Synthetic Spider Silk Research Articles

Artificial Spider Silk Materials: From Molecular Design, Mesoscopic Assembly, to Macroscopic Performances

AbstractSpider silk is one of the strongest and toughest fibrous materials available in nature. Its superior properties make it a good candidate for applications in various fields ranging from protective body armor to scaffolds for medical implants. However, harvesting a substantial amount of natural spider silk remains challenging because spiders cannot be easily bred. With the development of synthetic biology and its integration with materials science, considerable research has been directed toward engineering and production of synthetic spider silk materials. Here the study overviews general strategies on molecular design, mesoscopic assembly, and macroscopic regulation of artificial spider silk materials. The insights into the correlation between silk protein sequences, mesoscopic assemblies, and macroscopic material properties are provided for guiding de novo design and engineering of next‐generation spider silk materials. This review also emphasizes the challenges and future perspectives for advancing the translational research on these designer functional materials for diverse applications.

Biomimetic Spider Silk by Crosslinking and Functionalization with Multiarm Polyethylene Glycol

AbstractSpider silk is renowned for its exceptional mechanical properties, surpassing those of other natural and many synthetic fibers. Yet, replicating its remarkable properties through synthetic production remains a challenge. The variability in the mechanical properties of synthetic spider silks lacking protective coatings, exacerbated by factors such as spinning conditions and humidity levels, poses an additional challenge, impacting their application potential. Bioconjugation offers a versatile synthetic method to modify protein structures, enhancing their pharmacokinetics, solubility, stability, and immune response. In particular, polyethylene glycol (PEG)‐ylation has emerged as a successful strategy with numerous marketed PEG–protein conjugates. This study introduces synthetic spider silk—multiarm PEG bioconjugates, facilitating spidroin crosslinking, and chemical functionalization while retaining a biomimetic spinning approach. Two different examples demonstrate the potential of this approach to improve the fiber's tensile strength and extensibility, respectively, both leading to an increased toughness modulus. Furthermore, the approach could allow the tuning of fiber mechanical properties without developing a new mini‐spidroin construct and fiber coating with lipids attached to multiarm PEG, potentially mitigating the impact of environmental conditions on synthetic spider silk fibers.

Quantitative Shotgun Proteomic Analysis of Bacteria after Overexpression of Recombinant Spider Miniature Spidroin, MaSp1.

Spider silk has extraordinary mechanical properties, displaying high tensile strength, elasticity, and toughness. Given the high performance of natural fibers, one of the long-term goals of the silk community is to manufacture large-scale synthetic spider silk. This process requires vast quantities of recombinant proteins for wet-spinning applications. Attempts to synthesize large amounts of native size recombinant spidroins in diverse cell types have been unsuccessful. In these studies, we design and express recombinant miniature black widow MaSp1 spidroins in bacteria that incorporate the N-terminal and C-terminal domain (NTD and CTD), along with varying numbers of codon-optimized internal block repeats. Following spidroin overexpression, we perform quantitative analysis of the bacterial proteome to identify proteins associated with spidroin synthesis. Liquid chromatography with tandem mass spectrometry (LC MS/MS) reveals a list of molecular targets that are differentially expressed after enforced mini-spidroin production. This list included proteins involved in energy management, proteostasis, translation, cell wall biosynthesis, and oxidative stress. Taken together, the purpose of this study was to identify genes within the genome of Escherichia coli for molecular targeting to overcome bottlenecks that throttle spidroin overexpression in microorganisms.

Research Progress on Spider‐Inspired Tough Fibers†

Comprehensive SummarySpider silk has attracted increasing attention due to its fascinating combination of ultra‐high tenacity high strength, and excellent elasticity. Based on the fundamental biological studies on spider silk, significant research efforts have been devoted to biotechnology and chemical synthesis to mimic or even exceed the properties of natural spider silk fibers. Moreover, the natural spider silk fiber has been simulated with the burgeoning development of numerous spinning technologies, including wet spinning, dry spinning, electrostatic spinning, and microfluidic spinning, which continuously help to optimize the properties of synthetic spider silk. The unique characteristics of natural spider silk include high refraction transmission, heat resistance, antimicrobial properties, biocompatibility, and super shrinking. Biconical recreation of spider silk with special features and extraordinary capabilities demonstrates potential applications in biomedicine, smart wearables, artificial muscles and sensors, aerospace and other domains.

Microstructure similarity analysis between synthetic phase-separated block copolymers and natural spider silk

This papers primary purpose is to explore why the microstructure of synthetic block copolymers and natural spider silk is similar at the nanoscale. This paper analyses the main chain segments and secondary structures of natural spider silk, clarifies the aggregation order, and introduces the contribution of secondary systems to the properties of natural block copolymers. Secondly, combined with the synthesis mechanism of natural spider silk, this paper summarizes and analyzes the general process of the synthesized block copolymer. Finally, the conditions of self-assembly of artificial fragments to form the structure are analyzed by employing mean-field theory and a phase diagram. Natural and ideal synthetic spider silk block copolymers have high similarity in performance. To achieve this, scientists can only start from the primary segment to understand their arrangement order in the secondary structure. Then the influence of each block on the properties of the silk fiber is analyzed. At the same time, the size and shape of self-assembled block copolymers are controlled at the micro-scale, thereby changing their properties. The mechanism above shows that the synthesized spider silk block copolymer is similar to natural spider silk in microscopic morphology.

The Application of al-Intiqal (the transition) on Synthetically Modified Organisms (SMOs): An Analysis from Shariah and Science Approaches

Synthetic biology, or SynBio, is a fast-growing technology, that combines biology, chemistry, computer science, and engineering, producing synthetically modified organisms (SMOs), in which organisms for which a large part or the entire genome has been designed using computer-aided design tool and chemically synthesized. Concern arises when (deoxyribonucleic acid) DNA constructs are made up of impure (najs) substances in Shariah as raw material, like transfer of engineered pig cells with modified genomes. One of alternative verification methods of Shariah law, namely al-Intiqal (the transition), can assist Muslim jurists in determining legal rulings on particular Halal contemporary issues, as there is no explicit information about genetic modified technology in the Qur’an or Hadith. This study aims to determine the theory of al-Intiqal (the transition) from Shariah and science approaches and apply the structure and division of al-Intiqal (the transition) on SMOs productions, like synthetic spider silk and humanized pig organ. Qualitative methods were applied including library research, fiqh adaptation (al-takyif al-fiqhi), and semi structured in-depth interview. Study shows that two divisions of al-Intiqal (the transition) can be applied within SMOs productions, which are al-Intiqal al-sahih (the accepted transition) and al-Intiqal al-fasid (the damaged transition). Study concludes that DNA synthesizer as conversion agent within SMOs production process can be considered permissible (Halal), while synthetic spider silk is also permissible (Halal) as the raw material is considered pure and its transition process had completely occurred in Shariah, nonetheless humanized pig organ is impermissible (Haram) as the raw material and its recipient organism are impermissible (Haram) and its transition process had not completely occurred in Shariah, except for emergency (darurah) case with some provisions.

Evonik to make spider silk for AMSilk

Evonik Industries has signed a manufacturing contract to supply AMSilk, a German developer of synthetic spider silk proteins, with product made from the fermentation of renewable raw materials. The agreement is for industrial quantities of the protein, which can be produced as a powder, hydrogel, fiber, or coating. Applications include clothing and coatings for medical implants. Evonik will produce the proteins at its fermentation facility in Slovakia. In 2021, AMSilk raised $35 million in series C funding.

Blood, sweat, and tears: extraterrestrial regolith biocomposites with in vivo binders.

The proverbial phrase ‘you can’t get blood from a stone’ is used to describe a task that is practically impossible regardless of how much force or effort is exerted. This phrase is well-suited to humanity’s first crewed mission to Mars, which will likely be the most difficult and technologically challenging human endeavor ever undertaken. The high cost and significant time delay associated with delivering payloads to the Martian surface means that exploitation of resources in situ — including inorganic rock and dust (regolith), water deposits, and atmospheric gases — will be an important part of any crewed mission to the Red Planet. Yet there is one significant, but chronically overlooked, source of natural resources that will — by definition — also be available on any crewed mission to Mars: the crew themselves. In this work, we explore the use of human serum albumin (HSA) — a common protein obtained from blood plasma — as a binder for simulated Lunar and Martian regolith to produce so-called ‘extraterrestrial regolith biocomposites (ERBs).’ In essence, HSA produced by astronauts in vivo could be extracted on a semi-continuous basis and combined with Lunar or Martian regolith to ‘get stone from blood’, to rephrase the proverb. Employing a simple fabrication strategy, HSA-based ERBs were produced and displayed compressive strengths as high as 25.0 MPa. For comparison, standard concrete typically has a compressive strength ranging between 20 and 32 MPa. The incorporation of urea — which could be extracted from the urine, sweat, or tears of astronauts — could further increase the compressive strength by over 300% in some instances, with the best-performing formulation having an average compressive strength of 39.7 MPa. Furthermore, we demonstrate that HSA-ERBs have the potential to be 3D-printed, opening up an interesting potential avenue for extraterrestrial construction using human-derived feedstocks. The mechanism of adhesion was investigated and attributed to the dehydration-induced reorganization of the protein secondary structure into a densely hydrogen-bonded, supramolecular β-sheet network — analogous to the cohesion mechanism of spider silk. For comparison, synthetic spider silk and bovine serum albumin (BSA) were also investigated as regolith binders — which could also feasibly be produced on a Martian colony with future advancements in biomanufacturing technology.

Large scale production of synthetic spider silk proteins in Escherichia coli

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.

The effect of terminal globular domains on the response of recombinant mini-spidroins to fiber spinning triggers

Spider silk spidroins consist of long repetitive protein strands, flanked by globular terminal domains. The globular domains are often omitted in recombinant spidroins, but are thought to be essential for the spiders’ natural spinning process. Mimicking this spinning process could be an essential step towards producing strong synthetic spider silk. Here we describe the production of a range of mini-spidroins with both terminal domains, and characterize their response to a number of biomimetic spinning triggers. Our results suggest that mini-spidroins which are able to form protein micelles due to the addition of both terminal domains exhibit shear-thinning, a property which native spidroins also show. Furthermore, our data also suggest that a pH drop alone is insufficient to trigger assembly in a wet-spinning process, and must be combined with salting-out for effective fiber formation. With these insights, we applied these assembly triggers for relatively biomimetic wet spinning. This work adds to the foundation of literature for developing improved biomimetic spinning techniques, which ought to result in synthetic silk that more closely approximates the unique properties of native spider silk.

Tensile properties of synthetic pyriform spider silk fibers depend on the number of repetitive units as well as the presence of N- and C-terminal domains

Spiders can spin seven different types of silk, some of which are well characterized, but studies on natural and synthetic pyriform silks are few. In this study, recombinant spidroins composed of one to three pyriform repeat units from Araneus ventricosus, in some cases flanked with non-repetitive N- and C-terminal domains (NT and CT), were produced and spun into continuous silk fibers using a wet-spinning process in organic solvents. All the fibers showed high and similar tensile strain (60–80%), but the Young's modulus, stress and toughness of fibers increased with increasing number of repeat units and in the presence of NT and CT as well. Systematic studies of the secondary structure contents of the different spinning dopes and spun fibers revealed no major differences between the different types of recombinant spidroins. This suggests that optimal tensile properties of artificial spider silks require the presence of several repetitive units as well as terminal domains and that secondary structure content of silk dope and fibers have limited correlation with mechanical behaviors.

Molecular Dynamics Investigation of Self- Association of Synthetic Collagen and Spider Silk Composite System for Biomaterial Applications

ABSTRACTBio-composite materials with optimal mechanical and structural properties and capability of cell differentiation are crucial for tissue engineering. Synthetic collagen proteins with lengths of approximately 10 nanometers, along with natural spider silk proteins provide an opportunity for development of an optimal biomaterial for scaffolding applications in tissue engineering. This combines unique mechanical, structural and biological properties of two of the nature’s best polymer proteins, albeit the collagen used in the present work is of the synthetic nature, it mimics the properties and advantages of natural collagen itself. In the present work, we study the binding capability of these proteins at a molecular level via molecular dynamics modeling. Spider silk and synthetic collagen protein molecular models in biophysical saline conditions under standard pressure and temperature are investigated to understand if natural binding occurs between the two without any other external factors. An initial minimum separation of 10 angstroms between the proteins was used. Binding was observed between the two proteins throughout the dynamic simulation of 100 nanoseconds. The radius of gyration and minimum distance between the proteins shows a decreasing separation between the two proteins until a stable distance of 2.5 nanometers and 0.2 nanometers respectively, is achieved. Binding is further observed between the proteins via formation of strong and stable hydrogen bonds. A hydrogen bond between collagen Proline-31 and silk Serine-96 was observed to be the most stable and frequent bond between the single collagen and silk system. Results clearly indicate a self-assembly behavior of these two systems illustrating their potential as a biomaterial for tissue engineering.

Towards engineering and production of artificial spider silk using tools of synthetic biology.

Spider silk is one of the strongest biomaterials available in nature. Its mechanical properties make it a good candidate for applications in various fields ranging from protective armour to bandages for wound dressing to coatings for medical implants. Spider silk is formed by an intricate arrangement of spidroins, which are extremely large proteins containing long stretches of repeating segments rich in alanine and glycine. A large amount of research has been directed towards harnessing the spectacular potential of spider silks and using them for different applications. The interdisciplinary approach of synthetic biology is an ideal tool to study these spider silk proteins and work towards the engineering and production of synthetic spider silk. This review aims to highlight the recent progress that has been made in the study of spider silk proteins using different branches of synthetic biology. Here, the authors discuss the different computational approaches, directed evolution techniques and various expression platforms that have been tested for the successful production of spider silk. Future challenges facing the field and possible solutions offered by synthetic biology are also discussed.

Fishing for uranium

The world’s oceans contain more than 4 billion metric tons of dissolved uranium, a reserve 1,000 times as great as terrestrial deposits. Scientists would like to tap this almost-limitless fuel supply for nuclear reactors, but uranium’s low concentration in seawater—just 3.3 ppb—makes it a formidable challenge to extract the valuable element. Researchers in China hope that some new fishing gear will help land this prize catch. Ning Wang at Hainan University and his colleagues have combined synthetic spider silk and a uranium-adsorbing protein to make strong threads with the highest uranium-uptake capacity of any fiber adsorbents tested in natural seawater. The material also works more quickly than previous systems, taking days rather than weeks to reel in its quarry (Angew. Chem., Int. Ed. 2019, DOI: 10.1002/anie.201906191). The ideal adsorbent should snag lots of uranyl ions in a short time and shun competing metal ions such as vanadyl (VO2+). It should

Microwave Assisted Sol-Gel Synthesis of Silica-Spider Silk Composites.

This study introduces a simple and environmentally friendly method to synthesize silica-protein nanocomposite materials using microwave energy to solubilize hydrophobic protein in an aqueous solution of pre-hydrolyzed organo- or fluoro-silane. Sol-gel functionality can be enhanced through biomacromolecule incorporation to tune mechanical properties, surface energy, and biocompatibility. Here, synthetic spider silk protein and organo- and fluoro-silane precursors were dissolved and mixed in weakly acidic aqueous solution using microwave technology. Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) images revealed the formation of spherical nanoparticles with sizes ranging from 100 to 500 nm depending, in part, on silane fluoro- or organo-side chain chemistry. The silane-protein interaction in the nanocomposite was assessed through infrared spectroscopy. Deconvoluted ATR-FTIR (Attenuated total reflectance Fourier-transform infrared spectroscopy) spectra revealed silane chemistry-specific conformational changes in the protein-silane nanocomposites. Relative to microwave-solubilized spider silk protein, the β structure content increased by 14% in the spider silk-organo-silica nanocomposites, but decreased by a net 20% in the spider silk-fluoro-silica nanocomposites. Methods of tuning the secondary structures, and in particular β-sheets that are the cross-linking moieties in spider silks and other self-assembling fibrillar proteins, may provide a unique means to promote protein interactions, favor subsequent epitaxial growth process, and enhance the properties of the protein-silane nanocomposites.

Method for the Destruction of Endotoxin in Synthetic Spider Silk Proteins

Although synthetic spider silk has impressive potential as a biomaterial, endotoxin contamination of the spider silk proteins is a concern, regardless of the production method. The purpose of this research was to establish a standardized method to either remove or destroy the endotoxins present in synthetic spider silk proteins, such that the endotoxin level was consistently equal to or less than 0.25 EU/mL, the FDA limit for similar implant materials. Although dry heat is generally the preferred method for endotoxin destruction, heating the silk proteins to the necessary temperatures led to compromised mechanical properties in the resultant materials. In light of this, other endotoxin destruction methods were investigated, including caustic rinses and autoclaving. It was found that autoclaving synthetic spider silk protein dopes three times in a row consistently decreased the endotoxin level 10–20 fold, achieving levels at or below the desired level of 0.25 EU/mL. Products made from triple autoclaved silk dopes maintained mechanical properties comparable to products from untreated dopes while still maintaining low endotoxin levels. Triple autoclaving is an effective and scalable method for preparing synthetic spider silk proteins with endotoxin levels sufficiently low for use as biomaterials without compromising the mechanical properties of the materials.

Piezoresponse, Mechanical, and Electrical Characteristics of Synthetic Spider Silk Nanofibers.

This work presents electrospun nanofibers from synthetic spider silk protein, and their application as both a mechanical vibration and humidity sensor. Spider silk solution was synthesized from minor ampullate silk protein (MaSp) and then electrospun into nanofibers with a mean diameter of less than 100 nm. Then, mechanical vibrations were detected through piezoelectric characteristics analysis using a piezo force microscope and a dynamic mechanical analyzer with a voltage probe. The piezoelectric coefficient (d33) was determined to be 3.62 pC/N. During humidity sensing, both mechanical and electric resistance properties of spider silk nanofibers were evaluated at varying high-level humidity, beyond a relative humidity of 70%. The mechanical characterizations of the nanofibers show promising results, with Young’s modulus and maximum strain of up to 4.32 MPa and 40.90%, respectively. One more interesting feature is the electric resistivity of the spider silk nanofibers, which were observed to be decaying with humidity over time, showing a cyclic effect in both the absence and presence of humidity due to the cyclic shrinkage/expansion of the protein chains. The synthesized nanocomposite can be useful for further biomedical applications, such as nerve cell regrowth and drug delivery.

Economic feasibility and environmental impact of synthetic spider silk production from escherichia coli

Major ampullate spider silk represents a promising protein-based biomaterial with diverse commercial potential ranging from textiles to medical devices due to its excellent physical and thermal properties. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli). This study specifically investigates the economic feasibility and environmental impact of synthetic spider silk manufacturing. Pilot scale data was used to validate an engineering process model that includes all of the required sub-processing steps for synthetic fiber manufacture: production, harvesting, purification, drying, and spinning. Modeling was constructed modularly to support assessment of alternative downstream processing technologies. The techno-economic analysis indicates a minimum sale price from pioneer and optimized E. coli plants of $761 kg−1 and $23 kg−1 with greenhouse gas emissions of 572 kg CO2-eq. kg−1 and 55 kg CO2-eq. kg−1, respectively. Elevated costs and emissions from the pioneer plant can be directly tied to the high material consumption and low protein yield. Decreased production costs associated with the optimized plant includes improved protein yield, process optimization, and an Nth plant assumption. Discussion focuses on the commercial potential of spider silk, the production performance requirements for commercialization, and the impact of alternative technologies on the system.

Predicting rates of in vivo degradation of recombinant spider silk proteins.

Developing fundamental tools and insight into biomaterial designs for predictive functional outcomes remains critical for the field. Silk is a promising candidate as a biomaterial for tissue engineering scaffolds, particularly where high mechanical loads or slow rates of degradation are desirable. Although bioinspired synthetic spider silks are feasible biomaterials for this purpose, insight into how well the degradation rate can be programmed by fine tuning the sequence remains to be determined. Here we integrated experimental approaches and computational modelling to investigate the degradation of two bioengineered spider silk block copolymers, H(AB)2 and H(AB)12 , which were designed based on the consensus domains of Nephila clavipes dragline silk. The effect of protein chain length and secondary structure on degradation was analysed in vivo. The degradation rate of H(AB)12 , the silk with longer chain length/higher molecular weight, and higher crystallinity, was slower when compared to H(AB)2 . Using full atomistic modelling, it was determined that the faster degradation of H(AB)2 was due to the lower folded molecular structure of the silk and the greater accessibility to solvent. Comparison of the specific surface areas of proteins via modelling showed that higher exposure of random coil and lower exposure of ordered domains in H(AB)2 led to the more reactive silk with a higher degradation rate when compared with H(AB)12 , as validated by the experimental results. The study, based on two simple silk designs demonstrated that the control of sequence can lead to programmable degradation rates for these biomaterials, providing a suitable model system with which to study variables in protein polymer design to predict degradation rates in vivo. This approach should reduce the use of animal screening, while also accelerating translation of such biomaterials for repair and regenerative systems. Copyright © 2016 John Wiley & Sons, Ltd.

Thermophysical properties of thin fibers via photothermal quantum dot fluorescence spectral shape-based thermometry

To improve predictions of composite behavior under thermal loads, there is a need to measure the axial thermophysical properties of thin fibers. Current methods to accomplish this have prohibitively long lead times due to extensive sample preparation. This work details the use of quantum dots thermomarkers to measure the surface temperature of thin fibers in a non-contact manner and determine the fibers’ thermal diffusivity. Neural networks are trained on extracting the temperature of a sample from fluorescence spectra in calibrated, steady-state conditions, based on different spectral features such as peak intensity and peak wavelength. The trained neural networks are then used to reconstruct the evolution of the surface temperature in transient heating experiments. In order to determine the thermal properties of a thin fiber, modulated laser heating is applied and an FFT-based method is used to extract the phase and amplitude response of the temperature field at the modulation frequency. The spatiotemporal dependence of the fluorescence signal, obtained by scanning the distance between the excitation and detection laser spots and varying the frequency response due to an axial scan and a frequency scan, is then curve-fit to the resulting decay curves by a photothermal model in order to determine the thermal diffusivity of the fiber. The measured thermal diffusivity (3.3±0.8×10−7m2s−1) of a synthetic spider silk fiber by the current method has similar properties to other synthetic silk fibers, and demonstrates the ability of the current method to more rapidly measure thermophysical properties of thin fibers.