***p = 0.0004 and e yield stress (n = 3) **p = 0.0061, **p = 0.003, one-way ANOVA followed by Dunnett’s test. f Printed line diameter as a function of feed rates ranging from 2 to 10 mm s⁻¹ at 20 psi pressure (n > 10). g Images of filament collapse test and h. the plot of deflection angle versus pillar gap distances (n = 3). Experimental data: solid line, theoretically predicted data: dotted line. Data represented as mean ± standard deviation. **p ≤ 0.01, ***p ≤ 0.001, one-way ANOVA followed by Dunnett’s test. 3D printed structures using the microbial ink CsgA-αγ i single-layer grid, j 10-layer square, k 10-layer circle, and l 21-layer solid cone. Insets in (j–l). are corresponding top views. Scale bar 1 mm.

Fig. 4. 3D printing of functional living materials.

a Genetic design of E. coli (PQN4-Azu) cells, programmed to secrete an anticancer biologic drug azurin along with image of printed living material (top), and the incorporation of PQN4-Azu cells into the CsgA-αγ microbial ink (middle). Western blot (bottom) shows the difference in azurin detected in the supernatant of the printed structure with and without IPTG induction. b Genetic design of E. coli (PQN4-BPA), programmed to produce extracellular fibers displaying BPA-binding peptide (CsgA-BPABP) along with image of printed living material (top), and incorporation of CsgA-BPABP biofilm into microbial ink (middle). After 12 and 24 h, the BPA concentration (bottom) in the supernatant of the printed structure was analyzed by LCMS. Dotted line represents the initial BPA concentration of 1 mM. n = 3. c Genetic design of E. coli (PQN4-MazF) cells, programmed to express an endoribonuclease MazF, that inhibits/arrests cell growth along with image of printed living material (top), and incorporation of PQN4-MazF cells into microbial ink (middle). CFU count from printed structure over time, with and without IPTG induction (bottom). n = 3. Scale bar 5 mm. Data represented as mean ±  standard deviation.