Team:KULeuven/Safety

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HOW SAFE IS “SAFE ENOUGH”?

1. Introduction

Synthetic biology is a way of engineering organisms that do not exist in nature, by simplifying the complexity of biological systems using standard building blocks, called Biobricks. While creating an organism, researchers and students have to think twice whether their actions are safe for the environment and public.

While engineering E.D. Frosti we added several mechanisms to ensure biosafety. We thought about the dangers E.D. Frosti could entail for the environment and even for mankind if something would go wrong. But the question remains: How safe is safe enough?

2. Safety in the lab

The Irish novelist S. Lover said:” It’s better to be safe than sorry”. While working in the lab, this is the key sentence that the K.U. Leuven iGEM team 2011 kept in mind. We are working in a laboratory in a laminar flow with biosafety level 1 because the host organisms we use are non-infectious. Every student working in the laboratory attended and passed the course ‘safety and laboratory practice'.^[1] We apply the standard rules of ‘Good Laboratory Practice’; we always wear a lab coat, safety goggles and if using chemicals or handling organisms, we wear gloves. In addition, a member of the safety institution of our university ‘Health-Safety-Environment’ (HSE) is standby and the advisors are constantly present to guide, assist and help us. We reported the project to our department by filling out the forms ‘risk assessment for experiment with hazardous biological materials’. In this form we considered the danger of working with genetically modified organisms.

3. Safety and the environment

Marcus Schmidt wrote a chapter in his book ‘The technoscience and its societal consequences’: “Do I understand what I can create?” ^[2] Synthetic biology opened a whole new world for scientists where they can create everything they have ever dreamed of. There are many remarkable ideas, like an organism that detects when people become ill and cures them immediately, such as Dr. coli, to bacteria that fill micro cracks in concrete such as the Bacillafilla. ^{[3, 4]} The aim of these projects was to help people by improving the quality of their life.

But we have to keep in mind that there are also people who may not have the best intentions when creating a genetically manipulated organism. Although biosecurity – the prevention of intentional release of pathogens and toxins – is not something we can control, it is important to think about what happens if our creation falls in the hands of the wrong people. ^[2] And finally, we consider that even when we have good intentions, we can still, unintentionally, create something very dangerous.
The strain of E. coli that we are using, MG1655, is a derivate of K-12 and has the same properties. E.coli K-12 normally doesn’t colonize the human intestine. This strain is commercially used and has not any known adverse effects on humans, animals or the environment. If these bacteria are exposed to the environment, they would have low survival chance. Indeed, they would have to compete with other organisms for nutritional sources and E. coli would lose this competition since the other organisms are adapted to their habitat, while E. coli is adapted to live in the mammalian digestive system. ^{[5, 6, 7]}

The basic idea of our project is to make the bacteria E.D. Frosti, which makes ice nucleating proteins or anti-freeze proteins, after which it kills itself. Afterwards, we would add the bacteria with the ice nucleating proteins on their membrane to cold water, (the bacteria only act as a physical carrier of the proteins) which allows the water to freeze faster, and crystalize at a higher temperature. On the other hand, bacteria coated with the anti-freeze protein can be used to induce ice melting, so that they can replace sprinkling salt on the roads. But if something went wrong in our project, this could have major consequences for nature and mankind. We worked out our biosafety issues with the ‘Fault Tree Analysis theory’. This method defines unwanted scenarios and traces them back to the necessary precautions that are needed to avoid these failures. ^[2] We’ll demonstrate this theory with some examples relevant to our project.

Apocalyptic scenario 1: creation of the everlasting Ice age.

If the bacteria would make nonstop ice nucleating proteins and find their way into the ocean, the whole ocean could freeze. Frozen canals, rivers and lakes can lead to frozen oceans. We would need new technologies to defrost water so we can drink, shower, etc. But it doesn’t stop there: the ice cap will reflect more sunbeams and the temperature on earth will get lower. So eventually, the whole planet would turn to ice. This scenario would speed up the snow ball earth hypothesis. ^[8]

Apocalyptic scenario 2: the final meltdown.

Instead of the ice nucleating protein staying active, imagine that the anti-freeze protein stays active and the bacteria reach the arctic. If an enormous amount of anti-freeze protein reaches that point, it could cause a glacial meltdown. This scenario is comparable to an acceleration of the global warming, leading to more floods, tsunamis, tornado, etc. and this could lead to the extinction of human race. ^[9]

Luckily, we have taken several measures to make sure that the cells cannot overgrow the environment, as stated above. We engineered E.D. Frosti to induce cell death slowly when encountering cold temperatures. Even when the cells are dead the proteins remain attached to the cell membrane and carry on their function during an acceptable amount of time. After the first stimulus (transcription and translation of the ice nucleating protein or the anti-freeze protein), the DNase activity is induced when the cells encounter low temperatures. The DNase activity degrades the DNA and the cell dies. Thus, as mentioned before, the cells only function as a physical carrier for the proteins and most of the cells will die when exposed to a cold environment.

Deep ocean water has average temperatures between 0-3°C. ^[10] At these temperatures ice nuclear proteins are able to crystalize water ^[11], however E. coli is unable to survive or multiply and at low temperature cell death will be induced by the engineered cell death mechanism. The same goes for E.D. Frosti expressing the anti-freeze protein which would reach the cold arctic. With this information we conclude that the probability of both the "everlasting ice age" and the “final meltdown” scenario, happening are small. The amount of bacteria needed for either scenario are that huge that the only way this could happen is if it were to be done on purpose. This is a biosecurity problem.

4. Safety and the public

Apocalyptic scenario 3: Freezing from the inside out.

Scattering bacteria on the road instead of salt has a lot of advantages for the environment. Salt induces corrosion of car parts and when it floods into the surrounding natural environment, the change in salinity of the soils can greatly impact the local fauna and flora. A disadvantage however is that bacteria remain in the environment and could contaminate the water. If we ingest the bacteria by drinking very cold water and the ice nucleating protein is still active, ice crystallization in our body could cause organ damage.

If we assume the E.D. Frosti spreads throughout someone’s body and if that person drinks a lot of cold water, there is a high probability that the bacteria would instantly freeze the cold water. This could reduce the overall temperature and eventually freeze the whole human body.

The possibility of this scenario happening is insignificant. When we swallow the bacteria, they are able to survive until they get into the abdomen. In the stomach the pH lies between 1 and 2, causing the E. D. Frosti and it’s proteins to denaturize and break down into peptides. ^[5] Surviving bacteria are unable to colonize human intestine; as stated above. If the bacteria don’t enter the stomach, the INP will try to induce ice crystal formation. The protein will not succeed because the body temperature is too high to induce significant ice crystallization.

Toxicity- “Prevention is better than cure”

During the initial design of our project we planned to use copper ion as the inducer of the INP protein pathway. Although this system would work very well as a proof of concept, we quickly realized that eventually the copper would be found in high toxic levels in the cell debris affecting people, animals, and the environment.

Copper toxicity leads to vomiting, low blood pressure, coma, jaundice, vomiting of blood, melena and gastrointestinal distress. Living organisms like mammals have well defined and optimized processes to regulate the excess of copper within certain ranges. However the body becomes poisoned when faced with too high concentrations. There are general safety standards concerning metal pollution in Europe and U.S. which enforce strict regulations on the amount of copper in fumes, particles and aerosols. While identifying these risks we decided to use sugar based inducers, such as lactose and L-arabinose, to trigger the INP pathway. These do not pose any toxicity risks.

The ice nucleating protein occurs naturally in the bacterium Pseudomonas syringae. Scientists did research on the toxicity of this bacterium and proved that this bacterium is not a pathogen for humans or animals. ^[12] Yet we decided not to use this organism because it is proven to be a plant pathogen. ^[13] Pseudomonas syringae is adapted to cold temperatures, we believe if this strain was able to survive the high pressure at the surface layer of the ocean it would be able to freeze a part of it.

5. Danger of exchange of DNA

Apocalyptic Scenario 4: The 'X-men revolution'.

E. coli naturally occurs in our intestinal flora. The optimal temperature for growth is 37°C and pH is 7-8. Because E.D. Frosti is genetically engineered starting from E. coli we assume the bacteria will have the same characteristics. If we were to swallow the bacteria and we would take in the foreign DNA, this could cause the ‘X-men revolution’. “The powers” someone would gain after this, are difficult to foresee. It could result in people who can control ice formation and defrosting by self-secreting the ice nucleating protein and the anti-freeze protein.

For this to take place the DNA would have to find a way to escape or survive the acidity (pH 1-2) of the stomach, get through the cell membrane and the nucleus membrane. Given that the inside of the cell membrane is hydrophobic and DNA is a big hydrophilic molecule, this scenario is highly improbable. A more probable scenario is that other bacteria or the E. coli inside our body exchange DNA with E.D. Frosti. To avoid this, we added DNase activity in our project. We chose the DNase activity as it degrades the DNA without cell lysis and also shuts down its own induction. The degraded DNA stays in the cell, which makes the exchange of DNA with other organism less probable to occur. Cell debris are found all over the world and we believe that the cell debris won’t cause any problems, because the entire DNA is already degraded and no toxic compounds are found inside the cell.

6. Conclusion

“How safe is safe enough?” We will never know the answer to this question. The only thing we can do is to try to protect ourselves and the environment as much as possible and in case something goes wrong, try to minimize the impact of the outcome. Until now reports of major health problems with genetic manipulated micro organisms are scarce, if not absent. We believe that this is the result of the extensive risk analysis that are made beforehand and the necessary precautions that are taken while experimenting. We intend to make an organism that freezes and defreezes water. It is important that this organism doesn’t overgrow the environment or have toxic properties. We have taken enough safety precautions in our project to minimize the potential negative effects.

The discussed safety problems can be used by people who are opposed to genetic manipulated organisms to scare lay people (people who don’t know anything about this subject). It is important to explain them that the probability of these scenarios happening is very low. How to do this is further discussed in bio-ethics and education section.

References

1. Safety and laboratory practice link, 05/07/2011
2. Schmidt M., Kelle A., Ganguli-Mitra A and Vriend H., (2009) Synthetic biology - 2009 The technoscience and its societal consequences, 81-97 (chapter 6)
3. Dr. coli, link, 06/07/2011
4. Bacilla filla, link, 06/07/2011
5. Hickman Jr. C., Roberts L., Keen S., Larson A., I'Anson H., Eisenhour D., (2008) Integrated Principles of Zoology, McGraw Hill Higher Education 14th edition
6. Biotechnology Program under the Toxic Substances Control Act (TSCA), (1997) Escherichia coli K-12 derivatives final risk assessment, 12/07/2011
7. University of Wisconsin – Madison, E. coli genome project, link
8. Hoffman F. P. and Schrag P. D., (2002) The snowball Earth hypothesis: testing the limits of global change, Look for another paper about global warming, Blackwell Science 14, 129-155
9. National Academy of Sciences, National Academy of Engineering, Institute of Medicine, National Research Council, (2008) Understanding and responding to climate change - Hightlights of National Academi es Reports
10. Windows to the universe, link, 11/07/2011
11. Wu Z., Qin L. and Walker V. K., (2009) Characterization and recombinant expression of a divergent ice nucleation protein from ‘Pseudomonas borealis’, Society for general microbiology 155, 1164-1169
12. Goodnow R.A., Katz G., Haines D.C. and Terill J.B., (1990) Subacute inhalation toxicity study of an ice-nucleation-active Pseudomonas syringae administered as a respirable aerosol to rats, Toxicology Letters 54, 157-167
13. Wrzaczek M. , Brosché M., Salojärvi J., Kangasjärvi S., Idänheimo N., Mersmann S., Robatzek S., Karpiński S., Karpińska B. and Kangasjärvi J. (2010) Transcriptional regulation of the CRK/DUF26 group of Receptor-like protein kinases by ozone and plant hormones in Arabidopsis , Biomedcentral Plant Biology 10