Cell-Free Protein Expression in Polymer Materials.

While synthetic biology has advanced complex capabilities such as sensing and molecular synthesis in aqueous solutions, important applications may also be pursued for biological systems in solid materials. Harsh processing conditions used to produce many synthetic materials such as plastics make the incorporation of biological functionality challenging. One technology that shows promise in circumventing these issues is cell-free protein synthesis (CFPS), where core cellular functionality is reconstituted outside the cell. CFPS enables genetic functions to be implemented without the complications of membrane transport or concerns over the cellular viability or release of genetically modified organisms. Here, we demonstrate that dried CFPS reactions have remarkable tolerance to heat and organic solvent exposure during the casting processes for polymer materials. We demonstrate the utility of this observation by creating plastics that have spatially patterned genetic functionality, produce antimicrobials in situ, and perform sensing reactions. The resulting materials unlock the potential to deliver DNA-programmable biofunctionality in a ubiquitous class of synthetic materials.

Microbially Induced Calcium Carbonate Precipitation by Sporosarcina pasteurii: a Case Study in Optimizing Biological CaCO3 Precipitation.

Current production of traditional concrete requires enormous energy investment that accounts for approximately 5 to 8% of the world's annual CO2 production. Biocement is a building material that is already in industrial use and has the potential to rival traditional concrete as a more convenient and more environmentally friendly alternative. Biocement relies on biological structures (enzymes, cells, and/or cellular superstructures) to mineralize and bind particles in aggregate materials (e.g., sand and soil particles). Sporosarcina pasteurii is a workhorse organism for biocementation, but most research to date has focused on S. pasteurii as a building material rather than a biological system. In this review, we synthesize available materials science, microbiology, biochemistry, and cell biology evidence regarding biological CaCO3 precipitation and the role of microbes in microbially induced calcium carbonate precipitation (MICP) with a focus on S. pasteurii. Based on the available information, we provide a model that describes the molecular and cellular processes involved in converting feedstock material (urea and Ca2+) into cement. The model provides a foundational framework that we use to highlight particular targets for researchers as they proceed into optimizing the biology of MICP for biocement production.

Particle-Based Microrheology As a Tool for Characterizing Protein-Based Materials.

Microrheology based on video microscopy of embedded tracer particles has the potential to be used for high-throughput protein-based materials characterization. This potential is due to a number of characteristics of the techniques, including the suitability for measurement of low sample volumes, noninvasive and noncontact measurements, and the ability to set up a large number of samples for facile, sequential measurement. In addition to characterization of the bulk rheological properties of proteins in solution, for example, viscosity, microrheology can provide insight into the dynamics and self-assembly of protein-based materials as well as heterogeneities in the microenvironment being probed. Specifically, passive microrheology in the form of multiple particle tracking and differential dynamic microscopy holds promise for applications in high-throughput characterization because of the lack of user interaction required while making measurements. Herein, recent developments in the use of multiple particle tracking and differential dynamic microscopy are reviewed for protein characterization and their potential to be applied in a high-throughput, automatable setting.

Engineering Gelation Kinetics in Living Silk Hydrogels by Differential Dynamic Microscopy Microrheology and Machine Learning.

Microbes embedded in hydrogels comprise one form of living material. Discovering formulations that balance potentially competing for mechanical and biological properties in living hydrogels-for example, gel time of the hydrogel formulation and viability of the embedded organisms-can be challenging. In this study, a pipeline is developed to automate the characterization of the gel time of hydrogel formulations. Using this pipeline, living materials comprised of enzymatically crosslinked silk and embedded E. coli-formulated from within a 4D parameter space-are engineered to gel within a pre-selected timeframe. Gelation time is estimated using a novel adaptation of microrheology analysis using differential dynamic microscopy (DDM). In order to expedite the discovery of gelation regime boundaries, Bayesian machine learning models are deployed with optimal decision-making under uncertainty. The rate of learning is observed to vary between artificial intelligence (AI)-assisted planning and human planning, with the fastest rate occurring during AI-assisted planning following a round of human planning. For a subset of formulations gelling within a targeted timeframe of 5-15 min, fluorophore production within the embedded cells is substantially similar across treatments, evidencing that gel time can be tuned independent of other material properties-at least over a finite range-while maintaining biological activity.

Biomorphic Ceramics: Synthesis and Characterization of Preceramic Polymer-Modified Melanin.

Recent efforts have demonstrated that the morphology of ceramics can be manipulated to control both their deformation mechanism and mechanical performance. However, precise control of the ceramic nanostructure is still difficult to achieve. Biotemplating, leading to biomorphic materials, provides a facile route to manipulate the nanostructure of the resulting materials, and the use of melanin as a coating provides a new route to biotemplated materials. Melanin is underutilized for structural materials partly due to the cost of procuring it from natural sources and the inability to control the shape and sizes of melanin particles. Taking a combined synthetic biology and chemical synthesis approach, we report the melanization of Escherichia coli and its subsequent silanization and functionalization with preceramic polymers to make novel biomorphic silicon-based ceramic materials. Graft-to and graft-from reactions were used to append preceramic polymers to the melanin, followed by pyrolysis under argon. Samples were analyzed by FTIR, XRD, XPS, and TEM and found to retain the shape and size of the original cells with high fidelity. The homogeneity of coverage and yield of the resulting ceramic materials depended on the type of grafting reaction. This work provides a promising proof-of-concept that bacterial-templated ceramics can be readily made and opens a host of possibilities for further studies and applications.

Marine Structural Protein Stability Induced by Hofmeister Salt Annealing and Enzymatic Cross-Linking.

The Humboldt squid is one of the fiercest marine predators thanks in part to its sucker ring teeth that are biopolymer blends of a protein isoform family called suckerin with compression strength that rivals silkworm silk. Here, we focus on the popular suckerin-12 isoform to understand what makes the secondary structure of this biopolymer different in water and the potential role of diverse physical and chemical cross-linkings. By choosing a salt post-treatment, in accordance with the Hofmeister series, we achieved film stability with salt annealing that is comparable to chemical cross-links. By correlating the film morphology with the protein secondary structure changes, suckerin-12 films were shown to contract upon treatment with kosmotropic salts and exhibited increased stability in water. These changes are related to the rearrangement of suckerin-12 secondary structure from random coils and helices to ß-sheets. Overall, understanding secondary structure changes caused by aqueous and ionic environments can be instructive for the tuning of the suckerin film sclerotization, its conversion to a tough biological material, and to ultimately produce the natural squid sucker ring teeth.

Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12.

Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined ß-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ∼100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and ß-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

Lyophilized Cell-Free Systems Display Tolerance to Organic Solvent Exposure.

Cell-free systems offer a powerful way to deliver biochemical activity to the field without cold chain storage. These systems are capable of sensing as well as biosynthesis of useful molecules at the point of need. So far, cell-free protein synthesis (CFPS) reactions have been studied as aqueous solutions in test tubes or absorbed into paper or cloth. Embedding biological functionality into broadly used materials, such as plastic polymers, represents an attractive goal. Unfortunately, this goal has for the most part remained out of reach, presumably due to the fragility of biological systems outside of aqueous environments. Here, we describe a surprising and useful feature of lyophilized cell-free lysate systems: tolerance to a variety of organic solvents. Screens of individual CFPS reagents and different CFPS methods reveal that solvent tolerance varies by CFPS reagent composition. Tolerance to suspension in organic solvents may facilitate the use of polymers to deliver dry cell-free reactions in the form of coatings or fibers, or allow dosing of analytes or substrates dissolved in nonaqueous solvents, among other processing possibilities.

Silk fibroin as an additive for cell-free protein synthesis.

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

Corrigendum to "3D printed bionic nanodevices" [Nano Today 11 (2016) 330-350].

[This corrects the article PMC5016035.].

Anion-Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels.

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin-12, is used to create enzymatically crosslinked, macro-scale hydrogels. Upon exposure to specific salt conditions, suckerin-12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin-12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin-12 hydrogels. The potential for suckerins to add new properties to materials based on naturally-derived proteins is highlighted.

Disulfide Crosslinked Hydrogels Made From the Hydra Stinging Cell Protein, Minicollagen-1.

Minicollagens from cnidarian nematocysts are attractive potential building blocks for the creation of strong, lightweight and tough polymeric materials with the potential for dynamic and reconfigurable crosslinking to modulate functionality. In this study, the Hydra magnipapillata minicollagen-1 isoform was recombinantly expressed in bacteria, and a high throughput purification protocol was developed to generate milligram levels of pure protein without column chromatography. The resulting minicollagen-1 preparation demonstrated spectral properties similar to those observed with collagen and polyproline sequences as well as the ability to self-assemble into oriented fibers and bundles. Photo-crosslinking with Ru(II) ( bpy ) 3 2 + was used to create robust hydrogels that were analyzed by mechanical testing. Interestingly, the minicollagen-1 hydrogels could be dissolved with reducing agents, indicating that ruthenium-mediated photo-crosslinking was able to induce disulfide metathesis to create the hydrogels. Together, this work is an important first step in creating minicollagen-based materials whose properties can be manipulated through static and reconfigurable post-translational modifications.

Programmable Mechanical Properties from a Worm Jaw-Derived Biopolymer through Hierarchical Ion Exposure.

Mechanisms of biomaterial sclerotization in natural systems promise new insights into how the mechanical properties of engineered materials may be dynamically modulated. One such example involves the proteinaceous jaw of the marine sandworm, Nereis virens. Previously, the mechanical properties of the N. virens jaw were shown to be modulated by Zn binding, a property that was proposed to be enabled by the presence of the histidine-rich jaw protein, Nvjp-1. Here we demonstrate the creation of Nvjp-1-based hydrogels and show that progressive sclerotization of these hydrogels can be accomplished with hierarchical exposure to metal cations and anions. Divalent Zn cations are capable of reversibly sclerotizing the hydrogels through the formation of coordinate cross-links, an effect that is shown to be remarkably specific for Zn. Additionally, the degree of Zn-induced sclerotization is strongly influenced by the identity of the anion present in the hydrogel. Thus, the viscoelastic properties of Nvjp-1 hydrogels can be modulated through programmed, hierarchical exposure to specific cations and anions present in the sclerotizing salts. These observations have resulted in new hydrogel capabilities, such as the creation of anion-controlled shape-memory polymers, and will add to the number of control parameters that can be used to tune the properties of functional hydrogels in a dynamic manner.

Bio-Optics and Bio-Inspired Optical Materials.

Through the use of the limited materials palette, optimally designed micro- and nanostructures, and tightly regulated processes, nature demonstrates exquisite control of light-matter interactions at various length scales. In fact, control of light-matter interactions is an important element in the evolutionary arms race and has led to highly engineered optical materials and systems. In this review, we present a detailed summary of various optical effects found in nature with a particular emphasis on the materials and optical design aspects responsible for their optical functionality. Using several representative examples, we discuss various optical phenomena, including absorption and transparency, diffraction, interference, reflection and antireflection, scattering, light harvesting, wave guiding and lensing, camouflage, and bioluminescence, that are responsible for the unique optical properties of materials and structures found in nature and biology. Great strides in understanding the design principles adapted by nature have led to a tremendous progress in realizing biomimetic and bioinspired optical materials and photonic devices. We discuss the various micro- and nanofabrication techniques that have been employed for realizing advanced biomimetic optical structures.

Ion Effect and Metal-Coordinated Cross-Linking for Multiscale Design of Nereis Jaw Inspired Mechanomutable Materials.

The Nvjp-1 protein is a key component in the jaws of Nereis virens, a species of marine worm. It contains over 25 mol % of histidine, which is believed to play a key role in the metal-coordinated cross-linking responsible for the structural stability and exceptional mechanical performance of the worm jaw. Understanding the nanoscale mechanism behind this cross-linking and its pathway in affecting the macroscopic mechanical behavior of the material is crucial to develop bioinspired mechanomutable materials based on Nvjp-1. Here, we use a combination of multiscale modeling and experimental synthesis to understand the behavior of this heterologous-expressed protein from the nano- to the macroscale. We have built a bottom-up molecular-based model, which includes electronic-based density functional theory calculations, atomistic simulation of the nanoscale properties with replica exchange molecular dynamics, and an elastic network model for describing the macroscale behavior at different pHs. This multiscale modeling supports the experimental synthesis of a photo-cross-linked Nvjp-1 hydrogel by proving both the nanoscale mechanisms and mechanical behavior predictions. Our theoretical results agree well with the experimental observations, showing that Nvjp-1 forms a more compact structure in the presence of Zn2+ ions with a suitable pH environment, leading to the formation of more stable intramolecular metal-coordinated cross-links. These metal-coordinated cross-links induce nanoscale aggregation of Nvjp-1, which is responsible for the hydrogel contraction observed in experiments and predicted by the model.

3D Printed Bionic Nanodevices.

The ability to three-dimensionally interweave biological and functional materials could enable the creation of bionic devices possessing unique and compelling geometries, properties, and functionalities. Indeed, interfacing high performance active devices with biology could impact a variety of fields, including regenerative bioelectronic medicines, smart prosthetics, medical robotics, and human-machine interfaces. Biology, from the molecular scale of DNA and proteins, to the macroscopic scale of tissues and organs, is three-dimensional, often soft and stretchable, and temperature sensitive. This renders most biological platforms incompatible with the fabrication and materials processing methods that have been developed and optimized for functional electronics, which are typically planar, rigid and brittle. A number of strategies have been developed to overcome these dichotomies. One particularly novel approach is the use of extrusion-based multi-material 3D printing, which is an additive manufacturing technology that offers a freeform fabrication strategy. This approach addresses the dichotomies presented above by (1) using 3D printing and imaging for customized, hierarchical, and interwoven device architectures; (2) employing nanotechnology as an enabling route for introducing high performance materials, with the potential for exhibiting properties not found in the bulk; and (3) 3D printing a range of soft and nanoscale materials to enable the integration of a diverse palette of high quality functional nanomaterials with biology. Further, 3D printing is a multi-scale platform, allowing for the incorporation of functional nanoscale inks, the printing of microscale features, and ultimately the creation of macroscale devices. This blending of 3D printing, novel nanomaterial properties, and 'living' platforms may enable next-generation bionic systems. In this review, we highlight this synergistic integration of the unique properties of nanomaterials with the versatility of extrusion-based 3D printing technologies to interweave nanomaterials and fabricate novel bionic devices.

Plasmonic Biofoam: A Versatile Optically Active Material.

Owing to their ability to confine and manipulate light at the nanoscale, plasmonic nanostructures are highly attractive for a broad range of applications. While tremendous progress has been made in the synthesis of size- and shape-controlled plasmonic nanostructures, their integration with other materials and application in solid-state is primarily through their assembly on rigid two-dimensional (2D) substrates, which limits the plasmonically active space to a few nanometers above the substrate. In this work, we demonstrate a simple method to create plasmonically active three-dimensional biofoams by integrating plasmonic nanostructures with highly porous biomaterial aerogels. We demonstrate that plasmonic biofoam is a versatile optically active platform that can be harnessed for numerous applications including (i) ultrasensitive chemical detection using surface-enhanced Raman scattering; (ii) highly efficient energy harvesting and steam generation through plasmonic photothermal heating; and (iii) optical control of enzymatic activity by triggered release of biomolecules encapsulated within the aerogel. Our results demonstrate that 3D plasmonic biofoam exhibits significantly higher sensing, photothermal, and loading efficiency compared to conventional 2D counterparts. The design principles and processing methodology of plasmonic aerogels demonstrated here can be broadly applied in the fabrication of other functional foams.

3D Printed Programmable Release Capsules.

The development of methods for achieving precise spatiotemporal control over chemical and biomolecular gradients could enable significant advances in areas such as synthetic tissue engineering, biotic-abiotic interfaces, and bionanotechnology. Living organisms guide tissue development through highly orchestrated gradients of biomolecules that direct cell growth, migration, and differentiation. While numerous methods have been developed to manipulate and implement biomolecular gradients, integrating gradients into multiplexed, three-dimensional (3D) matrices remains a critical challenge. Here we present a method to 3D print stimuli-responsive core/shell capsules for programmable release of multiplexed gradients within hydrogel matrices. These capsules are composed of an aqueous core, which can be formulated to maintain the activity of payload biomolecules, and a poly(lactic-co-glycolic) acid (PLGA, an FDA approved polymer) shell. Importantly, the shell can be loaded with plasmonic gold nanorods (AuNRs), which permits selective rupturing of the capsule when irradiated with a laser wavelength specifically determined by the lengths of the nanorods. This precise control over space, time, and selectivity allows for the ability to pattern 2D and 3D multiplexed arrays of enzyme-loaded capsules along with tunable laser-triggered rupture and release of active enzymes into a hydrogel ambient. The advantages of this 3D printing-based method include (1) highly monodisperse capsules, (2) efficient encapsulation of biomolecular payloads, (3) precise spatial patterning of capsule arrays, (4) "on the fly" programmable reconfiguration of gradients, and (5) versatility for incorporation in hierarchical architectures. Indeed, 3D printing of programmable release capsules may represent a powerful new tool to enable spatiotemporal control over biomolecular gradients.

The incidence of over-the-counter medication use in patients with midgut neuroendocrine tumors.

Recent increases in the use of over-the-counter (OTC) medicines and the underreporting of the use of these medications to physicians have sparked interest in the number and types of "supportive" therapies used by patients with neuroendocrine tumors (NETS). Patients with NETS are of special interest as a result of the potential interactions/interferences between tumor-associated peptide and amine production and OTC supplements. A prospective analysis of patients with primary small bowel NETS between 1998 and 2012 was conducted to define and catalog each patient's prescription and OTC medication use at each clinic visit. The most recently recorded patient medications were used for this analysis. Three hundred sixty-two patients with small bowel primary NETS were studied. One hundred eighty-seven patients (51.6%) were taking nutritional supplements. Of these taking supplements, the per cent of patients taking one, two, three, or more than three supplements was 28.3, 24.1, 22.5, and 25.1 per cent, respectively. Females (n = 109) were more likely to take supplements in comparison to males (n = 78; P = 0.037). Fifty one patients (14.1%) took proton pump inhibitors and 31 patients (8.6%) took loperamide. OTC supplements were used by 50 per cent of patients with primary small bowel NETS in this study. Over one-third of our patients reported using three or more OTC medicines daily. These medicines have the potential to interact with the metabolism of prescribed medicines, modify ability to clot during surgery, exacerbate NET symptoms, and alter NET markers. Given the prevalence of OTC medications and their potential actions, it is important to carefully catalog their use.