Department of Biological Sciences, University of the Pacific
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Hsia, Y., Gnesa, E., Pacheco, R., Kohler, K., Jeffery, F., Vierra, C. Synthetic Spider Silk Production on a Laboratory Scale. J. Vis. Exp. (65), e4191, doi:10.3791/4191 (2012).
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.
Spider silk has extraordinary mechanical properties that out performs several manmade materials, including high-tensile steel, Kevlar and Nylon.1 Spiders spin at least 6-7 different fiber types that display diverse mechanical properties, each designed with varying amounts of tensile strength and extensibility to perform specific biological tasks.2 Research scientists are rapidly pursuing the use of spider silks as next generation biomaterials because of their outstanding mechanical properties, their biocompatibility, and their non-toxic and green-material nature.3,4 Because of the cannibalistic and venomous nature of arachnids, harvesting spider silks through farming is not a practical strategy to meet the demands necessary for industrial scale manufacturing. Therefore, scientists have turned to the production of recombinant silk proteins in transgenic organisms coupled with in vitro spinning of synthetic fibers from these purified proteins.5-8 Expression of full-length recombinant spider silk proteins has been technically difficult given the intrinsic properties of their gene sequences, which include their highly repetitive nature and physical lengths (>15 kb), GC-rich content and biased alanine and glycine codon usage.9-11 To date, most labs have focused on expressing truncated forms of the major ampullate silk proteins MaSp1 or MaSp2 using partial cDNA sequences or synthetic genes.12-15 Spinning synthetic spider silks is a challenging process that requires mastery and knowledge in several scientific disciplines, and the intricacies of the spinning process have not been fully revealed to the general public by video representation. In fact, only a handful of labs across the globe have the expertise to express the spider silk cDNAs, purify the silk proteins, spin synthetic fibers and perform post-spin draw, and then finally test their biomaterial properties.8, 16,17 Different approaches for spinning synthetic fibers have encompassed wet and dry spinning as well as electrospinning methods.16,18,19 All procedures have one goal in common - development of a protocol that produces synthetic spider silk with mechanical properties that rival natural threads for large-scale commercial manufacturing processes.
Here we describe the procedure to generate artificial spider silks on a laboratory scale using a wet-spinning methodology. Relative to other spinning methods, wet spinning has produced the most consistent results for fiber analysis. We outline this procedure beginning with the expression of the recombinant silk proteins in bacteria, followed by their purification, and then describe the protein preparation steps for spinning, including a post-spin draw methodology applied to "as-spun" fibers that yields threads with material properties that approach the quality of natural spider silks. Our methodology is designed to closely mimic the natural spinning process of silk fibers and it draws heavily upon our expertise of the architecture and function of the silk-producing glands from orb- and cob-weaving spiders.20-22 Furthermore, we conclude with the necessary steps to determine the material properties of the synthetic fibers using a tensometer to plot stress-strain curves, which allow investigators to calculate the ultimate strength, ultimate strain, and toughness of fibers. Lastly, but of significant value, the spinning, spooling, and drawing apparatuses can be home-built using commercially available parts, rather than purchasing elaborate and costly customized equipment.
Graphical Overview: Biomimicry of the Spinning Process
Biomimicry of the natural spider silk production pathway: a route to manufacture synthetic silk. This image shows the major ampullate gland from the golden orb weaver, Nephila clavipes, and the components utilized for natural silk production (white text). The tail region synthesizes large quantities of silk proteins that are transported to the ampulla, a storage region for the spinning dope. This concentrated dope is extruded through the spinning duct where the solution experiences ion exchanges and dehydration prior to fiber extrusion. The biomimetic processes used in our laboratory are indicated by the red text. Recombinant silk production is generated using transgenic bacteria, followed by protein purification using chromatography. Next, the purified protein is subject to lyophilization to concentrate the material. Lastly, the protein is re-dissolved in HFIP and extruded from a syringe needle into an isopropanol bath.
1. Plasmid Construction and Bacterial Cell Culture Preparation
2. Cell Lysis
3. Protein Purification: Ni-NTA Affinity Column Chromatography
4. Dialysis and Lyophilization
5. Spinning Dope Preparation
6. Syringe Preparation and Apparatus Setup
7. Post-spin Draw and Sample Collection
8. Tensile Testing
9. Representative Results
From step 3, the different fractions should be analyzed by SDS-PAGE analysis and the proteins visualized with silver or Coomassie Brilliant Blue R-250. From a standard Ni-NTA column conditions, elution fractions with >90% purity can be obtained (Fig. 9). Small contaminating proteins can be further removed by extensive dialysis. Using 25 μL of spinning dope at 20% (w/v), at least 30 separate fiber samples can be collected from the continuous wound fiber on the spool (assumes an initial length of 13 mm is used). The mechanical properties can be analyzed by tensometer tests (Fig. 10). Depending on the recombinant silk protein used for the spinning process, the maximum post spin draw ratios will need to be empirically determined. In general, post spin draw ratios of 4.0x can be achieved without fiber failure (Fig. 10). Spun fibers, before or after post spin draw, can be analyzed with a scanning electron microscope to visualize the ultrastructure (Fig. 11A,B). Spun fibers can also be used for mechanical testing, displaying results that with low variation within a post spin draw ratio sample group (Fig. 10).
Figure 1. Expression of the spider silk cDNA in bacteria. A) The pBAD TOPO/Thio vector containing the spider silk cDNA of interest is transformed into competent E. coli cells. B) A single colony is inoculated into 200 mL of LB and grown to saturation overnight. Following inoculation, 800 mL of fresh LB is added and the culture is induced for expression using arabinose. C) At the conclusion of induction, the culture is pelleted by centrifugation. Click here to view larger figure.
Figure 2. Lysis of bacterial cells after spider silk protein induction. A) Twenty milliliters of 1x lysis buffer and DNase is added to the cell pellet and placed on an orbital shaker and sonicated to lyse the cells. B) The cell lysate is spun in a centrifuge to clear the supernatant of cellular debris, and the supernatant is collected. Click here to view larger figure.
Figure 3. Purification of spider silk recombinant proteins using affinity chromatography. A) The cell lysate supernatant and Ni-NTA beads are added to a chromatography column and incubated for 1 hour. B) After the flowthrough is collected, 20 mL of wash buffer and 20 mL of elution buffer are used in sequence and collected in 5 mL fractions. C) The different fractions are analyzed by SDS-PAGE; the pure samples containing the target protein are transferred to a dialysis bag and dialyzed against DI water to completion. Click here to view larger figure.
Figure 4. Preparation of the purified spider silk protein for wet-spinning. A) The dialyzed product is transferred to pre-weighed centrifuge tubes in 1 mL aliquots. B) The 1 mL aliquots are flash frozen with liquid nitrogen. C) The frozen samples are lyophilized and more dialysis sample is added. D) Dried mass is calculated and HFIP is added to the dry powder to produce a 20% (w/v) spinning dope. Click here to view larger figure.
Figure 5. Loading of the spinning dope into the glass syringe for wet-spinning. A) While holding the syringe vertically, the spinning dope is pushed to the top of the syringe column, removing air bubbles. B) The loaded syringe is attached to the syringe pump and lowered into the 95% isopropanol bath so the tip is just breaking the surface of the bath.
Figure 6. Spooling of the synthetic spider silk fibers onto a custom reeling device. A) The spooling device is constructed from a digital caliper with attached metal combs. Double sided tape is applied to both sides of the comb to attach the fiber ends. B) The spool is attached to the slow speed motor using an alligator clip. C) The fiber is slowly pulled from the alcohol bath and wound around the spool. D) Glue is applied to the edge of each fiber segment to hold them in place. Shown are two different fibers spun from different proteins.
Figure 7. Post-spin draw of the synthetic fibers using a homemade apparatus. A) The spooling device is attached to the linear actuator setup using alligator clips. B) After a post spin draw step, the spool is lifted from the bath. Isopropanol droplets are allowed to evaporate before fiber collection.
Figure 8. Mounting of the synthetic silk fibers onto a cardstock for mechanical studies. A) Collected fibers are mounted on cardstock frames with a 1" x 1" cutout. The fibers are initially held in place with double sided tape, and then fixed with glue. B) The cardstock frame is fixed onto a tensometer. The sides are then cut so the tension is only running though the fiber.
Figure 9. Size fractionation of purified recombinant MaSp1 protein fractions using SDS-PAGE analysis followed by visualization with silver staining. Protein ladder is depicted in kDa. The two wash samples show non-specific binding to the beads, while the elution samples reveal the need for 6 collections to ensure total protein recovery.
Figure 10. Stress strain curves of fibers spun from recombinant TuSp1 proteins.8 Colors show fibers that were subject to different post spin draw ratios, ranging from 2.5x to 6x. Fibers show low variation within their ratio group; as post spin draw ratios increase, the strength of the fiber is increased while extensibility is decreased.
Figure 11. Scanning electron microscopy images of fibers spun from recombinant TuSp1 proteins. A) At 500x magnification, the smooth external surface can be seen. B) At 5000x, the dense interior core can be observed from a natural break of fiber.
Synthetic fibers spun from this methodology are mechanically on the same order of magnitude compared to the natural fibers. By decreasing the amount of human error by mechanizing the spooling and post spin draw processes, the experimental variation between samples are more controlled and greatly reduced.
Our methodology offers the potential to investigate the mechanical properties of other fibers that are spun from recombinant proteins encoded from the cDNAs of other members of spider gene family. Potentially, it could determine the mechanical role of different protein modules within a fibroin, or between the different fibroin types.23 It also allows for the testing of silk fibers as composites, as more than one silk protein or other molecules can be combined or added and spun as a protein mixture.
This spinning methodology is a platform for other laboratories to easily expand upon, as the apparatuses can be constructed in a facile manner. This also allows for adjusting parameters along each step to optimize or customize the design of specific fibers with unique mechanical properties. As the need for green biomaterials for the future increases, this methodology can be adapted to produce fibers that serve a host of next generation applications.
No conflicts of interest declared.
This work was supported by NSF RUI Grants MCB-0950372 and DMR-1105310 entitled "Molecular Characterization of Black Widow Spider Silks and Mechanical Behavior of Spider Glue Silks," respectively.
|pBAD/TOPO ThioFusion Expression Kit||Invitrogen||K370-01|
|FastBreak Cell Lysis Reagent, 10x||Promega||V857C|
|Ni-NTA Agarose||Qiagen||30210||Includes instructions for buffers|
|ProteoSilver Silver Stain Kit||Sigma-Aldrich||PROTSIL1-1KT|
|FreeZone Lyophilizer||Labconco||7960041||FreeZone 12Plus|
|Needle||Hamilton||7780-01||26s Gauge, Blunt end removable needle|
|Syringe Pump||Harvard Apparatus||702208||11Plus|
|Digital Caliper||Carrera||CP5906||0-150 mm range|
|Stainless steel forceps||World Precision Instruments||501764||Mini Dumont #M5S|
|Motor||Nature Mill||7090529||12VDC, 2 rpm speed|
|Linear Actuator||Warner Electric||01-D024-0050-A06-LP-IP65||24VDC, 6 inch range|
|Dissecting microscope||Leica Microsystems||Leica MZ16|
|Digital microscope camera||Leica Microsystems||DFC320||Software: Leica Application Suite v2.8.1|
|Vannas scissors||World Precision Instruments||500260|
|Microtensometer||Aurora Scientific||310C||5N Dual-Mode System|