Programmable and printable Bacillus subtilis biofilms as engineered living materials

Abstract

Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

Physical confinement impacts cellular phenotype within living materials

“…To address this question, we performed a computational analysis on the acquired SEM images as described in the materials and methods section. During this analysis, cell size (in µm 3 ) parameter was utilized to investigate the effects of physical confinement on the cells in hydrogels in comparison to suspension cells. We evaluated cell size differences after 48 h in the aforementioned samples (used in Figure 4).…”

“…1 Self-assembling block copolymer hydrogels have been demonstrated for extrusion-based 3D printing, and offer exciting opportunities to create synthetic polymer hydrogel networks that can immobilize microbial cells and recapitulate the environment of a biofilm. 2,3 These microbe-laden hydrogels form living materials (LMs) that are permissive for metabolic activity and can provide significant improvement with respect to robustness, reproducibility, and scale-up over traditional immobilization methods using natural biopolymers. 4 The multiscale properties of hydrogels of such polymers allow their applications in diverse fields, such as drug delivery 5 , tissue engineering 6 , and biotechnology 4,7 .…”

Resilient Living Materials Built By Printing Bacterial Spores

“…Studart and co-workers designed a bioink based on a shear-thinning hydrogel (hyaluronic acid, k-carrageenan, and fumed silica) that was shown to be able to print Bacillus subtilis, Pseudomonas putida and Acetobacter xylinum 40 . A bioink based on B. subtilis has also been developed that utilizes its natural biofilm amyloids fibers to produce 2D shapes that can survive at 4 o C for 5 weeks 41 .…”

Engineered Living Materials based on Adhesin-mediated Trapping of Programmable Cells

“…Programmable interactions between GMMs and their surroundings have recently gained interest as the design of multicellular structures such as engineered biofilms enables a higher control over shape and functionality of ELMs [12,13]. Inducible secretion of fibrous proteins drives the emergence of molecular architectures supporting the artificial biofilms.…”

“…Hybrid micro-patterned devices combining layers of elastomer and microporous hydrogel enabled the exchange of information with surrounding environment via diffusion of chemical inducers and their sensing by GMMs while displaying high mechanical resilience [10]. Nevertheless, the low porosity of the physical barriers mitigating bacterial escape also significantly hampers the diffusion of macromolecules, reducing the repertoire of synthetic biology applications to biosensing and release of small therapeutic molecules.Programmable interactions between GMMs and their surroundings have recently gained interest as the design of multicellular structures such as engineered biofilms enables a higher control over shape and functionality of ELMs [12,13]. Inducible secretion of fibrous proteins drives the emergence of molecular architectures supporting the artificial biofilms.…”