Spider Silk Research Articles

Production and secretion of recombinant spider silk in Bacillus megaterium

BackgroundSilk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry.ResultsIn this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures.ConclusionIt is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Determination of Stokes Vector from a Single Image Acquisition

AbstractFour Stokes parameters (1852) define the polarization state of light. Measured changes of the Stokes vector of light traversing an inhomogeneous sample are linked to the local anisotropies of absorption and refraction and are harnessed over an increasing range of applications in photonics, material, and space/earth observation. Several independent polarization sensitive measurements are usually required for determination of the all four Stokes parameters, which makes such characterization procedure time‐consuming or requires complex setups. Here, a single‐snapshot approach to Stokes polarimetry in transmission is introduced by use of a four‐polarization camera with the on‐chip integrated polarizers. A quarter‐waveplate is added in front of the sample and is illuminated by a linearly polarized light. This approach is demonstrated by measuring birefringence of spider silk of only m‐diameter using microscopy, however, due to its generic nature, it is transferable to other spectral ranges and imaging applications, e.g., imaging from a fast moving satellite or drone or monitoring fast changing events such as phase transitions.

Construction of cellulose nanofiber/carbon nanotube synergistic network on carbon fiber surface to enhance mechanical properties and thermal conductivity of composites

Drawing inspiration from the robustness and resilience of spider silk, this study harnessed a combination of polydopamine (PDA), cellulose nanofibers (CNFs), polyvinyl alcohol (PVA), and aminated carbon nanotubes (CNTs) to create a synergistic network through hydrogen and covalent bonding on the surface of carbon fibers (CFs). This strategy significantly bolstered the interfacial properties of the resulting composites. The enhancements in flexural strength, flexural modulus, interlaminar shear strength (ILSS), tensile strength, and interfacial shear strength (IFSS) were measured at 47.8 %, 75.2 %, 54.3 %, 55.6 %, and 51.8 %, respectively. These improvements are attributed to the formation of robust covalent and hydrogen bonds among PDA, CNF, PVA, and aminated CNTs. Furthermore, the thermal conductivity of the CF composites saw a 59.7 % increase, owing to the CNFs and CNTs. These nanofillers are instrumental in forming an efficient thermal conductivity pathway within the resin matrix, which contributes to the improved thermal conductivity. This study introduces a straightforward, eco-friendly, and innovative approach for the production of high-performance CF composites.

Superfast Gelation of Spider Silk-Based Artificial Silk Protein.

Spider silk proteins (spidroins) have garnered attention in biomaterials research due to their ability to self-assemble into hydrogels. However, reported spidroin hydrogels require high protein concentration and prolonged gelation time. Our study engineered an artificial spidroin that exhibits unprecedented rapid self-assembly into hydrogels at physiologically relevant conditions, achieving gelation at a low concentration of 6 mg/mL at 37 °C without external additives. Remarkably, at a 30 mg/mL concentration, our engineered protein forms hydrogels within 30 s, a feature we termed "superfast gelation". This rapid formation is modulated by ions, pH, and temperature, offering versatility in biomedical applications. The hydrogel's capacity to encapsulate proteins and support E. coli growth while inducing RFP expression provides a novel platform for drug delivery and bioengineering applications. Our findings introduce a superfast, highly adaptable, and cytocompatible hydrogel that self-assembles under mild conditions, underscoring the practical implication of rapid gelation in biomedical research and clinical applications.

Replicating shear-mediated self-assembly of spider silk through microfluidics

The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of β-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers.

High-strength and ultra-tough supramolecular polyamide spider silk fibers assembled via specific covalent and reversible hydrogen bonds

Achieving ultra-high tensile strength and exceptional toughness is a longstanding goal for structural materials. However, previous attempts using covalent and non-covalent bonds have failed, leading to the belief that these two properties are mutually exclusive. Consequently, commercial fibers have been forced to compromise between tensile strength and toughness, as seen in the differences between nylon and Kevlar. To address this challenge, we drew inspiration from the disparate tensile strength and toughness of nylon and Kevlar, both of which are polyamide fibers, and developed an innovative approach that combines specific intermolecular disulfide bonds and reversible hydrogen bonds to create ultra-strong and ultra-tough polyamide spider silk fibers. Our resulting Supramolecular polyamide spider silk, which has a maximum molecular weight of 1084 kDa, exhibits high tensile strength (1180 MPa) and extraordinary toughness (433 MJ/m3), surpassing Kevlar's toughness 8-fold. This breakthrough presents a new opportunity for the sustainable development of spider silk as an environmentally friendly alternative to synthetic commercial fibers, as spider silk is composed of amino acids. Future research could explore the use of these techniques and fundamental knowledge to develop other super materials in various mechanical fields, with the potential to improve people's lives in many ways. Statement of significance• By emulating synthetic commercial fibers such as nylon and polyethylene, we have successfully produced supramolecular-weight polyamide spider silk fibers with a molecular weight of 1084 kDa through a unique covalent bond-mediated linear polymerization reaction of spider silk protein molecules. This greatly surpasses the previous record of a maximum molecular weight of 556 kDa. • We obtained supramolecular polyamide spider silk fibers with both high-tensile strength and toughness. The stress at break is 1180 MPa, and the toughness is 8 times that of kevlar, reaching 433 MJ/m3. • Our results challenge the notion that it is impossible to manufacture fibers with both ultra-high tensile strength and ultra-toughness, and provide theoretical guidance for developing environmentally friendly and sustainable structural materials that meet industrial needs.

A trade-off in evolution: the adaptive landscape of spiders without venom glands.

Venom glands play a key role in the predation and defense strategies of almost all spider groups. However, the spider family Uloboridae lacks venom glands and has evolved an adaptive strategy: they excessively wrap their prey directly with spider silk instead of paralyzing it first with toxins. This shift in survival strategy is very fascinating, but the genetic underpinnings behind it are poorly understood. Spanning multiple spider groups, we conducted multiomics analyses on Octonoba sinensis and described the adaptive evolution of the Uloboridae family at the genome level. We observed the coding genes of myosin and twitchin in muscles are under positive selection, energy metabolism functions are enhanced, and gene families related to tracheal development and tissue mechanical strength are expanded or emerged, all of which are related to the unique anatomical structure and predatory behavior of spiders in the family Uloboridae. In addition, we also scanned the elements that are absent or under relaxed purifying selection, as well as toxin gene homologs in the genomes of 2 species in this family. The results show that the absence of regions and regions under relaxed selection in these spiders' genomes are concentrated in areas related to development and neurosystem. The search for toxin homologs reveals possible gene function shift between toxins and nontoxins and confirms that there are no reliable toxin genes in the genome of this group. This study demonstrates the trade-off between different predation strategies in spiders, using either chemical or physical strategy, and provides insights into the possible mechanism underlying this trade-off. Venomless spiders need to mobilize multiple developmental and metabolic pathways related to motor function and limb mechanical strength to cover the decline in adaptability caused by the absence of venom glands.

Molecular Engineering of a Spider Silk and Mussel Foot Hybrid Protein Gives a Strong and Tough Biomimetic Adhesive

AbstractHigh performance bio‐based materials are an important part of future sustainable technology, and engineered proteins provide excellent possibilities as functional polymers. Adhesives are widely needed for composite materials and biomimetic structures. In biological adhesives, two features have emerged as especially interesting—the role of coacervation and the presence of 3,4‐dihydroxyphenylalanine (DOPA). To study these, protein engineering is used to construct a hybrid silk‐mussel foot protein (mfp) adhesive. Tyr residues in the purified mfp are oxidized to DOPA and an encoded SpyCatcher‐Tag system allowed easy click‐chemistry to couple silk and mfp and to study the parts separately. The combined silk‐mfp protein have a strong tendency to coacervate. DOPA affected the properties of coacervates and increased adhesion by several ways of measuring. In lap shear testing, the combined mfp‐silk protein is superior to any of the components studied separately. Coacervation is suggested to contribute to the adhesion of silk‐mfp, and shows several features suggested to lead to the strength and toughness of natural adhesives. In the lap shear system, coacervation have a stronger overall effect on adhesion than the presence of DOPA. The results show that protein design provides a route toward high performance biosynthetic polymers and future sustainable materials.

Spider silk enhanced tissue engineering of cartilage tissue: Approach of a novel bioreactor model using adipose derived stromal cells.

Human cartilage tissue remains a challenge for the development of therapeutic options due to its poor vascularization and reduced regenerative capacities. There are a variety of research approaches dealing with cartilage tissue engineering. In addition to different biomaterials, numerous cell populations have been investigated in bioreactor-supported experimental setups to improve cartilage tissue engineering. The concept of the present study was to investigate spider silk cocoons as scaffold seeded with adipose-derived stromal cells (ASC) in a custom-made bioreactor model using cyclic axial compression to engineer cartilage-like tissue. For chemical induction of differentiation, BMP-7 and TGF-β2 were added and changes in cell morphology and de-novo tissue formation were investigated using histological staining to verify chondrogenic differentiation. By seeding spider silk cocoons with ASC, a high colonization density and cell proliferation could be achieved. Mechanical induction of differentiation using a newly established bioreactor model led to a more roundish cell phenotype and new extracellular matrix formation, indicating a chondrogenic differentiation. The addition of BMP-7 and TGF-β2 enhanced the expression of cartilage specific markers in immunohistochemical staining. Overall, the present study can be seen as pilot study and valuable complementation to the published literature.

Bioinspired Composites a Review: Lessons from Nature for Materials Design and Performance

Bioinspired composites have become an increasingly popular area of research in materials science, as they offer a promising approach to developing high-performance materials. By drawing inspiration from the structures and properties of natural materials, researchers can design composites with enhanced mechanical, thermal, and other properties. This review article discusses the lessons that can be learned from nature for materials design and performance, with a focus on the structures and properties of biological materials such as bone, spider silk, and nacre. We explore the key mechanisms that give these materials their unique properties, including hierarchical structures, nanoscale building blocks, and interfacial interactions. By understanding these mechanisms, researchers can develop new materials with improved strength, toughness, and other desirable properties. We also discuss the potential applications of bioinspired composites in fields such as aerospace, engineering, and biomedical science. Overall, this review highlights the importance of nature as a source of inspiration for materials design and provides insights into the development of high-performance composites.

A juvenile Eastern Spinebill Acanthorhynchus tenuirostris escapes from entrapment after colliding with an Australian Golden Orb-weaving Spider Trichonephila edulis web

This note describes an observation of a juvenile Eastern Spinebill Acanthorhynchus tenuirostris escaping from spider silk twice in rapid succession. First, the bird collided with the web of an Australian Golden Orb-weaving Spider Trichonephila edulis, from which it was able to free itself. Then, it became temporarily affixed to a tree branch by a portion of silk still attached to its tail-feathers. Although this individual escaped both events, the mean body mass of the Eastern Spinebill (11 g) places the species within the size range of birds documented in the scientific literature to have succumbed to web entrapment.

Recombinant silk protein condensates show widely different properties depending on the sample background.

There is an increasing understanding that condensation is a crucial intermediate step in the assembly of biological materials and for a multitude of cellular processes. To apply and to understand these mechanisms, in vitro biophysical characterisation techniques are central. The formation and biophysical properties of protein condensates depend on a multitude of factors, such as protein concentration, pH, temperature, salt concentration, and presence of other biomolecules as well as protein purification and storage conditions. Here we show how critical the procedures for preparing protein samples for in vitro studies are. We compare two purification methods of the recombinant spider silk protein CBM-AQ12-CBM and study the effect of background molecules, such as DNA, on the formation and properties of the condensates. We characterize the condensates using aggregation induced emitters (AIEs), coalescence studies, and micropipette aspiration. The condensated sample containing background molecules exhibit a lower threshold concentration for condensate formation accompanied by a lower surface tension and longer coalescence time when compared to the pure protein condensates. Furthermore, the partitioning of small AIEs is enhanced in the presence of background molecules. Our results highlight that the purification method and remaining background molecules strongly affect the biophysical properties of spider silk condensates. Using the acquired knowledge about spider silk protein purification we derive guidelines for reproducible condensate formation that will foster the use of spider silk proteins as adhesives or carriers for biomedical applications.

The effect of ingested copper on the structural and cytotoxic properties of Steatoda grossa (Theridiidae) spider silk

Spiders, assigned to macroconcentrators of heavy metals, are particularly threatened by the toxic effects of these chemicals. Until now, it has not been specified to what extent metals alter the processes proceeding in silk glands and if such changes could consequently influence the chemical and structural properties of the spun web threads. In the present study selected biological properties of Steatoda grossa (Theridiidae) silk yarn after nutritional exposure to copper at sublethal doses (0.234 mM CuSO4) were assessed. It was determined both changes in ultrastructure of ampullate glands and hunting web’s architecture as well the cytotoxic effect in model cells (fibroblasts: line ATCC® CCL–1 NCTC clone 929), that were in contact with the analyzed biomaterial. The exposure of spiders to copper caused the occurrence of apoptotic cells in the ampullate glands as well as a significant reduction in the diameter of single fibers in double and multiple connection complexes as compared with control. At both 24 and 72 h of incubation, intensification of apoptotic and necrotic processes was observed in the fibroblast cultures that were remaining in indirect contact with the webs produced by copper–contaminated individuals. In the case of fibroblasts in direct contact with silk from the copper group, a clear cytotoxic effect resulting in an increased frequency of necrosis was observed after 72 h of incubation. The results indicated that copper may change the biological properties of spider silk and compromise its biomaterial properties.

Durability improvement strategies for wettable fog harvesting devices inspired by spider silk fibers: a review.

Water scarcity is a persistent challenge, and in this case, the freshwater content in the air and water collection phenomena observed in nature provide ideas for fog harvesting. The fog-harvesting capabilities of natural spider silk have long attracted attention. Thus, researchers have undertaken significant efforts for the preparation of wettable biomimetic knotted fibers. However, the fragility of their chemical coating and the susceptibility of spun fibers to damage often present substantial challenges in the durability of fog harvesting equipment. Herein, from a bioengineering perspective, we review the improvement strategies for enhancing the mechanical properties of wettable biomimetic spider silk fibers based on the dense nanoconfined hydrogen-bond array crystalline regions and uniformly embedded amorphous regions of natural wettable spider silk fibers. These strategies aim to achieve high tensile strength, good fracture toughness, and corrosion resistance. Additionally, by incorporating UV inhibitors during spinning, the effects of sunlight can be mitigated or shielded, thereby greatly enhancing the mechanical durability of fog-harvesting devices under harsh realistic conditions.

Functional oligo- and polypeptide assemblies for photochemical, optical and electronic applications.

The primary and secondary structures of peptides are useful as scaffolds to sequentially arrange functional groups of molecules. In this review, we review self-assembled functional peptides, whereby peptides with appropriate amino acid sequences can assemble using functional groups on their side chains. First, we apply our design strategies for the synthesis of peptide-based materials with sequenced side chains with polar moieties, organic dyes and metal complexes. The synthetic oligopeptides thus obtained exhibit inherent photoinduced charge separation and electrochemical redox activities, as well as responses to bio-sequences. Next, catalytic and photocatalytic oxidation reduction reactions and hydrogen evolution reactions are shown by utilizing the peptides with separated functionalities on both sides of β-sheets by hybridizing with electro- and photoactive graphene oxide and metal nanoparticles. Finally, the self-assembled natural proteins that form micrometre-scale spherical geometry and fibres are utilized for optical and electronic applications. The silk fibroin forms well-defined microspheres with smooth surface morphology, leading to properties suitable for use in optical resonators, which can sense external humidity because of the hygroscopic nature of silk fibroin. Dragline silk fibres can act as optical waveguides that can perform intermediate natural polymer-based optical logic operations. These functional peptides are utilizable for various applications in catalysis, optics and electronics.

Spider Web-Inspired Additive Manufacturing: Unleashing the Potential of Lightweight Support Structures

This paper explores the methodology for the utilization of spider web-inspired additive manufacturing to enhance overhang support structures in 3D printing. Inspired by the strength and flexibility of spider silk, we propose an approach that reduces material consumption and postprocessing efforts. The methodology includes 3D printing spider webs, addressing key questions on silk production, web strength, and printing path generation. Experimental results demonstrate substantial weight reduction in printed objects, showcasing the efficiency of spider web-inspired support compared to traditional methods. The potential applications extend to hollow shell printing and efficient mass production.

Spider-silk inspired ultrafast alkali-induced molecular aggregation for 3D printing arbitrary tubular hydrogels.

Fabricating tubular hydrogel models with arbitrary structural complexity and controllable diameters using an ultrafast, facile yet universal method is desirable for vascular prototypes yet still a great challenge. Herein, inspired by the denaturing ability of spider silks, a novel strategy to induce complexation via applying highly concentrated alkali into a polyvinyl alcohol/ionic liquid (PVA/IL) solution, i.e., alkali-induced molecular aggregation (AMA), is proposed to achieve such purpose. This strategy enables the rapid and facile fabrication of tubular hydrogel architectures with tunable diameters, controllable thicknesses, and excellent mechanical performance with a tensile strength of up to 1.1 MPa and stretchability exceeding 600%. Importantly, this novel strategy combined with 3D printing facilitates the rapid fabrication of a variety of precise tubular hydrogel models with connected cavity structures which are difficult to achieve using current methods. This ultrafast solidification strategy could also be extended to various alkalis, cations and anions to build different hydrogels, showcasing its versatility and universality. Hence, this strategy can be pioneering to rapidly fabricate complex three-dimensional and hollow enclosed hydrogel models for simulating endovascular interventional therapy.

Biomechanics: Rain yields tougher spider silks

Broad ecological sampling of spider silks from multiple species shows that the biomechanical properties of spider silk reflect the habitat in which their orb webs are built. Silk toughness is highest in habitats with dense rain.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.

Harmonizing Thickness and Permeability in Bone Tissue Engineering: A Novel Silk Fibroin Membrane Inspired by Spider Silk Dynamics.

Guided bone regeneration gathers significant interest in the realm of bone tissue engineering; however, the interplay between membrane thickness and permeability continues to pose a challenge that can be addressed by the water-collecting mechanism of spider silk, where water droplets efficiently move from smooth filaments to rough conical nodules. Inspired by the natural design of spider silk, an innovative silk fibroin membrane is developed featuring directional fluid transportation via harmoniously integrating a smooth, dense layer with a rough, loose layer; conical microchannels are engineered in the smooth and compact layer. Consequently, double-layered membranes with cone-shaped microporous passageways (CSMP-DSF membrane) are designed for in situ bone repair. Through extensive in vitro testing, it is noted that the CSMP-DSF membrane guides liquid flow from the compact layer's surface to the loose layer, enabling rapid diffusion. Remarkably, the CSMP-DSF membrane demonstrates superior mechanical properties and resistance to bacterial adhesion. When applied in vivo, the CSMP-DSF membrane achieves results on par with the commercial Bio-Gide collagen membranes. This innovative integration of a cross-thickness wetting gradient structure offers a novel solution, harmonizing the often-conflicting requirements of material transport, mechanical strength, and barrier effectiveness, while also addressing issues related to tissue engineering scaffold perfusion.