840.119:Biosteel

Project Description
What if it was possible to produce stronger body armor for the military, or even biodegradeable containers? One company, Nexia Biotechnologies has set out to do just that, with the use of recombinant spider silk proteins. Spider silk is one of the strongest naturally made fibers in the world, so the ability to produce this in fabric form could be remarkable. BioSteel, the name of this potential product, could mean anything from stronger bulletproof vests, to eye sutures, or even aircraft equipment. The first group of scientists to successfully obtain dragline silk from goat's milk was done by Lazaris[1]. Currently, other methods of producing dragline silk outside of the spider are being pursued through transgenesis in potatos, tobacco plants, and bacteria; though problems with these alternative methods exist. Hence, the main focus still remains on the production in mammary goat cells. Currently there is no work being done on the use of mammary cells and spider silk. The company has lost a lot of money and is working under a new president. These factors have forced the company to look elsewhere for profits and are now currently working with a major oil and gas company to alleviate financial problems.

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. There will also be information on associated risks and ethical issues associated with the product that the public should be aware of. This is not meant to persuade anyone to accept this, but rather, this website is to be viewed only as information purposes of the results from limited sources on this topic. Most of the information present came from one scientific article and various wesites which helped to determine information in the ethical issues portion of the website. This website is meant to be informative and interesting, enjoy!

Why is this needed?
There is no major need for this type of Biotechnology, but the results could potentially change many lives. This product started out with the help of the army to produce stronger body armor, but has evolved to have the potential for many other uses as well. The following products are possible with spider silk but not limited to these. 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 produced by spiders. Two spiders that are being looked at are Araneus diadematus and Nephila clavipes The silk is simply a long chain of proteins, with a unique sidechain construction consisting mainly of Alanine and Glycine. The sequencing of these provide unique inter-chain bonding that give the dragline silk it's characteristic strength while still being lightweight and flexible. The protein is also a natural polypeptide, similar to collagen and keratin (found in hair and nails), evidence for it's biodegradability, strength, and flexibility.

Why goats?
If spider silk is the product needed, then why not go to the source, why use goats? Unlike the silkworms, spiders are not easy to control in mass numbers for farming purposes. Also, spiders are terrestrial carnivores; they will eat each other if they are in close proximity to one another. Ok, so why mammary cells. Mammary cells are chosen because female mammals are nature's protein factories, and milk production is basically protein synthesis. These cells are also cheaper to utilize than more invasive techniques. Once the genes are incorporated into the DNA of the goats, the silk will be naturally produced inside the goat, without any harmful effects, and the proteins will then be extracted from the milk of the goat.

Scientific Approach
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. It is assumed that this milk is still edible dispite the fact that there are spider silk proteins in the milk. 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 to the dragline silk producing glands within the spiders. Hamster cells were also picked in order to determine if the experiment would be successful in other organisms. The experimental usage of these specific cells would help determine if the cells could produce spider silks in the milk of other potential transgenic animals, including goats. It was also found that the baby hamster kidney cells fared better at producing the silk. Though the reason for this is currently unknown, these cells may have a more similiar anatomy and physiology to that of the spider silk producing glands.

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. For a more in-depth description of the details about this experiment, please refer to the supplementary material for the experiment[2]. 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 ADF-3 His fibers produced from MAC-T and BHK cells obtained were brittle and difficult to manipulate[1], so they decided to focus on the ADF-3 recombinant spider silk protein in the BHK cells, since hamster cells are often used to test the effectiveness of experiments. 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[1], illustrates the results of the silks obtained compared to the natural spider silk produced. M, 75% methanol; W, water; NA, not applicable. 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[1]. For single draw, fibers were exposed to 70-80% methanol, while double drawn fibers were also exposed to water after methanol[1]. This higher draw helped develop a fiber that showcased higher toughness, tenacity, and modulus values[1]. Water allowed the silks to plasticize which allowed for stability in postspinning draw by the packing and folding of the beta sheets[1]. Tenacity is the silk's resistance to breaking or bending, while modulus is the tendency for the silk to be deformed when a force is applied. Each of these areas displayed similar numbers to the natural spider silk, except for the tenacity values. Tenacity values have the potential to increase with further optimization of the spinning process[1].

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.

With this project currently in limbo, it is unclear as to which route is the most feasible for producing spider silk. Because plants have been successfully used as a medium for producing silk as well as transgenic animals, part of the dilemma for this project may be a conflict consisting of which medium would be the most efficient for producing this product. Since the BHK cells showed to produce the silk more effectively, these cells can be used in other transgenic animals. Perhaps there is a better choice of mammal than the goat for producing silk within milk. One concern is which animal would be best suited for mass production of the silk. However, this is just speculation.

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
With any new advancement in Biotechnology, there will always be some risks. Most of the risks deal specifically with ethical issues related to the usage of animals. This experiment displayed no effects on the goats. The goats that produced the proteins were just like all other goats, except that they produced another protein. Although, this will be further explained in the next section. There is also the risk that this information could leak out and possibly abused by individuals not familiar with this technique. Obviously there needs to be regulations in order to prevent this from occuring. A great amount of testing will also need to be done through trial and error. This may take years, even after the fibers are spun into a fabric. It's a good thing that the silk is biodegradable, so there will not be a large amount of waste material. Since the material is biodegradable, how do we know that this silk will last long enough to be useful for body armor and aircraft. Will the proteins degrade while in use, and how often will a person have to replace such items made from the material? How long will eye sutures last after a surgery? Will the sutures need to be replaced often, or will they degrade the same time that the wound is healing? These are all important questions to answer before any of this product is to produced and sold to the public.

Ethical issues
Overall, there should not be many ethical issues associated with this area of Biotechnology. Yet, there will always be those few people who are not well educated and think that everything done in Biotechnology is scientists trying to play God. This is far from the truth. Granted there are those out there who wish to take advantage of new techniques, but many advancements like this one are being developed to make the world a better place, that will have more concern about the safety of individuals and the effects on the environment. Protective gear will benefit the lives of people around the world, and aircraft made from this spider silk will help protect those leaving the atmosphere from intense heat. As mentioned above, there are individuals who only look for the bad in a good situation. One problem that may be an issue is the altering of another life. There are more ethical issues with doing this with humans, and 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 develop a tendency to eat flies. Another concern is that this ability to exchange genes from one animal 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. And one must remember that scientists are not interested in taking the position of God, they are simply using available materials to produce better products from human consumption.

Scientific articles
1) Lazaris et al., 2002-01-18. Science. Vol. 295:472-476

2) http://www.sciencemag.org/cgi/content/full/295/5554/472/DC1

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