The authors would like to gratefully acknowledge funding from the Utah Science and Technology Research (USTAR) initiative.

1. Recombinant spider silk mixture preparation from lyophilized protein stocks
<ol>
	<li>Determine the necessary formulation and volume required for the intended material formations. Typical formulations range from 3% (w/v) up to 15% (w/v). Using this selection, calculate the appropriate rSSp, concentrations, and ratios.
	<ol>
		<li>Use the following respective formulations to prepare each material described in this protocol: hydrogels/sponges/lyogels, 6% (w/v) 50:50 MaSp1:MaSp2; films/coatings, 5% (w/v) 80:20 MaSp1:MaSp2; adhesives, 12% (w/v) 50:50 MaSp1:MaSp2; fibers, 12.5% (w/v) 80:20 MaSp1:MaSp2. 
		NOTE: Even though most formulations are better fitted for specific forms and materials, there is a wide range of formulations that can often overlap. Additionally, the final rSSp materials can also be tailored during formation and processing to produce the desired properties. Generally, each protein will require investigation into appropriate or useful parameters.</li>
	</ol>
	</li>
	<li>Select a clean and new 8 mL autoclavable borosilicate glass culture vial with a rubber lined screw cap.</li>
	<li>Remove the cap and place the empty vial on an analytical balance. Tare the mass of the empty vial so that the balance reads zero mass.</li>
	<li>Add the desired lyophilized rSSp powder to the empty vial for each specific material.
	<ol>
		<li>Use these specific masses of each protein type for each material, when preparing a 2 mL of solution: hydrogels/sponges/lyogels, 60 mg of MaSp1 and 60 mg of MaSp2; films/coatings, 80 mg of MaSp1 and 20 mg of MaSp2; adhesives, 120 mg of MaSp1 and 120 mg of MaSp2; fibers, 200 mg of MaSp1 and 50 mg of MaSp2.</li>
	</ol>
	</li>
	<li>Add the desired amount of ultrapure water, at least 2 mL, to the vial that already contains the weighted rSSp powders. 
	NOTE: A minimal volume of 2 mL is recommended for all solvation procedures.</li>
	<li>Seal the vial cap and briskly vortex the contents to create a dispersed, and homogenous, rSSp mixture that is now ready for the solvation procedure. Additional homogenization approaches such as sonication or impeller mixing can be employed with, or in addition, the vortex mixing.</li>
</ol>
2. Recombinant spider silk solvation
CAUTION: High heats and pressures are generated during the solvation procedure. Proper personal protective equipment, especially goggles, long sleeves, and heat resistant gloves are required for this process.
<ol>
	<li>Perform a final check of the vial, or vessel, cap to ensure that it has been firmly and securely tightened. Then transfer the suspended rSSp mixture to a conventional microwave oven. 
	NOTE: Microwave units within the power range of 700 to 1,500 Watts, possessing smaller internal chamber capacities, and rotating platforms are recommended to provide better solvation conditions.</li>
	<li>Begin operation of the microwave with 5 s bursts at full power. After each burst briefly open the door and carefully mix/swirl the vial to prevent settling and maintain the suspended mixture.</li>
	<li>Repeat this microwave process until the mixture and/or solution has obtained a temperature of at least 130 &#176;C, when measured with an infrared thermometer directly against the solution containing portions of the vial. Repeat this process until all of the solid particulates have been completely dissolved and are no longer visible. 
	NOTE: It is suggested to allow the vial and solution to cool occasionally, especially if the formulation has a high concentration of rSSp present. Temperatures exceeding 200 &#176;C increase the risk of vial seal failure. Special attention must also be given to prevent the superheated mixture/solution from touching the seal, which will also result in vial containment failure.</li>
	<li>After successfully solvating the rSSp mixture into a solution allow the temperature of the solution and the vial cap to cool below 100 &#176;C (boiling point) before opening.</li>
</ol>
3. Hydrogels
<ol>
	<li>Prepare a hydrogel from the solution after removing it from the microwave and allowing it to cool and set. Cast the hydrogel into specific geometries prior to allowing it to fully cool. 
	NOTE: Different rSSps will require varied amounts of times to transition to a hydrogel. For example, MaSp2-like sequences tend to form hydrogels more rapidly in comparison to MaSp1-like sequences. Protein concentrations, salinity, and pH also directly affect the rate of transition to a hydrogel.</li>
</ol>
4. Sponges
<ol>
	<li>Prepare a rSSp sponges by first allowing the primary solvated solution to form a hydrogel.</li>
	<li>Place the hydrogel in a water bath, place this bath in the freezer at -20 &#176;C, and wait until the bath is frozen completely.</li>
	<li>Complete the sponge formation process by removing the frozen hydrogel and water bath from the freezer and thawing at 25 &#176;C. The resultant sponge can now be removed from the thawed water.</li>
</ol>
5. Lyogel
<ol>
	<li>Prepare a rSSp lyogel by directly freezing a formed hydrogel, either with or without a water bath, and transferring the frozen hydrogel sample to a lyophilizer (freeze dryer).</li>
	<li>Remove the final lyophilized gel material from the vessel that the moisture sublimation occurred in.</li>
</ol>
6. Films and coatings
<ol>
	<li>Use one of the three following methods: solution casting, solution spraying, or dip coating to produce films or coatings of rSSp.
	<ol>
		<li>Cast the solubilized silk solution into/onto PDMS forms of the desired shape.</li>
		<li>Pour and spread 200 &#181;L of the film solution and allow this to dry before peeling them off of the PDMS substrate for testing or treatment.</li>
		<li>After allowing these to dry, remove the formed films for mechanical testing or post-treat the films to improve the mechanical properties.</li>
	</ol>
	</li>
	<li>To prepare a coating, or a film that cannot be removed from the substrate, use either spray or dip coating to produce a thin film layer. 
	NOTE: To spray coat, this protocol has found success with a master airbrush model paint sprayer.
	<ol>
		<li>Form a dip coating by simply submerging the substrate of choice into the solubilized rSSp and repeat after drying to achieve the desired thickness.</li>
		<li>Perform an initial spray coat before applying a dip coat to increase the consistency and effectiveness of the final coating.</li>
	</ol>
	</li>
</ol>
7. Adhesives
NOTE: The formation of adhesives is achieved through one of the following methods.
<ol>
	<li>Directly add the solubilized rSSp onto a substrate and then apply a second substrate over the top of the solution. Firmly clamp the pieces together and then dry the samples in an oven with a minimal temperature of 25 &#176;C for at least 16 h.</li>
	<li>Alternatively, spray the two substrate surfaces with a spray coating and then clamp the substrates together.</li>
	<li>Applying the rSSp through the dip method of coating the substrates and sticking the substrates can also be used to prepare and adhesive.</li>
</ol>
8. Wet-Spun fibers
<ol>
	<li>Load the solubilized dope solution into a concentric syringe with Luer-Lok tip through a 19 G glide needle. Eject air bubbles and let the dope sit at the Luer-Lok end of the syringe.</li>
	<li>Insert at least 25 mm of PEEK tubing, internal diameter 0.01 inch, into the PEEK tubing&#8217;s one-piece finger tight Fittings for 1/16 inch OD and 10/32 cone. Attach this fitting to a PEEK tubing to Luer-Lok female adapter.
	<ol>
		<li>Replace the 19 gauge needle with this set up on the loaded syringe.</li>
	</ol>
	</li>
	<li>Fill a tall, clear glass bath with 99% pure isopropanol to use for coagulation bath.
	<ol>
		<li>Fill the stretch baths, located below the stretch godets. These will have 80:20 isopropanol: distilled water in the first stretch bath, and 20:80 isopropanol: distilled water in the second stretch bath.</li>
	</ol>
	</li>
	<li>Set the godet stretch system such that the first godet after the coagulation bath and the first godet in the first stretch bath are rotating at the same speed.
	<ol>
		<li>Initiate the first stretch by adjusting the speeds of the final godet in stretch bath 1, the middle upper godet, and the first godet in stretch bath 2 to the same speed. This speed will be 2x as fast as the initial fiber removal speed.</li>
		<li>Initiate the second stretch by adjusting the speeds of the final godet in stretch bath 2, the last upper godet, and the winder to the same speed. This speed will be 2x as fast as the speed used for the first stretch or 4x the initial fibers removal speed.</li>
		<li>Place nitrile gloves on the outside of the intermediate godets to keep the fiber from slipping.</li>
	</ol>
	</li>
	<li>Start to slowly extrude the solution into the coagulation bath. In an automated system set the extrusion rate to match a removal speed of 10 mm/s.
	<ol>
		<li>Allow fiber extrusion to become uniform before pulling the fibers out of the bath using with a thin metal hook or forceps. Verify removing the fiber from the bath created a loop between the PEEK tubing tip and path the fiber leaving the bath the bath.</li>
	</ol>
	</li>
	<li>Guide the retrieved fiber through the series of godets such that the fiber is submerged in the stretch baths but drying in the air between the stretch baths and before going onto a spool. This drying is achieved by the higher placed intermediate godets. 
	NOTE: The fiber removal rate and/or extrusion rate will need to be adjusted based upon the protein concentration, additives, and protein type to allow ample coagulation time without pooling fibers on the bottom of the coagulation bath.</li>
	<li>Attach the fully stretched fiber to the spool on the winder mechanism using tape.</li>
</ol>

<ol>
	<li>Huemmerich, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Structure+Elements+of+Spider+Dragline+Silks+and+Their+Contribution+to+Protein+Solubility.">Primary Structure Elements of Spider Dragline Silks and Their Contribution to Protein Solubility.</a> Biochemistry. 43 (42), 13604-13612 (2004).</li><li>Schacht, K., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Hydrogel+Formation+of+a+Recombinant+Spider+Silk+Protein.">Controlled Hydrogel Formation of a Recombinant Spider Silk Protein.</a> Biomacromolecules. 12 (7), 2488-2495 (2011).</li><li>Jones, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+Than+Just+Fibers:+An+Aqueous+Method+for+the+Production+of+Innovative+Recombinant+Spider+Silk+Protein+Materials.">More Than Just Fibers: An Aqueous Method for the Production of Innovative Recombinant Spider Silk Protein Materials.</a> Biomacromolecules. 16 (4), 1418-1425 (2015).</li><li>Tucker, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+Physical+Properties+of+Recombinant+Spider+Silk+Films+Using+Organic+and+Aqueous+Solvents.">Mechanical and Physical Properties of Recombinant Spider Silk Films Using Organic and Aqueous Solvents.</a> Biomacromolecules. 15 (8), 3158-3170 (2014).</li><li>Harris, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sticky+Situation:+An+Investigation+of+Robust+Aqueous-Based+Recombinant+Spider+Silk+Protein+Coatings+and+Adhesives.">A Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives.</a> Biomacromolecules. 17 (11), 3761-3772 (2016).</li><li>Jones, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+Heat+and+Pressure+for+Solubilization+of+Recombinant+Spider+Silk+Proteins+in+Aqueous+Solution.">Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.</a> International Journal of Molecular Sciences. 17 (11), 1955 (2016).</li><li>Copeland, C. G., Bell, B. E., Christensen, C. D., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Process+for+the+Spinning+of+Synthetic+Spider+Silk.">Development of a Process for the Spinning of Synthetic Spider Silk.</a> ACS Biomaterials Science and Engineering. 1 (7), 557-584 (2015).</li><li>Arcidiacono, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+Processing+and+Fiber+Spinning+of+Recombinant+Spider+Silks.">Aqueous Processing and Fiber Spinning of Recombinant Spider Silks.</a> Macromolecules. 35 (4), 1262-1266 (2002).</li><li>Work, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Major+Ampullate+Silk+Fiber+Formation+by+Orb-Web-Spinning+Spiders.">Mechanisms of Major Ampullate Silk Fiber Formation by Orb-Web-Spinning Spiders.</a> Transactions of the American Microscopical Society. 96 (2), 170-189 (1977).</li><li>Decker, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Destruction+of+Endotoxin+in+Synthetic+Spider+Silk+Proteins.">Method for the Destruction of Endotoxin in Synthetic Spider Silk Proteins.</a> Scientific Reports. 8 (12166), 1-6 (2018).</li></ol>

The authors declare no conflict of interest.

After recombinant spider silk proteins are purified they have to then be prepared in a solution that can be used for material formation. By mixing lyophilized spider silk protein with water and exposing this mixture to microwave irradiation, to generate heat and pressure, it possible to prepare a rSSp solution. A wide variety of material forms can be produced from this simple and efficient method of rSSp solubilization. Each material has to be uniquely prepared and processed to achieve the desired outcome and properties. With minor alterations to the initial formulations, formation conditions, and/or processing parameters, each material can be easily adjusted using this method. There are more forms than are presented here and through further investigations by others in the field, these materials will continue to evolve to explore new material forms using this technique.
Provided that the solution is comprised of primarily water and protein (additives can be utilized to delay gelation and improve stability of the solutions) the possibility for functionalization with biologically active components is greatly improved in comparison to HFIP based rSSp solutions. A variety of components, but not exhaustive samplings have been included in the dopes and thus material forms including: antibiotics, antimycotics, heparin, silver nanoparticles, and integrins for cell adhesion. In addition to additives, multiple recombinant spider silk proteins of various sizes, sequences, natures, and sources have been successfully solvated with this method and used in the formation of materials described in this protocol.
Further expanding the usefulness of this method of solubilization for not just rSSp but all proteins solvated in this method, is that the solutions are sterile provided the temperature and pressures inside the vial or chamber are sufficiently high. These solutions can be and have been taken directly to cell culture without contaminating the cultures.
If these materials are to be taken directly into in vivo systems, endotoxin levels must be addressed. A triple autoclave method that destroys endotoxins so that their levels are at, or below, the recommended 0.25 EU/mL has recently been reported10. While the autoclave is useful for destroying the endotoxin, its pressures and temperatures usually fail to reach the critical temperature or pressure required to completely solvate all of the rSSp samples attempted to date6. This necessitates microwaving or a temperature/pressure reactor necessary to complete the solvation.
Uniquely, removal of endotoxin and solvation of the material using heat and pressure does not degrade the protein or the mechanical ability of the resultant material form4,5,6,7. It is appreciated that there likely is a tipping point of obtaining too high of pressure and/or temperature and too many cycles of heat and pressure that results in degraded mechanical ability and/or destruction of the protein. This tipping point will likely vary for the type of rSSp solvated and, to some degree, the length of the rSSp utilized. However, with this basic method of solvation, several scouting solvation experiments can be performed in short order to delineate appropriate solvation temperature and pressures required for specific proteins.

Spider silks have garnered the interest of material scientists for their impressive combination of strength, elasticity, and biocompatibility. Recreating fibers has traditionally been the thrust of the research. This effort was hampered by the recombinant spider silk protein (rSSp) insolubility in water as well as the inability of traditional solvation techniques (chaotropic agents and detergents) to achieve aqueous solvation. Further, techniques that have been developed for solvating versions of rSSp do not work on all rSSp variants and also require substantial manipulation and time that often results in protein loss1,2. This has largely resulted in the field utilizing 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as a solvent from which to form fibers, and other limited material forms. The advantage being that all known rSSp are soluble in HFIP, providing data uniformity between each research group. The disadvantage is that HFIP is a toxic solvent that is expensive and impractical to scale due to health concerns and environmental considerations.
A novel approach to rSSp solvation was developed that bridged the technological gap between the harsh organic solvent HFIP and other techniques that selectively worked for rSSp solvation. The combination of specific heats and pressures was applied to suspensions of rSSp and water. The results were near 100% solvation and recovery of the rSSp, as well as high protein concentrations; a variety of materials forms were determined to be possible from these formulations that were not all achievable using HFIP or other organic solvents3,4,5,6. The objective of this approach is to efficiently and easily solubilize purified and dried recombinant spider proteins in an aqueous solution that can then be utilized for the production of a variety of material forms.
Fibers, films, coatings, adhesives, hydrogels, lyogels, microspheres, and sponge materials are all readily accomplishable from the same aqueous rSSp solution using this method. The continued evolution of this method, not only with additional rSSp but with other proteins, could lead to new material forms and alternative protein purification and solubilization avenues.

<table><tbody><tr><td>3 mL Syringe with Luer-Lok Tip</td><td>BD</td><td>309657</td><td>Other size syringes can be used but to keep the tips on, it is advised to use luer-lok tips</td></tr><tr><td>4 mL culture vial, clear with rubber lined cap</td><td>Wheaton</td><td>225142</td><td>Minimum dope volume is 1mL, max is 2mL</td></tr><tr><td>8 mL culture vial, clear with rubber lined cap</td><td>Wheaton</td><td>225144</td><td>Minimum dope volume is 2mL, max is 4mL</td></tr><tr><td>99% Isopropyl Alcohol, Reagent ACS/USP Grade</td><td>Pharmco-Aaper</td><td>231000099</td><td></td></tr><tr><td>Freezone 4.5 Plus</td><td>Labconco</td><td>7386030</td><td>Freeze Dryer</td></tr><tr><td>Luer Adapter Female Luer x 10-32 Female, Tefzel (ETFE)</td><td>IDEX</td><td>P-629</td><td></td></tr><tr><td>Microwave</td><td>Magic Chef</td><td>HMD1110B</td><td>120V, 60Hz AC; 1000 watts; 1.1 cu. ft. capacity; with glass turn table</td></tr><tr><td>One-Piece Fingertight 10-32 Coned, for 1/16&quot; OD</td><td>IDEX</td><td>F-120X</td><td></td></tr><tr><td>PEEK Tubing 1/16&quot; OD x 0.010&quot; ID</td><td>IDEX</td><td>1531B</td><td></td></tr><tr><td>Sprayer: Master Airbrush</td><td>Master Airbrush</td><td>TC-60</td><td></td></tr></tbody></table>

material formation of recombinant spider silks through aqueous solvation using heat and pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

From the described method of solubilization of rSSp, a variety of material forms can be achieved as seen in Figure 1. The method of solubilization is to apply heat and pressure, generated by a conventional microwave, to a suspension of rSSp and water. When critical temperatures and pressures are achieved, the protein will solubilize. From this solubilized rSSp solution, the required conditions are presented for seven material forms: hydrogels, lyogels, sponge, adhesives, coatings, films, and fibers. Hydrogels are prepared by allowing the solubilized rSSp to cool and naturally self-associate. A lyogel is prepared by lyophilizing the hydrogel. Sponge material is formed by freezing the hydrogel while it is immersed in water. Films can be prepared by casting the solubilized rSSp onto PDMS surfaces (and other amenable surfaces) and dried. The PDMS allows the film to be easily removed for post treatments or analysis. Coatings and adhesives are generated using either spray or dip methods or combinations of spray and dip. Fibers require the most extensive processing by extruding into a coagulation bath and then serially stretching the raw fiber in post-spin stretch baths. Fibers can be generated by extruding into a coagulation bath alone. However, the best mechanical ability in the fibers requires stretching in post-spin stretch baths3,7,8,9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59318/59318fig1.jpg" /> 
Figure 1: Aqueous solvation and rSSp materials. Representative pictures of the materials that have been formulated using this solubilization method of heat and pressure with rSSp solvated in water. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59318/59318fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

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5.1K Views.  Utah State University. Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Video: Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Biology

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Neuroscience

Formation and Attachment of FN-Silk Membranes to Inserts of Cell Culture 

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Author Spotlight: Enhancing In Vitro Cell Culture Models with Recombinant Functionalized Spider Silk Membranes

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Summary

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Chapters in this video

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Synthetic Spider Silk Production on a Laboratory Scale

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

Microdissection of Black Widow Spider Silk-producing Glands

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches