840.119:Biosteel

Project Description

The first group of scientists to successfully obtain dragline silk from goat's milk was done by Lazaris, et al, 2002. The project was successful in obtaining spider silk, but not enough to spin into a fiber. In the future, if scientists are successful in obtaining enough with the same strength as a natural spider web, the possibilities for its use would be limitless. In the experiment done by these scientists in 2002, they started out with the spider silk gene, and then placed that gene into the egg of a goat. Once the goat grew to an adult, it was cabable of producing silk within its own milk producing glands. Currently, other methods of producing dragline silk outside of the spider are being persued through transgenesis in potatos, tobacco plants, and bacteria; though problems with these alternative methods exist. Hence, the main focus still remains on the goat production method, though Nexia Biotechnologies has as of late put this new application of biotechnology on hold.

Objectives

The focus of this website is to inform the public about prior research using recombinant spider silk genes in mammary glands. The following information will describe the potential products from this silk and the results of one well known experiment in this area. Background information on the spider and their proteins will also be described.

State of the Art

Why is this needed?

This product could potentially be used for artificial tendons, and limbs; tissue repair; sutures for eye or neurosurgery. Using this product could potentially save hundreds of lives through stronger bulletproof vests, and its use as medical suturing. Because spider silk is biodegradable, this product could replace plastic bottles and nylon to make these products safer for the environment. Since silk is lighter than many synthetic fibers, it has the potential to be used for aircraft and cars

Spider Silk

Over 400 million years of evolution has perfected this amazing fiber. The silk has evolved into a fiber that is both durable and yet invisible in order to be successful at capturing prey. First, the silk fibers start to form within the silk gland, then the silk is draw out of the body; as the silk dries, it becomes taut, forming an insoluble and biodegradable thread. The dragline silk is of greater importance since it is the strongest of the silks that are porduced by spiders. Two of the spiders that are of interest and that produce this are the Araneus diadematus and Nephila clavipes The silk is simply a long chain of proteins, mainly Alanine and Glycine, which give the silk its elasticity and strength. The protein is a natural polypeptide, similar to collagen and keratin (found in hair and nails).

Why goats?

Spiders are terrestrial carnivores, so they will eat each other if they are in close proximity to one another. The reason mammary cells were chosen was because female mammals are nature's protein factories, and milk production is basically protein synthesis. The proteins that are put together are then easily obtained from the milk that is given out anyway.

Scientific Approach

Canada's Nexia Biotechnologies were the first to discover what would happen when a spider silk gene was inserted into a goat's genome. Because the spider silk producing gland is similiar to the goat mammary glands, they were able to inject the gene into an unfertilized egg. This 'biopolymer' is then produced in the specialized epithelial cells in the goats mammary glands. When the female goat goes through lactation, the spider gene will turn on and subsequently off once the female is done lactating. In one day, the goat can produce 1.5 liters of milk, with a substantial amount of this being the silk proteins. The silk can be harvested from the milk through intense heat; cooking off all other materials except for the silk which is strong enough to resist denaturing.

The arachnid genes used in this process are actually derived from two seperate spider species. These include Nephila clavipes, and Araneus diadematus. The specific genes used from these spiders are the ADF-3 and ADF4, derived from A. diadematus', as well as MaSpI and MaSpII from N. clavipes.

Methods

The following is taken from the Lazaris experiment which successfully obtained spider silk in mammary cells. Two cell lines were chosen for the expression of spider genes: MAC-T (bovine mammary epithelial alveolar cells) and BHK (baby hamster kidney cells). MAC-T cells were picked since they are secretary epithelial cells, which are similar in cell type within the spiders, and the experimental usage of them would help determine if the cells could produce spider silks in the milk of transgenic animals, such as goats.

Experiment

Each of the spider genes (ADF-3His, ADF-3, MaSpI, and MaSpII) had their own vectors designed specifically for them. E coli was the plasmid used in all cases. MAC-T and BHK were transfected, then allowed to sit for 7-8 days. The surviving colonies were expanded further.

Results indicate that BHK cells showed higher amounts of ADF-3 proteins than MAC-T cells. This demonstrates that hamsters have greater capabilities of incorporating the new genes rather than goats. The following table shows the results of the recombinant proteins and compares that information to natural silk proteins. All areas except for tenacity demonstrated similar results comparable to natural silk. Table 1. Taken from Lazaris experiment, illustrates the results of the silks obtained compared to the natural spider silk produced. M, 75% methanol; W, water; NA, not applicable.

Recombinant spider silks compared to natural spider silk

Sample	Draw	Draw ratio	Toughness	Modulus	%Strain break	Tenacity	n
ADF-3, sample 1	M/W	5	0.895	42.8	59.6	1.91	7
ADF-3, sample-2	M/W	5	0.850	110.6	43.4	2.26	7
ADF-3, sample-3	M	4	0.645	63.2	45.0	1.8	5
Araneus, dragline	NA	NA	0.6-1.3	38-76	19-30	7-11	20

Spun fibers achieved a diameter of 40 micrometers; after postspinning, the fibers lengthened up to five times longer than original length, decreasing the diameter to 20 micrometers.

Alternative Methods

Other methods of obtaining the dragline silk, includes using recombinant DNA technology in plants, specifically the tobacco leaves and potato tubers. Up to 2% of the total soluble protein in these plants endoplasmic reticulum is found to be the dragline silk transgenic product. The silk can be obtained from these plants through extreme heat purification, due to the large stability of the silk products. Problems with this method include producing manufacturable products from the purified silk intermediate.

Attempts with yeast and bacteria were also tried. However, the size of the silk produced was too large for the yeast and bacateria to house, due to their small size. Because of the repetitive nature as well as the unusual mRNA secondary structure, there was inefficient translation, thus limited size of the produced silk proteins.

Impacts

Advantages of using Biotechnology

Recombinant silk in mammalian cells is far more efficient then obtaining enough spiders to mass produce the amount of silk that would be needed to manufacture items. With enough silk fibers, a fabric could be produced that would be five to even 20 times stronger than steel, biodegradable, flexible, and extremely lightweight for its strength. The material could be used to produce flak jackets beyond the strength of current kevlar, and biodegradable products including sutures. The use of this product along with the science of the unique molecular make-up and inter-chain bonding of the dragline silk, could potentially create huge advancements in cable/rope strength and flexibility, that could potentially be used for anything from "space-age architecture" to suspension bridges to air and spacecraft.

Associated Risks

Currently there are no known problems with using transgenesis in goats. The goats that are born are like any other healthy goats, except that they produce silk within their milk.

Ethical issues

One problem that may be an issue is the altering of another life. There are more ethical issues with doing this with humans, but there are laws against cruelty to animals. People not familiar with transgenesis may not realize that this is safe for the animal. All that is being done is the introduction of another gene, that does not alter or ruin any other processes in the goat. Only one gene is being inserted into the many genes of the goat. This will not alter the physical appearances of the goat; it will not have eight legs nor the tendency to eat flies. Another concern is that this ability to exchange genes from one animale to another could be abused and used for the production of weird cross breeds to be showcased in some type of freakshow for people to come and laugh at. With any new advancement in biotechnology, there should always be regulations so that the public can rest assured that these will not be abused.

References

Scientific articles

Lazaris et al., 2002-01-18. Science. Vol. 295:472-476

Scheller, J. et al., 2001-06. Nature Biotechnology. Vol. 19:573-577

Weblinks

http://www.eurekalert.org/pub_releases/2002-01.nbi-nau011102.php

http://www.howstuffworks.com/news-item38.htm

http://arachnophiliac.co.uk/burrow/news/spinning_steel.htm

http://www.moaa.org/magazine/January2003/f_superwarriors.asp

http://www.spacedaily.com/news/materials-02a.html

http://news.bbc.co.uk/1/hi/sci/tech/889951.stm