Engineering Bacillus subtilis for the formation of a durable living biocomposite material

Abstract

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.

A de novo matrix for macroscopic living materials from bacteria

“…We suggest that cell-matrix interactions may be essential for BUD-ELMs to reach a macroscopic size. This idea is supported by previous literature that shows that neither cell-cell adhesion alone 31 , nor sparse cell-matrix interactions in the absence of additional forces 19 lead to microscopic cell aggregates. Additionally, we have demonstrated that nucleation of a pellicle at the liquid-air interface and hydrodynamically-driven coalescence and collapse of the pellicle are required to form macroscopic ELMs.…”

“…Engineering principles to achieve this are unknown 11,12 , so most ELMs are microscopic [13][14][15][16][17] and must be processed into macroscopic materials. The few macroscopic ELMs have been created by genetically modifying existing matrices 18 or genetically manipulating mineralization of inorganic matrices 19 . However, these approaches have afforded little genetic control over the matrix composition and only ~20-30% changes in material mechanics 18,19 , which is much more limited than the tunability of naturally-occurring and chemically synthesized materials.…”

Engineered Living Hydrogels

“…Researchers also engineered B. subtilis to secrete a protein-based hydrogel matrix that allows cell attachment and silica biomineralization. [141] The microbial biofilm is a ubiquitous form of aggregated biopolymers generated by microbial cells and composed of exopolysaccharides (for example, cellulose, alginate, and hyaluronate) (Figure 8a). [1,2] However, natural biofilms are usually weak and tend to lose their structural integrity under shear loading.…”

“…Cell-generated living hydrogels are regenerative, which means, they can be reused and expanded to additional living materials. [42,141,159,160] The autonomous expansion of engineered living hydrogels indicates their potential applications in cost-effective manufacturing and scalable building materials. [160,161] Another critical capability of cell-produced materials is to repair damaged materials.…”

“…[160,161] Another critical capability of cell-produced materials is to repair damaged materials. [26,141,160,[162][163][164][165][166] The metabolites involved in microbial cells can heal cracks in matrix materials. [162] For example, cracks in concrete can be repaired when the incorporated microbial spores recover their metabolic activity and induce calcium carbonate precipitation in the concrete, as reported by Ehrlich et al in 1996.…”