Bottom-up fabrication of nanoscale materials has been a significant focus in materials science for expanding our technological frontiers. This assembly concept, however, is old news to biology — all living organisms fabricate themselves using bottom-up principles through a vast self-organizing system of incredibly complex biomolecules, a marvelous dynamic that we are still attempting to unravel. Can we use what we have gleaned from biology thus far to illuminate alternative strategies for designer nanomaterial manufacturing? In the present review article, new synthetic biology efforts toward using bacterial biofilms as platforms for the synthesis and secretion of programmable nanomaterials are described. Particular focus is given to self-assembling functional amyloids found in bacterial biofilms as re-engineerable modular nanomolecular components. Potential applications and existing challenges for this technology are also explored. This novel approach for repurposing biofilm systems will enable future technologies for using engineered living systems to grow artificial nanomaterials.
Nanomaterials engineering holds the promise of novel materials through the programming of matter on the atomic or molecular scale. The resulting materials would have properties that are unattainable by current fabrication approaches. The vast majority of nanomaterial fabrication methods today employ physical or chemical manipulation of inorganic atomic assemblies to yield various supramolecular architectures, such as nanoparticles, nanotubes, nanowires, and nanosheets [1–5]. Although a chemical approach to the synthesis of these nanomaterials allows for facile synthesis and scale-up, the synthesis conditions often require harsh chemicals or extreme temperature/pressure conditions, and maintaining control over ordered self-assembly of the material through various hierarchical scales from the nanoscale up to macromolecular materials is difficult [6–8]. Biological approaches to nanoscale material fabrication is a growing area of research that attempts to leverage the unique self-assembling properties of living systems for the production of nanomaterials. Consider that the level of complexity for biological systems far exceeds what we can accomplish by purely synthetic means — for example, a single subunit of α-hemoglobin consists of over 1000 atoms that precisely co-ordinate their own spatial organization, driven only by atomic connectivity and ambient physical forces, to produce a nanomachine that can dynamically transport gases. Given that the human proteome is estimated to contain at least 20 000 different proteins , all of which must fold and interact properly for the optimal function of every cell in your body, we can see that biology harnesses nanomolecular manipulation to great effect. All living organisms can be viewed as incredibly complex assemblies of nanomachines and nanostructures. Exploring biological nanoscale self-assembly will guide us in the development of design principles for developing our own novel synthetic bionanomaterials.
Bioinspired engineering approaches can apply biological design principles to synthetic systems, such as synthetic polymer materials [10,11] or even robotics [12,13]. The use of biological materials, however, allows for the utilization of systems that have co-evolved in lockstep with these design principles. Numerous examples abound for the engineering of self-assembling proteins [14–17], DNA [18–21], and bacteriophage [22,23] for creating materials. The past decade has also seen great advances in synthetic biology, which utilizes modular biological parts for the creation of artificial biological pathways. Synthetic biology uses these parts to create artificial biological circuits that are capable of complex dynamics, such as gene expression oscillation , genetic timers , toggle switches , spatial patterning , and light-controlled gene expression . An integration of nanomaterials science and synthetic biology approaches could allow the development of nanomaterial-producing systems that are able to adapt dynamically to stimuli, modulate their properties over time, and heal themselves, much like tissues are capable of doing. Such engineered systems can be thought of as ‘engineered living materials’, materials that have integrated living and non-living components, respectively, the cells and the materials they generate. Herein, I review the use of engineered bacterial biofilms as a complete synthetic biological technology platform for the production of such living materials.
Bacterial biofilms: cells as a materials foundry
The design and engineering of biomaterials in the creation of self-assembling nanomaterials has in the past, primarily been executed in an in vitro fashion. Self-assembling peptides and proteins as the biomaterial are often used in a purified or extensively processed form and, in many cases, are subjected to precise conditions to initiate assembly of the material . Recently, DNA has also been explored as a route for the creation of programmable nanostructures capable of hierarchical assembly into micron-scale supramolecular structures, dynamic actions, and nanoparticle templating . Although incredibly promising, these studies have primarily used costly synthesized DNA and precise in vitro reaction conditions. Such in vitro approaches have many drawbacks — extensive purification and processing of the materials (e.g. synthetic DNA or purified proteins) adds on significant costs [31,32], and these materials, once assembled, often cannot be dynamically remodeled. Tissues offer inspiration for a different paradigm for designing artificial nanomaterials. In tissues, cells act as factories to produce, secrete, and co-ordinate self-assembling extracellular materials that bind the cells together and engender the distinct physical properties of the tissue material. The diverse expanse of biological tissues demonstrates the impressive range of large-scale materials that are capable of being generated: collagen and elastin matrices form the tough properties of leather, cellulose matrices form the bulk of plant structures, collagen forms flexible cartilage, protein-based hydrogels of the vitreous humor provide structure and transparency to the eye, keratin structures that form tough nails, hooves, and horns, interpenetrating polymer networks of cellulose and lignin polymers forms compression-resistant wood, extended protein macrofibers forming tough silks, polysaccharide or protein-based adhesives used by various organisms, and chitin polymers in arthropods enable rigid exoskeletons. Biological tissues can also harness inorganic molecules in the environment to create biomineralized structures, such as hydroxyapatite biomineralization onto collagen scaffolds to form resilient bone, dentin and enamel that forms hard teeth and tusks, optically transparent calcite lenses in primitive eyes, silica-based skeletons found in diatoms and some sponges, chitin-fiber reinforced crystalline magnetite of the chiton rasping organ, and biotemplated magnetite organelles. All of these materials are composed of nanoscale biomaterials that assemble hierarchically, in a genetically encoded manner. That the entire library of biomaterials composing the human body is encoded in every single one of our cells is impressive to consider.
Attempting to rationally engineer such materials for artificial applications using animal tissues would be quite difficult considering the complexity of eukaryotic genetic engineering and extracellular matrix production. A more facile system for manipulation can be found in bacterial biofilms, which can be considered a less complex analogue to eukaryotic tissues. Bacteria, the most abundant organisms on the planet, can exist in two modes of growth, planktonically as free floating cells, or as a biofilm, a sessile multicellular community of bacterial cells (Figure 1A). It is thought that the vast majority of bacteria in nature exist as biofilms  for protection and colonization of a favorable niche . These bacterial communities produce biofilm-specific extracellular matrices and display distinct spatial metabolic differentiation, indicating they should be regarded as multicellular organisms . Thus, biofilms can be considered as a proto-tissue, composed of bacteria and the materials they produce.
Synthetic biology for nanomaterials fabrication through engineered biofilms.
Biofilms are an ideal platform for the engineering and production of nanomaterials. First, genetic manipulation of bacterial cells is much easier than eukaryotic cells and synthetic biology tools are correspondingly more readily available for bacterial systems. Second, during biofilm formation, the bacterial cells are able to secrete/release a wide variety of biomolecules that assemble to form the biofilm extracellular matrix, including polysaccharides, proteins, and nucleic acids (Figure 1B). These existing pathways can be used as starting points for reengineering matrix components, rather than de novo design of such a system. Third, bacterial cells are much more robust and require less stringent growth conditions than eukaryotic cells, allowing the use of engineered biofilms in a wide variety of environments. Furthermore, these biofilms can use low-cost feedstocks to continually produce and assemble these nanomaterials which would be substantially cheaper than in vitro-based self-assembly that utilizes chemically synthesized and/or purified components. Lastly, bacteria have rapid growth times, enabling fast design and optimization cycles during engineering.
The biofilm is composed of the cells themselves and extracellular polymeric substances (herein referred to as EPS) that they produce collectively known as the matrix. The three main biomolecules that have been identified in biofilm EPS are polysaccharides, DNA, and proteins. All of these components contribute to the essential functions of a biofilm matrix: structural integrity, hydration, and adhesion to surfaces. Polysaccharides are typically the dominant component of most biofilms and provide the majority of the structure in the extracellular matrix. The composition of his matrix is highly heterogeneous across bacterial species, with polysaccharide networks being composed of glucan and fructan homopolymers, glucose/mannose/rhamnose heteropolymers (Pel and Psl polysaccharides), cellulose, alginate, colanic acid, and/or N-acetylglucosamine . Differences in the presence of these polysaccharides in the EPS dictate the physicochemical properties of the matrix, such as charge, hydrophobicity, and mechanical properties. Surprisingly, extracellular DNA (eDNA) has also been found to play a large role in the structural integrity of biofilms  as well as directing cellular migration in biofilms . The release of eDNA is thought to primarily occur through cellular lysis . Polymeric proteinaceous structures have been found to play an essential role in the structural stabilization of biofilms, either in the form in extracellular organelles, such as flagella , pili , outer membrane proteins , carbohydrate binding proteins , or through dedicated protein nanofibrils  that are expressed primarily during biofilm formation. In some species, these proteins can constitute the major fraction of the extracellular matrix . Such nanofibrils constitute the largest proteinaceous component of bacterial biofilms, and were first identified in Escherichia coli biofilms as ‘functional amyloids’ — self-assembling amyloid proteins that have evolved to play physiological (rather than pathological) roles.
Although polysaccharides constitute the majority of biofilm EPS mass, they are not highly programmable, as engineering of functionality would require complex pathway engineering for the chemical modification of the sugar monomers. DNA has seen a significant increase in use as a nanotechnology scaffold, but most of the eDNA present in biofilms is derived from cell lysis. Although DNA nanostructures produced in vivo and assembled extracellularly after cell lysis is an intriguing possibility, scaling up such a process would be challenging. Cell lysis circuits to release such engineered DNA nanomaterials would essentially destroy the bacteria that produce the materials, resulting in an energetically and mass-inefficient system for generating large-scale materials, in comparison with systems that continually secrete biomolecules such as polysaccharides and proteins from the cells while preserving their viability. Furthermore, most DNA nanostructures engineered to date require precise thermal annealing steps in order to attain the desired architecture , although recent advances demonstrate the in vivo assembly of simple structures . Furthermore, it would be difficult to engineer DNA-based nanomaterials with different chemical properties. In contrast, proteinaceous functional amyloids that form a key structural role in bacterial biofilms have many distinct advantages as a platform for nanomaterials engineering. Polypeptides are highly programmable, with structural, physicochemical, and catalytic functions that can be encoded by the amino acid sequence (Figure 1C). Also, the functional amyloid fibers in biofilms have dedicated extracellular export apparatuses — this allows the cells to continually fabricate the desired nanomaterial given the proper raw materials. The remainder of this review will focus on current synthetic biology efforts for reprogramming functional amyloid systems for nanomaterials production.
Self-assembling functional amyloids for extracellular matrix display
Curli are high aspect-ratio extracellular nanofibers that were first identified as virulence factors in E. coli . It was subsequently discovered that these protein polymers were in fact amyloids that serve an essential role in the structural integrity of E. coli biofilm EPS . The curli monomeric unit is a protein called CsgA and is secreted in a unfolded conformation by a dedicated secretion system, composed of an outer membrane porin and several chaperones that prevent intracellular self-assembly [49–51] (Figure 2A). Once CsgA is secreted into the extracellular milieu, it self-assembles into nanofibers by seeding onto a membrane-anchored nucleation protein, CsgB . The resulting curli nanofibers are only 4–7 nm in diameter and are highly stable, resistant to boiling, strong acids, bases, harsh organic chemicals, proteases, and detergents . They can reach tens of microns in length and form extended nanofibrous networks that provide structural integrity to the biofilm extracellular matrix [40,54] (Figure 2B,C).
Reengineering of the E. coli curli functional amyloid system.
The development of nanomaterials production using such functional amyloids is based on a straightforward concept. Functional peptides or proteins are appended to the amyloid monomer to create a chimeric protein, which is exported to the extracellular space via the dedicated secretion machinery. The amyloid domain facilitates self-assembly into a nanofiber network, while the fused peptide/protein domains serve to alter the bulk properties of the resulting material in a programmable manner. The result is a large-scale nanomaterial with programmable functionality that is continually generated and secreted by bacterial cells. The diversity of functionalization domains can be mined from previously engineered peptides that mediate adhesion to various materials [55–57], act as a template for nanoparticle formation [58,59], serve as covalent or noncovalent affinity tags [60,61], alter physical properties such as hydrophobicity , or potentially allow for the engineering of conductivity or optical properties. A large pool of such peptides can be mined from the current literature of cell- or phage-display studies Whole proteins could also be functionalized into the nanofiber network, to enable the fabrication of more complex nanomaterials, for example, that are capable of enzymatic catalysis. Furthermore, bacterial strains used for production could be selected that do not produce other EPS components such as polysaccharides, thus producing a biofilm matrix consisting of nearly pure protein-based nanomaterials.
This concept was demonstrated by appending 12 different peptide domains to the CsgA protein ranging in size from 7 to 59 amino acids and encompassing a variety of functions, such as binding to metals or minerals, templating nanoparticles or optically active quantum dots, biomineralization of bone, and a tag for covalent bond formation to larger proteins . Of these CsgA fusions, 11 of the 12 were able to be secreted and were competent for self-assembly into nanofibers, as demonstrated by binding to the amyloid-specific dye Congo red and by extensive ultramicroscopic analyses. The artificially programmed functions for three of the nanofiber materials, ranging from binding to steel, to silver nanoparticle templating, and also for site-specific covalent immobilization of large proteins (utilizing the binary SpyTag/SpyCatcher protein fragment system ) were tested and confirmed (Figure 2D–F). The authors specifically designated their platform BIND, for Biofilm-Integrated Nanofiber Display. An analogous system using curli nanofibers to display peptides was used to engineer temporal regulation to control the arrangement of the CsgA fusion proteins along the length of the nanofibers, enabling the genetic control of protein nanofiber block co-fibrils [64,65]. The authors also created electrically conductive and optically active biofilms by displaying an affinity tag on the curli nanofibers and then incubating the biofilm with functionalized gold nanoparticles or quantum dots, respectively (Figure 2G). A different study testing the limits of polypeptide fusions to CsgA in regard to its ability to be exported and self-assemble have indicated small proteins of up to 260 residues can be successfully displayed on curli nanofibers, greatly increasing the range of functions that can be programmed, although it is dependent on the folding properties of the particular protein . The functions of the BIND platform were recently expanded using the SpyTag/SpyCatcher covalent immobilization strategy to create biofilm matrices to which enzymes could be immobilized . The high surface area and extreme stability of the curli nanofiber network make it an ideal substrate for immobilizing enzymes for biocatalysis applications. Although utilizing self-assembling proteins to create customized nanomaterials has been extensively explored before, the key difference compared with the biofilm-produced materials described above is in the use of an EPS component that is fabricated and secreted by the cells, circumventing the need for purification. In contrast, previous numerous efforts employing self-assembling proteins used purified proteins and the assembly often occurs under highly controlled in vitro conditions [17,68]. The BIND strategy also allows for the flexibility of removing the bacterial factories after matrix production, if desired, to obtain highly pure nanofiber materials on a macroscopic scale by the simple use of detergent and salt washes to lyse and remove cellular material, as demonstrated recently . This would allow the production of large-scale abiotic nanomaterials for applications in which biocontainment is a concern.
Other functional amyloid systems
The modularity of the BIND concept extends beyond the diversity of the functional domains, with various self-assembly domains that can conceivably be utilized in lieu of the CsgA protein. It has been found that various amyloidogenic domains, including the yeast prion proteins Sup35, Rnq1, Cyc8, New1, Mss11, Pub1, or a polyQ fragment of the human huntingtin protein Htt, can be exported through the curli pathway by adding the CsgA translocation and secretion signals onto these domains and co-expressing only a few components of the curli export machinery in an E. coli csgA deletion strain . All of these non-native amyloids were successfully secreted and were able to form nanofibers once exported to the extracellular space. This greatly expands the repertoire of self-assembly domains that can be employed to tune the mechanical or biochemical characteristics of the biofilm-generated nanomaterials.
Adaptation of other amyloid domains for the E. coli curli system could be difficult due to incompatibilities between the domain and the export machinery, although it has been shown to be quite amenable for a large number of fusions. Alternatively, other bacterial biofilm systems can be employed. This also allows for a broad expansion of the environments in which these biofilms could grow. For example, deployment of engineered biofilms for environmental applications, such as a living material for toxin removal or sequestration, may require a robust bacterial species that thrives in soil such as Bacillus or Shewanella spp; or nanomaterials fabrication using a biofilm from a thermophile may be more advantageous for certain industrial applications requiring high temperatures. Although curli is the most studied biofilm-integrated functional amyloid, many other orthogonal amyloid systems have been identified in other bacterial biofilms over the past decade. Bacillus subtilis floating biofilms contain functional amyloid nanofibers self-assembled from TasA protein monomers with trace amounts of a TapA-anchoring protein that provide structural integrity to the biofilm [71,72]. The fap operon of Pseudomonas spp. generates extracellular amyloid nanofibers that are essential for cellular aggregation and biofilm integrity . Similarly, the Bap protein is secreted by Staphylococcus spp. and forms biofilm EPS amyloid nanofibers which assemble under acidic conditions . An interesting class of functional amyloids, called chaplins, are produced by Streptomyces spp., and display hierarchical supramolecular assembly at the air–liquid interface to form hydrophobic protein sheets composed of amyloid nanofibers . Bacterial plant pathogens are known to secrete proteins known as harpins, which self-assemble into amyloid spheres and fibrils that are interestingly cytotoxic to plant cells . Other as yet unidentified functional amyloid systems are thought to be widespread in bacteria, based on a phylogenetic analysis of 10 diverse metagenomes that identified an extensive the distribution of similar operons homologous to the curli-based operon . Of the non-curli orthologous functional amyloid systems, only TasA has been recently engineered in a similar concept as BIND, for the display of peptides and full proteins on the TasA nanofibers in Bacillus biofilms . Proposals to engineer the Staphylococcus Bap protein to obtain engineered extracellular nanofibers using the same strategy have also been put forth . The utility of these various bacterial amyloids for nanomaterials engineering endeavors will require further study of their physicochemical and mechanical properties, which are currently available for only CsgA and TasA nanofibers.
Applications of biofilm engineering
A ‘living materials’ technology that uses bacterial cells to drive the production and assembly of programmable nanomaterials can be utilized in many applications (Figure 3A). As described above, immobilized enzyme catalysis reactors can take advantage of the high surface area nanoporous matrix of these engineered amyloid biofilms. Given a proper bioreactor design, the bacteria could be designed to construct this matrix, immobilize the desired enzyme on the nanofibers, and maintain or replenish the catalytic structure as needed. Much of the laborious steps in setting up and maintaining catalytic bioreactors can thus be significantly reduced, by engineering these steps as autonomous synthetic biological programs.
Expanding the synthetic biology toolkit for biofilm materials.
Nanoscale or not, all materials undergo wear and tear due to usage or aging. Engineered living materials, in contrast, would enable designed advanced materials that could continually repair, rebuild, and dynamically alter itself using resources in the environment. This bioinspired strategy is encapsulated in an example found in nature — stromatolites are large macroscopic structures that could reach meters in size and are formed by cyanobacterial biofilm mats that continually deposit EPS matrix material . Deployed engineered biofilms could be used to generate healing structural materials, such as self-mineralizing biofilm matrices for the repair of cracks in concrete; or, the artificial biofilm could be used as a protective coating over a structure such as a building or a ship hull, with the matrix nanomaterial designed for adhesion to specific materials and the living material to provide some benefit, such as anti-corrosion capabilities. This could be done by using bacterial species that are known to prevent metal corrosion by forming biofilms that create a protective barrier on the metal, consuming oxygen at the metal interface, producing metal-protecting compounds, or inhibiting the growth of other bacteria which promote corrosion [81–86].
One could also envision robust engineered biofilms used in highly contaminated environmental sites for the removal or abatement of toxic materials, such as heavy metals or chemicals, where cell-based  and designer nanomaterials  approaches can be synergistically combined for remediation efforts. Related to these efforts are the potential use of programmable biofilms for waste water management, where current technology utilizes naturally occurring biofilms  for waste water processing. An on-demand engineered biofilm could greatly expand the efficiency of these processes by matching desired artificially designed properties to a particular waste water stream. Another area of application is in microbial fuel cell development, where bacterial communities are used for electricity generation. Interfacing of the bacterial cells with the electrodes in these fuel cells are a critical parameter for optimal fuel cell efficiency  and therefore the ability to engineer adhesive, physical, or mechanical properties into biofilm nanomaterials would be a very fruitful area to pursue. Biofilm engineering could also play a key role in recent efforts to combine waste water processing with electricity generation through microbial fuel cells .
Biofilm interfaces could also be used for sensing biotechnologies. There have been significant efforts in synthetic biology for the development of genetic circuits for bacterial biosensing and reporting . Engineered biofilm materials combined with these synthetic circuits would allow for robust bacterial systems that would enable persistence in harsh environments or dynamically respond to changing conditions to increase bacterial survival. Most of the output signals from designed bacterial biosensors often utilize reporter proteins that generate light output by fluorescence, luminescence, or a colorimetric output from enzyme activity. Engineered biofilms could allow novel outputs upon an environmental trigger, such as the programmed growth of a material structure or even an electrical interface to electronic circuits, if a means could be engineered to create inherently conductive biofilm nanofibers such as those that already exist in Geobacter sulfurreducens .
Biofilms are widely used by bacteria to persist in niches where they form complex symbiotic and mutualistic relationships with other organisms. With the explosion of interest and studies focused on microbiome ecosystems, engineered biofilm materials could play a central role in attempts to alter the properties and dynamics of such microbiomes. Of key importance is ensuring that the engineered biofilm nanofibers, such as the re-engineered self-assembling amyloids in the BIND technology, do not pose significant risks to the host organism. Biofilm formation is also a highly successful evolutionary strategy that allows bacteria to persist in harsh and changing conditions by converting local resources into an encapsulating material, essentially altering their local environment. A forward-looking application to exploit this property of biofilms could be the use of synthetic biology engineering of biofilms for use in space exploration and colonization . Engineered biofilms could be used to manufacture programmable nanomaterials on-site, in resource-poor environments such as off-planet colonization sites. For example, a photosynthetically driven version of the BIND system could generate designer nanomaterials largely from simple resources such as carbon dioxide, sunlight, and water — components which are present on Mars . Thus, materials can be generated on-site using local matter, using self-replicating biological systems as factories to assemble this matter into the desired nanomaterials. This self-sustaining strategy for material production, known as In Situ Resource Utilization (ISRU) [96–98], circumvents the prohibitive costs of transporting bulk materials into space by transporting instead the platforms that enable in situ material generation, such as that described in this review. Furthermore, engineerable biofilms could be a cornerstone for developing hardy synthetic biology approaches to biological terraforming endeavors .
Limitations and future directions for engineered living materials
The majority of the described and ongoing efforts for engineering biofilm systems for nanomaterials production uses the synthetic biology workhorse, E. coli and its cognate curli nanofiber pathway. BIND would be ideal for generating programmable nanomaterials for industrial purposes, due to the extensive knowledge and genetic tools available for engineering E. coli, as well as the fact that curli functional amyloids are the most widely studied of all the bacterial amyloids. However, the use of CsgA as the self-assembly domain does carry some significant caveats, as curli fibers are highly immunogenic , proinflammatory , promote mammalian cell invasion , and induce autoimmune diseases , and several studies have demonstrated that it promotes amyloid formation in animals, probably through a cross-seeding mechanism with native proteins that are aggregation prone [104–106]. Thus, any biomedical applications using a CsgA-based BIND system may be limited. In light of this, the continued exploration of different self-assembly domains and different bacterial species capable of exporting these domains is a critical area for diversifying the synthetic biology toolkit for biofilm nanomaterials engineering. In the future, the design and engineering of a biofilm nanomaterial could draw from libraries of nanomaterial scaffolds and bacterial nanofactories to optimally suit the parameters of a particular application (Figure 3B).
The few studies thus far that have explored programmable nanomaterials using biofilm functional amyloid systems have utilized common chemical inducer systems to control gene expression. One fertile area of exploration would the integration of more complex synthetic biology circuits toward creating engineered living materials that can operate autonomously and dynamically without extensive user intervention. A wide variety of synthetic biological sensors can be integrated into the BIND platform to drive nanomaterial production upon the exposure to light, toxins, metals, pH changes, temperature changes, or quorum sensing molecules from other species. Furthermore, complex circuits allowing a degree of biological computation could allow temporal and/or spatial regulation of these engineered living materials, allowing cyclical production or dynamic remodulation of the nanomaterial structure. With current studies elucidating the spatiotemporal control of amyloid production during biofilm formation [40,72,107–109], integrating these findings into engineered systems could allow greater control over expression and three-dimensional distribution of the resulting materials. Ongoing efforts uncovering the mechanisms used for regulated biofilm dispersal can be used to engineer circuits for dynamically changing living materials [110–113]. An exciting example from Wood and colleagues of such an engineered system allowed for the controlled formation and dispersion of biofilm consortia . One can envision libraries of different synthetic sensors and engineered amyloids integrated with such synthetic biology circuits, all encoded in a single bacterium. This engineered cell would have the capacity to match the fabrication of particular nanomaterial(s) in response to the environmental inputs.
The recent advances in engineering biofilm nanomaterials establish a new emerging area of bioengineering, leveraging all of our knowledge of systems and synthetic biology toward engineering the extracellular realm. Aside from the expansion of biological parts, many optimization efforts and questions remain regarding the development of this technology for practical applications. What are the limits of scalability — can we produce mass quantities of these programmed nanomaterials? Given that the functional amyloids used in the BIND strategy are known to be toxic to the cell if it is not exported expediently [50,115,116], how can we design systems that balance maximal production while eliminating any deleterious effects to the cell? What are the safeguards that need to be implemented for this technology, and what are the potential consequences of engineered biofilms escaping into the wild? Most the focus of this review has centered on engineering functional amyloid systems of bacteria, but can we develop extracellular protein polymer systems that are not based on amyloid scaffolds, thereby circumventing safety concerns? Can other biomolecular components of biofilms, such as polysaccharides and DNA, be successfully re-engineered as functionalizable scaffolds to create extracellular materials with multiple programmable components? With the exploration of these challenges combined with continued expansion of different bacterial foundries as chassis, self-assembling extracellular molecules as scaffolds, programmable functional domains, and efforts at complex circuit integration, we are poised to enter a future where advanced nanomaterials can be designed to grow themselves.
The Author declares that there are no competing interests associated with this manuscript.