Justin Lo

April 24, 2006

20.109 Module 3: Synthetic Biology Paper

 

Unveiling Synthetic Biology

            With the recent influx of biological knowledge concerning the underlying language and logic on which all living organisms operate, it is only natural that human understanding would cross a threshold beyond which it would be possible not only to dissect living functions but also to create novel ones.  The idea of modifying living organisms to satisfy a human need is hardly new – people have been experimenting with modifications to living creatures for many centuries, mostly concerning the creation of hybrids (e.g. donkeys), pure-breeds (dogs), and organisms with extreme features (corn and grapefruit).  What is new is the ability to delve deeper into organisms and specify a particular function by engineering DNA and proteins.  Just as working with compiled, packaged programs severely limits one’s ability to produce a specific desired output, looking only at phenotypes limits the creative powers of a biological engineer.  In a sense, the ability to modify DNA and, by extension, proteins and protein networks of all kinds is akin to being taught a new programming language. 

            It is this ability to consciously and rationally engineer life from its essential components to produce a particular result that composes the heart of synthetic biology.  This work could be performed at various levels: mixing and matching various full genes, generating fusion proteins, or even just writing DNA from scratch.  The direct interaction of living organisms with the physical world, combined with the vast library of existing Nature-created functions that can be used as tutorials, makes synthetic biology a potentially powerful method for getting the job done.

            Of course, it would be irresponsible to simply fool around with life as if it were a set of Legos in the hands of a child.  The goals of synthetic biology are specific and poised to greatly benefit humankind: making manufacturing safer and more cost-effective, improving medical devices and treatments, and generally improving the status of living by automating basic processes with intelligent and intelligently-designed organisms.

            In many ways, Nature’s technology is light-years ahead of people’s, despite the vast number of human brains that exist and have existed.  However, there is no reason to stand around and fume in jealousy when Nature is a perfectly willing teacher.

            We now know that there are countless enzymes in the world that effectively catalyze a myriad of organic and inorganic chemical and physical reactions, all at ambient or near-ambient temperature, without the use of harmful additives and usually without producing toxic by-products.  These enzymes not only deal with the processing of sugar or the cleavage of polypeptides, but also have functions in assembling silicates that can focus light on particular points1, degrading complex hydrocarbons2, and copying DNA at the phenomenal rate of over 9000 bases per minute3.  With the recent push for the minimization of occupational hazards and for green solutions to industrial challenges, synthetic biology can be the answer.  Unlike many other environmentally friendly systems, synthetic biology is neither inherently expensive nor work-intensive.  Self-assembly, self-replication, and self-improvement are features that mean minimal work, effort, and up-keep.  Current technology can only dream of machinery that builds itself, only using dirt, air, and water.

            Additionally, living systems are naturally much more biocompatible than artificial, inorganic ones.  For this reason, biologically engineered solutions seem to be the answer for effective disease treatment, organ replacement, and many other medical applications.

            A useful application of synthetic biology that is feasible and simple in design is to facilitate the production of frosted or otherwise resurfaced or recut glass.  Frosted glass, which is ordinary glass (silicon dioxide SiO2) whose surface has been modified to maintain translucency while eliminating transparency, is used mostly in home construction (e.g., for admitting light while not sacrificing privacy) and in decoration.  Because of glass’s tendency to be inert in most reactions, harsh and possibly dangerous methods are employed to produce frosted glass.  Traditionally, frosted glass has been produced in two chief methods: acid frosting and sand-blasting, with the former giving the more pleasant visual experience4.  Acid frosting involves dipping the glass in liquid hydrofluoric acid or blasting gaseous hydrofluoric acid onto its surface – inherently dangerous processes given that hydrofluoric acid can deal serious internal tissue damage upon contact and, in severe cases of exposure, death5, 6.  Sand-blasting is safer but the process removes the glass surface by physically breaking off tiny particulates that can be dangerous if inhaled7.

            Thus, to reduce the occupational risk of frosting glass while also increasing the availability of this useful technique, I propose to employ bacteria in this setting to directly perform the frosting process.  The new frosting bacteria would be outfitted with two basic functions: first, adhesion to a new substance, and second, a novel engineered enzyme, expressed conditionally, that would degrade glass at a moderate speed.  Although adhesion to glass is possible -  such behavior is observed in transformed cell lines, especially – it may not be advantageous to have the bacteria sticking to all glass, which may result in inadvertent degradation of designated non-frosted regions or even the factory windows.  In the acid glass-frosting process, the regions to be protected from frosting are coated with a substance to prevent acid action8.  Such a marking system is key to mass-manufacturing of the same design or pattern, but in this case, it may be advantageous to “stamp” the areas that are to be frosted using the newly-conferred adhesion substance rather than those that are not, which could be for instance an oil, just something that would not dissolve in water.  A flexible stencil would facilitate the application of this substance in a regular, reproducible way.

 

 

 

 

 

 

 

 

 


            The stamped glass would be dunked into a bacterial solution and then rinsed to remove bacteria that inadvertently stuck to the non-stamped glass.  The ideal situation would be to have the glass-degradation enzyme activate when the cell detects surface contact with the stamping substance (Fig. 1, right), but programming such a mechanism would be more difficult than simply having the enzyme tied to a repressor that is produced in the presence of light (Fig. 1, left).  A method for employing light-deactivated production of a protein is described in a paper by Levskaya et. al 9.  It requires part BBa_R0082 (ompC promoter) followed by the glass-degrading enzyme, as well as a light sensor-ompR fusion protein.  The promoter and other basic DNA parts are available in the Registry of Standard Biological Parts (http://parts.mit.edu).  As a side note, it may also be possible to regulate the action of the degradation enzyme through pH without requiring dangerous levels of acids or bases11.

            The bulk of preparatory work, and indeed the part that requires the most synthetic biology, involves the production of a glass-degradation enzyme that does not, to my knowledge, exist in nature.  Fortunately, some organisms provide a clue as to how to produce this enzyme: diatoms and sponges, among other aquatic organisms, regularly deal with silicates (specifically, (ortho)silicic acid, H4SiO4) in order to build their skeletons1.  Although silicic acid is not exactly the same molecule as silicon dioxide, a couple rounds of affinity-based selection on relevant enzymes mutated randomly at the active site may yield a sequence with an affinity for SiO2. 

A prime example of an enzyme that handles silicates is silicatein alpha, found in certain sponges.  A silicatein alpha protein generator is available in the Registry of Standard Parts as BBa_T9502.  Silicatein α catalyzes the polymerization of silicic acid to form the spicules which give siliceous sponges their characteristic shape and skeleton10.  The mechanism of its action remains rather unclear, but it has been determined that it produces the needle-like spicules by self-assembling into long, fractal-like fibers and then catalyzing the polymerization of silicic acid molecules caught by the fiber11.  The self-assembly in fact occurs independently of any outside factors, and this feature could prove useful in ensuring even frosting across the desired region. 

Interestingly enough, silicatein α hails from the Cathepsin-L class of proteases, with forty-five percent amino acid sequence homology and seventy-five percent conservation of residues with similar side-groups10.  In fact, just one residue change may be responsible for silicatein’s loss of esterase activity10.       Esterases are very important in cleaving ester-bond-dependent polymers.  Although glass is not usually a polymer (although some modern methods of glass-making actually do use polymers), it may still be possible to isolate a version of the enzyme that would help split the Si ··· O – Si association that forms the glass structures, which sort of resembles an ester.

After the frosting process has been run for an appropriate amount of time (a time to frosting-depth equation would have to be empirically determined), the adhesion substrate would be removed by detergent or soap, and the now clean glass would be complete.

Frosted glass itself is a very small niche, but with the very same bacteria, whole other glass-related applications could become possible.  Through a combination of glass-polymerizing bacteria and glass-degrading bacteria, it would become possible to shape natural silicates to form custom-shaped structures (e.g. for use inside the human body), and glass-degrading bacteria could also work in conjunction with existing bioactive glasses (SiO2 with a larger proportion of lime (CaO)).  Glass etching, currently achieved using a thin rotating metal disk, could also be achieved using glass-degrading bacteria.  Lastly, it has been noted that silaceous sponges actually produce optically superior glass fibers, and work is underway to take advantage of the fibrous nature of silicatein alpha, in its wild-type form or very near it, to produce fiber optics1.

            Of course, there are plenty of other potential applications of synthetic biology – to industry, public amenities, household utilities, medicine, and so forth.  For example, one might imagine super-decomposer bacteria capable of efficiently breaking down all carbon-based trash in a landfill and producing natural gas (methane) as a by-product of this decomposition.  The two current methods of dealing with common, non-recycled waste are insufficient for both getting rid of waste and recovering potential energy.  Landfills in principle should be able to allow bacteria to break down biodegradable components in trash; unfortunately, compaction, moisture minimization, and the sheer rate at which new trash covers up old trash all contribute to lowering natural degradation to a crawl, as the bacteria, fungi, and plants fail to receive the necessary air, moisture, and light to operate12.  Incinerators do get rid of waste volume and produce electricity, but at the cost of air quality and the expelling of concentrated toxic leftovers.  Bacteria designed to operate under unique landfill conditions, and equipped to break down plastics, would be instrumental in safely reducing waste volume while also potentially producing large amounts of methane (similar to anaerobic methane-producing bacteria in swamps), which could be then collected and used to power homes and bunsen burners.  The main caveat is that sufficient enclosure and emergency fire prevention measures would be necessary precautions.  The escape of super-degrading bacteria would present a crisis due to promiscuous degradation, and the risk of spontaneous methane combustion due to heat or lightning is probably the reason why landfill design actively discourages decomposition.

            A second potential application is to thermostat technology.  Despite the advances in thermostat systems, a large number of temperature-regulating devices simply fail to react appropriately to temperature changes.  In contrast, the mammalian thermoregulation system is so precise that an increase of just one degree is considered an anomaly indicating illness.  Whereas it would be impractical to export the entire hypothalamus and thermoreceptor system for installation into homes, the incorporation of a simplified version may suffice for living quarters.  The basic concept behind thermoreceptors is very simple: there are “cold” and “hot” receptor neurons with differing conduction speeds.  Depending on which type is activated, different fluorescent proteins could be produced, which would be read by an inorganic sensor that would activate the heater, A/C, or neither.  Differing intensities of fluorescence could correspond to differing degrees of heating/cooling.  Maintenance of the cells could prove to be a burden, however, and failing to maintain them well could lead to bizarre and inconvenient results.

            Finally, synthetic biology could be the basis for a new electricity-independent flashlight.  The package would be sold with two units: the light itself and a recharger.  The recharger would simply be a super-stable colony of algae to be placed outside that has been modified to release some of its generated glucose at a steady rate.  The light unit would have non-dividing luminescent bacteria (similar to those found in flashlightfish) packed densely inside a very strong plastic shield.  Regarding the non-dividing feature of the bacteria: although the factory would have some compound that could induce division, it would be unpleasant to deal with an expanding colony of cells living inside a flashlight.  The resulting variations in output and requirements for input would be difficult to tackle. 

            The flashlight could be activated by a switch that simply fills or unfills a membraned cavity with fluid containing some ligand that would bind weakly with the bacteria and induce a cascade of light production.  An opaque black shield would be provided as back-up, and the flashlight could be recharged by hooking it up to the recharger and allowing sugar to drip in, which would feed the bacteria.  As with the thermostat, however, there are still minor issues of maintenance and possible clean-up issues (if a flashlight breaks) to consider.

            As seen in the above examples of glass frosting, trash disposal, room “homeostasis,” and organic flashlights, each newly created organism comes with its own risks.  How serious these risks would be in reality is up to debate because no household applications of the technology have yet been made available to the public.

            Nevertheless, it may be instructive to examine past technologies to weigh their benefits as well as the intentional or unintentional harmful consequences of their inception.  As pessimistic as it may sound, it is necessary to examine the worst-case scenario because of humans’ unfortunate track record of using almost anything and everything possible as an instrument of destruction.  This is not to say that technology must be banned if it can cause people harm; it is simply a good practice to put hypothetical advances and benefits in perspective before delving headfirst into its development.

            Most technology provides one of the two following services: first, making life easier by adding convenience and safety while reducing required effort in order to accomplish a task; and/or second, granting people greater power over the natural elements and each other.  Neither convenience nor power is inherently good or bad, and by extension, technology itself is neither good nor bad either.  Its role, then, depends on the person using it and the rules governing its use.

            With nuclear fission (the second variety: power-giving) came an amazing source of energy that now powers 78% of France without adding any greenhouse gases to the atmosphere and without any meltdowns within the country13.  Yet the science behind it was only pioneered for the sake of the two bombs that ended, between them, over 300,000 lives14.  Likewise, airplanes (the first variety: convenience-adding) are now key to moving people and freight around quickly for business and leisure – but they were fitted with guns and bombs (including the atomic bombs) during wartime before commercially transporting the public, and have since become a medium for large-scale terrorism as well.

            While those technologies were conceived and developed probably with knowledge of their ability to bring harm as well as good, the famous Alfred Nobel developed dynamite with purportedly pure intentions: to improve the safety of explosives by packaging them and providing a means of long-range detonation; the explosives would be used for removing hills in the way of construction or for mining.  Regarding war, he believed (at first at least) that such powerful explosives would “[make] it so horrible that no one would want to engage in it” 15.  It took just three years for dynamite to find its way into war, and it only disappeared when better bombs came to replace it16.  One can certainly find some parallels between the invention of dynamite and the introduction of synthetic biology, such as the power that each confers, the possibly unexpected results (accidental detonation of dynamite, accidental mutation in DNA), and the benign original intention.  But beyond those similarities, the two technologies really diverge.  Dynamite was a single invention; it received one patent for its design.  As such, a single law can govern its usage.  Synthetic biology is not a stand-alone invention, nor could it be covered by a single patent.  In other words, rather than being an end product, synthetic biology is an entire method of construction, the potential for future patents and a logic towards invention.

            Because of this fundamental difference in societal function, it is worth examining another historical technology in comparison to synthetic biology, this time, something that is also a method rather than a device.  Written language is a technology in its own right, and it encompasses not just a written code of letters and words, but a method of understanding it and of constructing it to create larger works that have never been produced before.  Just as DNA is the literal form of synthetic biology, words are the literal form of language, and neither DNA nor words represent the final comprehensive meaning or purpose for which they stand.

Once published, a work of writing is out of the original author’s hands and free to circulate and possibly mutate due to copying errors or misquotation.  For instance, in The Mourning Bride, William Congreve wrote “Music hath charms to soothe the savage breast, …”17.  The famous quotation has since become a proverb in the English language, but more often than not, it is quoted as “Music hath charms to soothe the savage beast” or “Music doth soothe the savage beast.”  The deletion of the “r,” while changing the meaning of the phrase, hardly affects society at all, but the fact that intention is not always preserved is one of the greatest risks of synthetic biology.  How does one ensure that mass-produced synthetic biology products will work in the same way, given the natural rate of mutation, which is especially high in the bacteria and viruses that are easiest with which to work?  Natural and inadvertent evolutionary selective pressures may aggravate this problem of inconsistency and unintended effects.  In this example, society has placed a “selective” pressure on the later form of the quote because it’s easier to say and comprehend.  Nature is notorious for trying to find ways to make things “easier” as long as the solution is energetically favorable.  If we make a super-bacterium that will can metabolize everything, but only on command, it wouldn’t be long before it figured out how to turn on its metabolites constitutively completely on accident. 

One may write instructions on how to make cheesecake or on how to make bombs.  One might write a letter declaring love or a letter encouraging a declaration of war.  Here, it becomes apparent that writing has one safeguard that synthetic biology does not: the human reading the writing may choose not to heed it if he or she so wishes.  The organisms that will carry out the DNA products of synthetic biology do not have this choice, this “voice of reason.”  Like a cell infected with a virus, the workhorse organisms would churn out the proteins according to the DNA just like a computer running a program.  Perhaps a background-check mechanism for the recipient cells of synthetic biology devices and systems will become necessary.

Even with this safeguard of reason in place, the power of writing must not be underestimated, for texts have certainly set the foundation for many revolutions and wars.  Manifestos and religious texts have been the primary written works that spurred these events.  Like plasmids, they are copied over and over again, like antibiotic or antipesticide resistance passing across populations and species.  The initial printing of the Communist Manifesto would not have caused the rise of the Soviet governments that came to control half of Europe and a good portion of Asia were it not for this proliferation long after Marx’s death.  Whether or not Marx would have even liked what happened is debatable, but the fact of the matter is that the USSR was a far cry from his original intent.

In summary, unlocking synthetic biology is not just making another dynamite.  Dynamite has been largely outmoded in present day and has been rather neatly regulated as well.  We have to be prepared for a “really cool bacterium” to become the next Communist Manifesto, or perhaps alternatively a New Testament, being overwhelmingly charitable, but also leading to a multitude of wars, Crusades, hangings, and purges that may easily continue millenia after the time of writing.

Governmental regulations can be implemented to address the malicious use of synthetic biology: treating dangerous sequences as classified information of the same rank as any confidential secret, requiring background screens of any reasonably long piece of DNA that is set to be synthesized, central regulation of DNA synthesizers, etc.  Although it is tempting to write off synthetic biology as the perfect tool for terrorists, it must be noted that there are already existing viruses that are deadly enough to deal considerable damage to entire populations.  The production of synthetic bioweapons would be difficult to do in a freelance, terrorist setting, for the slightest mishandling could lead to the end of the entire terror cell.

The greatest danger of synthetic biology, then, is probably its unpredictability.  The reason why it is so powerful is also why it is so alarming.   Whereas, up to this point in history, all regularly mass-manufactured goods have had basically a single template and many standard, identical products made from that template, synthetic biology is proposing to make two products from a template, then use those products in turn as the template.  The good part is that the product just might improve before one’s very eyes.  The bad part is that it might decide that it wants to retire from being a product and take up a new job.

Because change – or in the crudest terms, pure mistake – is so integral to the development of the biological solutions in the first place, it may turn out to be impossible to eliminate from the product.  As crazy as this may sound, maybe what needs to change is not synthetic biology, but the methods that we employ regulate the new technology.  Because organisms do change, the defenses we have against them must also change, should they escape our grasp.  That is, whenever a new bacterium is created, an antibody, drug, or even another organism that would kill it should also be created in conjunction.  Nature has taken the initiative here: for just about every organism, there is another organism that eats it.  This does not hold for human beings, but the human population situation is probably indication enough that natural predation is a key aspect of population control.  Introduced species such as the cane toads in Australia or kudzu in the United States have grown into epidemics because they have no natural antidote18.  To prevent this problem in the future, when every single synthetic biology product is essentially an alien species in its environment, an antidote is necessary.  It might even be possible to set up an hierarchy of living antidotes: specific predators made to hunt down this one product – say, stray luminescent bacteria that somehow escaped a broken flashlight – then a macrophage-type non-specific organism that would eat whatever specific predators are around and then die.  In brief summary, something brand new needs to be met with equally fresh ideas.

After all this hypothesizing, one might wonder just how real any of this technology actually is, at present.  In some ways, the present technology is in its most infant of stages, having very few useful applications ready for public consumption.  However, it  might also be said that, in analogy to the history of flight, Kittyhawk has already occurred.  Various systems have already been shown to work.  There are now bacteria capable of taking black and white photographs in response to exposure to light and a “negative” 9, bacteriophages displaying unnatural peptides such that they can nanoassemble silicon semiconductors on a simple level19, and bacteria that can autonomously produce concentric circles of fluorescence.

One interesting and basic use of synthetic biology is the production of better enzymes suited for industry.  Work by Y. Yoshikuni, T. Ferrin, and J. Keasling in the paper “Designed divergent evolution of enzyme function” shows how a non-specific (“promiscuous”) enzyme, here, sesquiterpene synthase, can be refined through simple mutations in the active site in order to yield highly specialized novel enzymes20.  Although this kind of screen is not a  new concept, it is essentially all that is needed to make, for instance, the glass-degrading enzyme in the glass-frosting bacterium proposal made earlier.

Wild-type sesquiterpene synthase catalyzes a whopping fifty-two ring-closing reactions, eight of which were considered by this group for profiling.  In essence, the group was able to create specific enzymes for seven of the eight candidates, showing that commercially useless, promiscuous enzymes are but one degree of separation away from being useful specific enzymes, which can even be stereospecific, unlike many current organic chemistry reactions.  A systematic method of custom-tailoring enzymes to new functions in this manner would greatly facilitate the production of much more complicated systems that will require unique, not-in-nature parts.

With all this groundwork laid down by recent work in synthetic biology, it seems that society is on the cusp of having biological gadgets at its disposal.  Only a few barriers remain to be surmounted by the proponents of synthetic biology.  First and foremost is that public fear, largely unfounded, of the new technology must be quelled by rigorous proof of the safety of synthetic biology and a flagship project that has clear benefits to society.  Laws, even if only provisional, that give an indication of control would also help with the public image of synthetic biology.

On the production end of things, successful synthetic biology in the near future will be dependent on the continuation of the trend in lowered prices for straight DNA synthesis, which would encourage more creative ideas and more experimentation.  There also has to be a “starter package” of sorts that would take care of the more routine tasks so that the engineer may focus on the larger picture.  This starter package would include a more extensive parts list (similar to the online protein databases, for instance), some sort of diagnostic tool on which models of systems and devices could be “debugged” prior to live-cell testing, and established protocols for directed evolution of enzymes.

As interest and faith in synthetic biology gradually increases, the real possibilities of the new technology will slowly become evident – it may be the case that all these hypothesized dreams are merely dreams in the end, while some completely unknown niche might be the perfect spot for synthetic biology to take root.  Despite the uncertainty of the new field, there is always reassurance to be found in the fact that synthetic biology is, in essence, three billion years in the making.  It is only a matter of time before the vast wealth of knowledge and capabilities accrued through the experience of all life on Earth will begin to bear fruit through the inauguration of synthetic biology.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Works Cited

1.  Wikipedia (2006).  Sponge.  Retrieved from http://en.wikipedia.org/wiki/Sponge.

 

2.  Röling, W. et al. (20 Aug 2002).  Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation.  Applications of Environmental Microbiology.  Vol. 68, Iss. 11, pp. 5537-5548.  Retrieved 24 Apr 2006 from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=129918.

 

3. Voet, D., Voet, J., and Pratt, C. (2006).  Fundamentals of Biochemistry: Life at the Molecular Level.  John Wiley & Sons: Hoboken, NJ.

 

4.  Jackson, B. (2005). Sand-blasted and acid-etched glass. Gold Reverre. Retrieved from http://www.goldreverre.com/technique/etched.html.

 

5.  Wikipedia (2006).  Hydrofluoric Acid.  Retrieved from http://en.wikipedia.org/wiki/Hydrofluoric_acid.

 

6.  Terry, A.  (12 Jan 1994).  Significant Incident Summary - Fatal Accident - Hydrofluoric Acid.  Retrieved from http://www.safetyline.wa.gov.au/pagebin/pg001383.htm.

 

7.  Wikipedia (2006).  Sandblasting.  Retrieved from http://en.wikipedia.org/wiki/Sandblasting.

 

8.  No Author.  Is it Etched?  Sandblasted?  Is it engraved?  Cut?  PatternGlass.com Glass Store.  Retrieved from http://www.patternglass.com/School/Lessons/ColorlessDecor.htm

 

9.  Levskaya, A. et al. (24 Nov 2005).  Synthetic biology: Engineering Escherichia coli to see light.  Nature.  Vol. 438, Iss. 7067, pp. 441-442.  Retrieved 24 Apr 2006 from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=129918.

 

10.  Shimizu, K. et al. (26 May 1998). Silicatein alpha : Cathepsin L-like protein in sponge biosilica. Proceedings of the National Academy of Sciences of the USA, Vol. 95, Issue 11, pp 6234-6238.  Retrieved 24 Apr 2006, from http://www.pnas.org/cgi/content/full/95/11/6234.

 

11. Murr, M. and Morse, D. (16 Aug 2005).  Fractal intermediates in the self-assembly of silicatein filaments.  Proceedings of the National Academy of Sciences of the USA, Vol. 102, Issue 33, pp 11657-11662.  Retrieved 24 Apr 2006 from http://www.pnas.org/cgi/content/full/102/33/11657.

 

12.  Environmental Protection Agency (EPA) – Office of Solid Waste (2006).  Solid Waste.  Retrieved from http://www.pollutionissues.com/Re-Sy/Solid-Waste.html.

 

13.  Uranium Information Centre, Ltd. (Apr 2006).  French Nuclear Power and Uranium Supply to France.  Retrieved from http://www.uic.com.au/nip28.htm.

 

14.  Wikipedia (2006).  Atomic Bombings of Hiroshima and Nagasaki.  Retrieved from  http://en.wikipedia.org/wiki/Atomic_bombings_of_Hiroshima_and_Nagasaki.

 

15.  Johansson, T. (2003).  Nobel turns 100: A century of honoring top achievers.  CNN In-Depth: Nobel Centennial.  Retrieved from http://www.cnn.com/SPECIALS/2001/nobel.100/overview.html.

 

16.  Tägil, S. (29 June 2005).    War and Peace in the Thinking of Alfred Nobel.  NobelPrize.org.  Retrieved from http://nobelprize.org/nobel/alfred-nobel/biographical/tagil/index.html.

 

17.  Bartleby.com (2006).  3183 - William Congreve (quotation).  Retrieved from http://www.bartleby.com/100/212.1.html

 

18.  Lewis, M. (Director). (1988). Cane Toads: An Unnatural History [Motion picture]. Australia.

 

19.  Belcher, Angela.  (2006).  Biomolecular materials lab.  Retrieved from http://belcher10.mit.edu/.

 

20.  Yoshikuni, Y., Ferrin, T., Keasling, J. (20 Apr 2006).  Designed Divergent Evolution of Enzyme Function.  Vol. 440, Iss. 7087, pp. 1078-1082.  Retrieved from http://www.nature.com/nature/journal/v440/n7087/abs/nature04607.html.