Methods+of+Harvesting+Spider+Silk

Traditionally, in order to investigate the properties of spider silk or attempt to develop any applications for it, researchers had to collect the silk directly from spiders. In order to mass produce spider silk innovative methods have been developed; these involve cloning the genes responsible for producing the more common dragline silk threads and inserting these genes into other organisms such as E.coli, silkworms, and goats.

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 =** Traditional Methods ** =  Spiders are highly territorial and aggressive creatures; for these reasons it is not possible to raise spiders together in the same environment. In order to collect spider silk directly from spiders, these would have to be captured from the wild and housed individually. //Nephila clavipes//, a golden orb-weaving spider, has been studied extensively by numerous groups worldwide because it is a larger spider, which makes routine operations and handling a little bit easier. [1] To illustrate the struggles encountered by traditional methods of harvesting spider silk the following examples are outlined below:
 * Dr. Carl Michal, from the University of British Colombia, extracts the silk from //Nephila clavipes// directly for studying. [2] Only 1.5 mg of silk can be collected per spider, which is a small, but significant, contribution towards the 50 mg needed to run their NMR tests.
 * An 11-foot by 4-foot textile, designed by Simon Peers and Nicholas Godley, took over 4 years to make from the silk of more than a million wild spiders. [3] Simon built a machine that would extract the silk from 24 spiders simultaneously. These spiders had to be collected daily, and this task was performed by 70 people during the 4 year period. Another dozen workers were required to carefully extract about 80 feet of silk filament from each of the arachnids. Fourteen thousand spiders yield only an ounce of silk, and the finished textile weighed about 2.6 pounds.

=** Artificial Biosynthesis ** =

As outlined above, direct extraction of silk from spiders is not feasible for commercial production. Researchers have developed methods to artificially produce the liquid silk precursor using other organisms. Some of these methods are reviewed below:

=** Chimeric Silkworms ** =

Silkworms don’t exhibit the territorialism and cannibalism as seen with spiders, and hence, can be cultivated in mass. This method of cultivating silk is less expensive to scale up the products, and the hosts are naturally equipped to spin silk fibers, hence less work is needed to process the assembled fibers. Normally, scientists need to use post-production spinning technologies such as extrusion in order to convert the liquid monomers into silk fibers, however these techniques are not yet reliable or effective. One caveat of this method of production is that silkworms still produce endogenous silk proteins, thus the resulting product is actually a combination of both silkworm and spider silk fibers. On the other hand the composite fibers have been shown to be tougher than the parental silkworm fibers, and as tough as the native dragline silk fibers. Another down fall is the low level of incorporation of chimeric spider silk fibers in the overall product.

Teule et al. (2011) [4] cloned 3 sequences of spider silk genes which are highly repetitive in the natural spider’s genome and products, and inserted these into a p//iggyBac// vector (See Kraig Biocraft Laboratories Biotechnology for more information). The sequences are flanked by a promoter, //Bombyx mori//, to initiate the translation of this gene once it is inserted into the silkworms’ genome, and silkworm silk genes, fhc, to help direct the assembly process of the chimeric silk with other native silkworm silk fibres. These vectors are tissue specific, and hence the recombinant protein is only produced in the silk-gland tissue. Lastly, Teule et al. also produced a second vector which contained the same sequences, as well as an enhanced green fluorescent protein, so that the chimeric silkworm-spider silk proteins could be monitored via fluorescence.

= **Transgenic goats ** =

Mammal cells have also been used as a host to produce spider silk monomers. Research in this field was first investigated by Nexia Biotechnogies, who took genes from spider dragline silk, flanked these with regulatory sequences, and then inserted them into the mammary gland cells which are responsible for the production of milk in female goats. As a result, the spider silk proteins are produced only in the milk of lactating goats.

Lazaris et al. (2002), published some of the first work accomplished by Nexia Biotechnologies on the successful harvesting and spinning of silk using bovine mammary epithelial alveolar cells and baby hamster kidney cells as expression systems[5]. These fibres were strong, but overall, were inferior to native spider silk. Later on in 2002 Nexia Biotechnologies announced its success with inserting the same genes into a goat’s embryo; this opened the doorway for mass production of spider silk using entire mammals.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Elices et al. (2011), who is also associated with Nexia Biotechnologies, recently published the progress that has been made in the synthesis of spider silk extracted from the milk of transgenic goats.<span style="color: #0000ff; font-family: 'Times New Roman','serif'; font-size: 16px;">[6] They used two recombinant proteins which were isolated from the milk to produce the polymers with a melt extrusion spinning apparatus. It was found that the tensile behaviour of the artificial polymer was very similar to that of the natural fibres, and that the composition of the artificial polymers had little effect on the tensile properties. Thus, further research in this area needs to shift towards developing better processing methods of the monomers in order to improve the characteristics of artificial silks.


 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Metabolically engineered //Escherichia coli// **

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Xia et al. (2010) has reported success with using //E.coli// as an expression system to produce spider silk proteins of similar molecular weight and mechanical properties as native spider silk.<span style="color: #0000ff; font-family: 'Times New Roman','serif'; font-size: 16px;">[7] The vector used was a plasmid, and it contained regulatory sequences, to promote the transcription of the gene, a His-tag, to aid in purification of the protein, as well as the spider silk gene sequence. This specific spider silk gene sequence results in proteins which are really high in glycine. Thus, to ensure efficient silk protein production, the glycyl-tRNA pool was elevated by inserting a second compatible plasmid into the bacteria, which effectively resulted in over expression of the glycyl-tRNA.



<span style="font-family: 'Times New Roman',Times,serif;"> [1] Gould, P. 2002. Exploiting Spiders’ Silk. Materials today. 5(12): 42-47. <span style="font-family: 'Times New Roman',Times,serif;"> [2] Simmons, A., et al., Science (1996) 271 (5245), 84-87 <span style="font-family: 'Times New Roman',Times,serif;"> [3] http://www.wired.com/wiredscience/2009/09/spider-silk/ <span style="font-family: 'Times New Roman',Times,serif;"> [4] Teuléa F, Miaob YG, Sohnc BH, Kimc YS, Hulla JJ, Fraser MJ, Lewisa RV, Jarvisa DL. 2012. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. PNAS. doi: 10.1073/pnas.1109420109. Accessed Feb 13, 2012. <span style="color: #0000ff; font-family: 'Times New Roman','serif'; font-size: 13.3333px;">[5] <span style="font-family: 'Times New Roman','serif'; font-size: 13.3333px;"> Lazaris A, Arcidiacono S, Huang Y, Zhou JF, Duguay F, Chretien N, Welsh EA, Soares JW, Karatzas CN. 2002. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 295 (5554): 472-476. <span style="color: #0000ff; font-family: 'Times New Roman','serif'; font-size: 13.3333px;">[6] <span style="font-family: 'Times New Roman','serif';">Elices M, Guinea GV, Plaza GR, Karatzas C, Reikel C, Agullo-Rueda F, Daza R, Perez-Rigueiro J. 2011. Bioinspired fibers follow the track of natural spider silk. Macromolecules 44: 1166-1176. <span style="color: #0000ff; font-family: 'Times New Roman','serif'; font-size: 13.3333px;">[7] <span style="font-family: 'Times New Roman','serif';"> Xia XX, Qiana ZG, Kib CS, Parkb YH, Kaplanc DL, Lee SY. 2010. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. PNAS 107 (32): 14059–14063.