Living Material Fuses Function and Scale

The future of sustainable materials may lie with microbiology where macroscale materials can be autonomously assembled, modular in function and physical property, genetically tunable, and seed new growth. Read on to learn about one such material.
Living Material Fuses Function and Scale
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As our population continues to grow and resources decrease, there is a pressing need to do more with less. A route forward for truly sustainable materials relies on biology, as cells are masters at molecular to macroscale synthesis from limited resources and skilled at recycling. Meeting the need for sustainable materials and the intriguing multifunctionality of such items as skin, shells, wood, and bone has lured many a microbiologist into the field of engineered living material (ELMs). We imagine a world where all materials have longer usage thanks to self-healing abilities, that any material can be programmed to biodegrade, that the material’s physical properties can be adapted as needed, and that all materials have expansive functions, ranging from colorimetric, catalytic, electric, acoustic, adsorptive, and more. However, teasing apart and controlling the methods at which cells simultaneously assemble into enormous structures, interface with their environment, and perform a myriad of functions is no easy task. Early ELM research was often forced into tradeoffs wherein a material could be tough but not functional, or functional but require assembly, or self-assembling but tiny. As the field matures, it slowly fuses together more desirable qualities.

As we explored the usage of surface-layer proteins for ion absorption, Dr. Caroline Ajo-Franklin and team conceived an unique method for controllable, bottom-up assembly of ELMs. Utilizing a microbial base otherwise unseen in ELMs, Caulobacter crescentus and its surface-layer (S-layer) RsaA, we felt we could achieve self-assembly, functionality, and scale. C. crescentus has many useful features, such as a bimodal cell life cycle, non-pathogenicity, and minimal resource needs compared to the more common bioengineering platform E. coli. RsaA creates an intricate lattice on the cell surface that is ideal for displaying high-density recombinant protein1 and involves secretion machinery that can be exploited for high-level export2. These two abilities are key for ELMs because self-interacting proteins displayed at ~10% the level of our recombinant RsaA results in only small aggregates3, and polysaccharides are the only secreted material that results in macroscale gelation of ELMs and they cannot be genetically engineered easily for alternative properties. Using the C. crescentus RsaA system, our team, led by Dr. Sara Molinari and PhD candidate Bobby Tesoriero, was able to grow a viscoelastic solid over 4 orders of magnitude, from micrometer cells to centimeter hydrogels, with physical properties tunable through genetic engineering and easy functionalization.

Images of hierarchical engineered living material from centimeter scale material in flask to confocal images of cells and matrix to atomic force microscopy showing brush-like matrix bound to cell surface. Images of material in syringe, extruded into a spiral and into a worm, and as a hard pellet.
Fig 1: a Hierarchical structure of our ELM demonstrated by images of increasing granularity: First is the material in a flask (far left). Next, confocal microscopy imaged at three magnifications (protein matrix stained with SpyCatcher-GFP (cyan) and cells producing mKate2 (yellow)). Finally, atomic force microscopy image of an individual cell surface showing the brush-like structure of the attached matrix protein as opposed to the usual RsaA hexagonal lattice (AFM taken of strain incubated separately). b ELM can be shaped: ELMs collected into a syringe (far left) and extruded using different-sized nozzles (two middle panels). ELM mixed with glass powder to form a firm paste that hardens when dehydrated (far right).

To achieve this, the team capitalized on the fact that the final 336 amino acids of the RsaA protein, which codes for secretion, spontaneously aggregates in solution when not properly bound and assembled on the cell surface4. Through genome engineering, we created a sequence in place of the original rsaA gene to produce recombinant protein including the RsaA surface-binding domain, a moiety for 60 repeats of the well-characterized hydrogel, elastin-like polypeptide (ELP60), a tag for functionalization, and the aggregation/secretion domain. Elastin-like polypeptides are known to self-associate in a concentration and sequence dependent manner and in this instance, were used to provide solution accessibility and add non-covalent interactions that could alter the material properties. The SpyTag5 peptide was used for functionization, as it spontaneously forms covalent bonds with its partner protein SpyCatcher in solution thus allowing us to bind any enzyme of interest to the material just by fusing it to SpyCatcher.

Incubated for a mere 24 hours, the engineered cells autonomously assemble into a centimeter scale material that when analyzed by microscopy and immunoblot, is a conglomerate of C. crescentus cells and recombinant protein. The material is formed over time by the cells growing freely in solution for 12 hours after which a thin skin appears at the air-water interface and gradually becomes more cell-dense. At around 24 hours, the final material sinks. The hydrophobicity and accessibility of the air-water interface was a key component to assembly and when disrupted either by surfactants or static conditions respectively, the material will not form. A model was developed to predict material size based on the speed, volume, and flask size. This method for material formation proved simple and highly reproducible as it was tested by multiple researchers across two labs in different states and only requires the strain, culture media, flask, temperature, and rotation.

A unique aspect to this material is its extensive tunability through genetic modification. By removal of either the surface binding domain or the ELP60 section, the storage modulus can be tuned over a range of 14-fold and the loss modulus 25-fold. The material can be extruded from syringes to form desired shapes, or mixed with glass powder and dried down to form a hard solid. We are already working to further expand these material properties through new variants that contain different modules with different characteristics and lengths. We hope to eventually develop design rules for the platform wherein a desired material property can be easily programmed based on known genetic alterations.

In addition to its tunable physical properties, the material is inherently functional as it was able to absorb 90% of cadmium in solution without needing further engineering, more than a solution of individual C. crescentus cells is capable of. When purposefully functionalized by incubating an oxidoreductase PQQ-glucose dehydrogenase, the material was able to enzymatically reduce an electron carrier. This shows that the material is capable of converting chemical energy into electrical energy. Finally, it can also regenerate. Fragments from material dried for three weeks can be inoculated into fresh medium to grow new material as a seed would.

As a whole, the field of ELMs is expanding rapidly with platforms capitalizing on curli fibers, cellulose, mycelium, mineral precipitation, and more. Future ELM products will meet needs in areas such as medicine, bioremediation, and construction, replacing current methods that are less environmentally friendly, have only a single function, or require constant upkeep. The greater the modularity of the ELM, the more readily it will supplant other technologies and we predict the platform described herein will be incredibly modular. There are potential control knobs in the relative cell and protein densities, environmental conditions, and enzymes added exogenously or recombinantly to imbue desirable optical, electrical, mechanical, thermal, transport, and catalytic properties.

These results were recently published in Nature Communications: Molinari, S., Tesoriero Jr., R.F., Li, D., Sridhar, S., Cai, R., Soman, J., Ryan, K.R., Ashby, P.D., & Ajo-Franklin, C.M., “A de Novo Matrix for Macroscopic Living Materials from Bacteria.” Nat. Comm. 13, 5544 (2022). https://doi.org/10.1038/s41467-022-33191-2 

References

    1. Charrier, M. et al. Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials. ACS Synth. Biol. 8, (2019).
    2. Orozco-Hidalgo, M. T., Charrier, M., et al. Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials. mSystems 6, e00903-20 (2021).
    3. Glass, D. S. & Riedel-Kruse, I. H. A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell 174, 649-658.e16 (2018).
    4. Bingle, W. H., et al. Secretion of the Caulobacter crescentusS-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins. J. Bacteriol. 182, 3298–3301 (2000).
    5. Zakeri, B., et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. USA. 20;109(12):E690-7 (2012).

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