Name: synthetic spider silk production on a laboratory scale
Uploaded: 2012-07-18T12:00:00
Description: Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

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.

<img alt="Figure 11" src="/files/ftp_upload/4191/4191fig1overview.jpg" /> 
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 t...

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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 fa...

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 (&gt;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.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>pBAD/TOPO ThioFusion Expression Kit</td>
 <td>Invitrogen</td>
 <td>K370-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FastBreak Cell Lysis Reagent, 10x</td>
 <td>Promega</td>
 <td>V857C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ni-NTA Agarose</td>
 <td>Qiagen</td>
 <td>30210</td>
 <td>Includes instructions for buffers</td>
 </tr>
 <tr>
 <td>ProteoSilver Silver Stain Kit </td>
 <td>Sigma-Aldrich</td>
 <td>PROTSIL1-1KT</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FreeZone Lyophilizer</td>
 <td>Labconco</td>
 <td>7960041</td>
 <td>FreeZone 12Plus</td>
 </tr>
 <tr>
 <td>Hexafluoroisopropanol (HFIP)</td>
 <td>Sigma-Aldrich</td>
 <td>52512</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>7657-01</td>
 <td>250 &mu;L</td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>Hamilton</td>
 <td>7780-01</td>
 <td>26s Gauge, Blunt end removable needle</td>
 </tr>
 <tr>
 <td>Syringe Pump</td>
 <td>Harvard Apparatus</td>
 <td>702208</td>
 <td>11Plus</td>
 </tr>
 <tr>
 <td>Digital Caliper</td>
 <td>Carrera</td>
 <td>CP5906</td>
 <td>0-150 mm range</td>
 </tr>
 <tr>
 <td>Stainless steel forceps</td>
 <td>World Precision Instruments</td>
 <td>501764</td>
 <td>Mini Dumont #M5S</td>
 </tr>
 <tr>
 <td>Motor</td>
 <td>Nature Mill</td>
 <td>7090529</td>
 <td>12VDC, 2 rpm speed</td>
 </tr>
 <tr>
 <td>Linear Actuator</td>
 <td>Warner Electric</td>
 <td>01-D024-0050-A06-LP-IP65</td>
 <td>24VDC, 6 inch range</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica Microsystems</td>
 <td>Leica MZ16</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital microscope camera</td>
 <td>Leica Microsystems</td>
 <td>DFC320</td>
 <td>Software: Leica Application Suite v2.8.1</td>
 </tr>
 <tr>
 <td>Vannas scissors</td>
 <td>World Precision Instruments</td>
 <td>500260</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microtensometer</td>
 <td>Aurora Scientific</td>
 <td>310C</td>
 <td>5N Dual-Mode System</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

Synthetic Spider Silk Production on a Laboratory Scale

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 ...

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