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Team:KULeuven/Safety
From 2011.igem.org
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 through abstraction using standard building blocks, called Biobricks. While creating an organism, the researchers and the students have to think twice whether their actions are safe for the environment and public.In E.D. Frosti we added several mechanisms to ensure the biosafety. We thought about the dangers E.D. Frosti could entail and the impact it would have on 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 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 Practices; 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) or the advisors are constantly present to guide, assist and help the students. 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: “Do I understand what I can create?” [2] Synthetic biology opened a whole new world for scientists where they can ‘try’ to create everything they have ever dreamed of. There are many remarkable ideas, from an organism that detects when people get sick and cure 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 and improve 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. Though 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 really dangerous.
The strain 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 used commercially and has not any known adverse effects on human, animals or the environment. If these bacteria were to be exposed to the environment, they would have little chance to survive. (1) 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, and then kills itself. Afterwards, we would add the bacteria with the ice nucleating proteins on their membrane in cold water, (the bacteria only acts as a physical carrier of the proteins) which allows the water to freeze faster, more crystallized and at a higher temperature. On the other hand, bacteria coated with the anti-freeze protein can be used to induce ice melting, so e.g. that instead of sprinkling salt on the roads, the bacteria can be sprinkled out. 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 backward to the necessary precautions that are needed to avoid these failures. [2]
Apocalyptic scenario 1: creation of the everlasting Ice age.
If the bacteria would make nonstop ice nucleating proteins and if the bacteria find their way into the ocean, the whole ocean could freeze. Frozen oceans lead to frozen canals, rivers and lakes. We would need new technologies for defrosting 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 gets to the arctic, 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 (1). In addition, we engineered E.D. Frosti so that it induces cell death slowly when it encounters 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 actually only function as a physical carrier for the proteins, and the most or all cells will die when exposed to a cold environment.
Deep ocean water has average temperatures between 0-3°C. [10] The ice nuclear proteins can crystalize water [11], but E. coli cannot survive or multiply at those temperatures and will be killed by the engineered cell death mechanism. The same goes for E.D. Frosti expressing the anti-freeze protein which would reach the cold artic. With this information we conclude that the probability of both the ‘’everlasting ice age’’ and the “final meltdown” scenario, happening are really small. An enormous amount of bacteria would be needed and the only way that amount could get there is if someone did it on purpose. This is a biosecurity problem.
3. Safety and the public
Scattering bacteria on the road instead of salt has a lot of advantages for the environment. Salt induces corrosion on car parts and when it floods into the surrounding environment, the change in salinity of the soils can greatly impact on local fauna. But the problem remains that the bacteria stay in the environment. The bacteria can contaminate the water and 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. Let us assume the E.D. Frosti spreads throughout the body of someone. If that person drinks a lot of cold water, in such a situation there is a huge 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, it perhaps can survive until it gets into the abdomen. In the stomach the pH is between 1 and 2, the E. D. Frosti is digested and its proteins are denatured and broken down into peptides. [5] Surviving bacteria are unable to colonize human intestine; as stated above. If the bacteria don’t get into 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.
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 copper would be in the cell debris which in high levels would cause toxicity affecting people, animals, and the environment.
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.
4. Danger of exchange of dna
