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

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.

Robust Self-Regeneratable Stiff Living Materials Fabricated from Microbial Cells.

Living systems have not only the exemplary capability to fabricate materials (e.g. wood, bone) under ambient conditions but they also consist of living cells that imbue them with properties like growth and self-regeneration. Like a seed that can grow into a sturdy living wood, we wondered: can living cells alone serve as the primary building block to fabricate stiff materials? Here we report the fabrication of stiff living materials (SLMs) produced entirely from microbial cells, without the incorporation of any structural biopolymers (e.g. cellulose, chitin, collagen) or biominerals (e.g. hydroxyapatite, calcium carbonate) that are known to impart stiffness to biological materials. Remarkably, SLMs are also lightweight, strong, resistant to organic solvents and can self-regenerate. This living materials technology can serve as a powerful biomanufacturing platform to design and develop advanced structural and cellular materials in a sustainable manner.

Water-processable, biodegradable and coatable aquaplastic from engineered biofilms.

Petrochemical-based plastics have not only contaminated all parts of the globe, but are also causing potentially irreversible damage to our ecosystem because of their non-biodegradability. As bioplastics are limited in number, there is an urgent need to design and develop more biodegradable alternatives to mitigate the plastic menace. In this regard, we report aquaplastic, a new class of microbial biofilm-based biodegradable bioplastic that is water-processable, robust, templatable and coatable. Here, Escherichia coli was genetically engineered to produce protein-based hydrogels, which are cast and dried under ambient conditions to produce aquaplastic, which can withstand strong acid/base and organic solvents. In addition, aquaplastic can be healed and welded to form three-dimensional architectures using water. The combination of straightforward microbial fabrication, water processability and biodegradability makes aquaplastic a unique material worthy of further exploration for packaging and coating applications.

Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut.

Mucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer Escherichia coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution. We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo, and is non-pathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.

Genetically Programmable Self-Regenerating Bacterial Hydrogels.

A notable challenge for the design of engineered living materials (ELMs) is programming a cellular system to assimilate resources from its surroundings and convert them into macroscopic materials with specific functions. Here, an ELM that uses Escherichia coli as its cellular chassis and engineered curli nanofibers as its extracellular matrix component is demonstrated. Cell-laden hydrogels are created by concentrating curli-producing cultures. The rheological properties of the living hydrogels are modulated by genetically encoded factors and processing steps. The hydrogels have the ability to grow and self-renew when placed under conditions that facilitate cell growth. Genetic programming enables the gels to be customized to interact with different tissues of the gastrointestinal tract selectively. This work lays a foundation for the application of ELMs with therapeutic functions and extended residence times in the gut.

Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

Fluorescence imaging using synthetic GFP chromophores.

Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.