Team:Toulouse France/HP/Silver

iGEM Toulouse 2016

Ethics, Safety and Women in science

The Human Practices are an integral and fundamental part of our project. It was mandatory for us to question what we were doing outside from laboratory manipulations and all the consequences of our commitment. We have so thought important issues related to very different questions such as Ethics, Safety, or the place of women in nowadays science.

ETHICS

At the heart of the synthetic biology iGEM competition is the modification of microorganisms and therefore the creation of genetically modified microorganisms (GMM), a sub-category of the genetically modified organisms (GMO) that also includes higher organisms such as animals and plants. The issues raised by GMO in the public mainly concern plants (GMP) and more recently animals (GMA) including Human because of the new ease to manipulate organisms with the powerful CRISPR-Cas9 tools. These issued made us think about the ethics of our iGEM project. [Photo 1: GMA, Photo2: GMP, Photo3: GMM]

General ethics:

General considerations.

The field of biology evolves very quickly thanks to the technological advances that we witness day by day. The definition of "biology" is also changing. Formerly simply meaning "study of the living", it is now more complex because we are able to transform the living. Indeed, the birth of synthetic biology has created a revolution in the world of biology as important as the discovery of the structure of DNA in 1953 by Watson and Crick or the first complete sequencing of a human genome in 2003. It is natural that suspicion emerges in the minds of the public when they only have a vague idea of what this new discipline is, and the safety measures implemented to minimize the risks. Scientists everywhere have to work with integrity, communicate with transparency on their results, their doubts and their failures so that the public can gain more confidence.

Because respecting the living is or at least should be a value rooted in each one of us, it is only natural to be reluctant to projects that aim to modify organisms and living systems. Scientists therefore need to carefully explain the purpose of their projects. However, before doing so, to prevent a blurry debate, some basic concepts comprehensible by everybody need to be explained. First of all, how do we define a living being?

The definitions are very diverse and evolve over time. Actually more than 300 definitions have been collected by the astrophysicist Radu Popa (2010). For example, Vikipédia (an encyclopedia for children aged from 8 to 13 years old) gives the following definition: “A living being is an organism that is endowed with life, that is to say that this organism: is born, grows, nurtures itself, reproduces itself and dies”. For the NASA, “life is an auto-maintained system, capable of Darwinian evolution” (Gerald Joyce, 1992). Some definitions exclude, for example, viruses from their definition when others don’t. The limit has never been clear and yet we know is something lives almost intuitively. James Lovelock even said “The detection of life is part of our natural mental equipment”.

From the start, it is complex to define what we work with. In spite of the absence of definite answer, we can question ourselves differently: when is an organism considered “modified”? Indeed, we are not capable of changing the whole system of an organism, we can only change some functions or metabolic pathways. Is it a lack of respect to nature and the living in general to insert a gene controlling insulin production into a bacterium if that means saving thousands of human lives? From an anthropocentrism point of view the answer might be no, however from a biocentrism point of view the answer might be yes.

Whatever would be the answer, we believe that respecting living beings first and foremost rests on scientific integrity and rigor, a realization of all the implications of the scientific project, the precise knowledge of its content, and finally the establishment of security measure. We should never consider Nature and living beings solely as work tools.

In the European Union, the legal definition of GMO is mentioned in the 2001/18 directive: a GMO (Genetically Modified Organism) is “an organism, with the exception of human beings, whose genetic makeup has been modified via methods that do not occur in nature and/or through traditional crossbreeding methods”.

However, the regulation regarding GMOs varies from a country to another as far as research, production, marketing and their use are concerned. The US consider that a GMO is an organism whose “genetic makeup has been modified, either using classic selection, recombinant DNA methods (also called gene splicing) or gene modification”. The regulation is based on other criteria than the ones applied inside the European Union. Indeed, American policy is based on the product of gene modification techniques, not on the process itself. The regulation is founded on verifiable risks and does not focus on the risks that were not proven (for more details see the precautionary principle).

As mentioned in the introduction, distinction must be done between different kinds of GMOs (GMMs, PGMs, etc…). As far as the iGEM competition is concerned, only genetically modified microorganisms are pertinent.

In France, the confined use of GMMs is subject, via the 2009/41/CE directive, to a system of notification and authorization. Even though MGMs in France are under a strict regulation because of their potential effects on health and their dissemination, they’re commonly used for medical purposes (insulin production by E. coli), or in the food industry (modified yeast for beer production).

Their irruption in the production processes went almost unnoticed. They were even perceived well since they were able to engender more reliable products, as it was the case with the production of the growth hormone. It is with the PGMs that the debate has emerged. The questions raised are often linked to them, but the confusion is present and the reluctance seems to affect all GMOs.

The risks are not the same for each category of GMOs, and to the best of our knowledge, for the moment no major health or environmental problems associated to MGMs have been reported despite more than 70 years of MGMs manipulation in academic or private laboratories. This observation has been recently confirmed to us by eminent scientist from various European ethics committees (Pr Père Puigdomenech; Dr Anne Cambon-Thomsen)

Precautionary principle in France.

A very interesting law was written in 1995: the Barnier’s law, more known as the precautionary principle. It states that “the lack of certainties, given the scientific and technical knowledge of the moment, shall not delay the adoption of effective and proportionate measures that aim to prevent a risk of severe and irreversible harm to the environment to a cost economically acceptable”. It was written in response to the problems related to contaminated blood and the mad cow disease. This law applies to a lot of areas like health, environmental health, or the governance of economy and stock exchange. Thus it enables taking safety precautions even if the risk in question is not demonstrated or proven. However, no state has given this law a constitutional rank, except in France, Germany and Brazil.

The philosopher Hans Jonas tackles this subject in his book “Le Principe de Responsabilité”. According to him, every technology that contains a risk –no matter how small- of destroying humanity should be forbidden. He adds that if there are many consequences possible when using a technology, one has to decide [to use the technology or not] according to the most pessimistic hypothesis.

In France, this principle is applied for the use of GMOs and allows avoiding to the maximum the risks endangering health and the environment. However, it is sometimes difficult to quantify the risks and visualize the limit between what could be really dangerous for humanity and what is not.

Thereby, thanks to the awareness raised to this debate, numerous precautions have been taken to avoid the dissemination of GMOs, because the scientific community does not know their exact effects on the environment. Thorough studies in order to bring answers are still necessary to adapt our security measures in an efficient manner. It is thus important to act responsibly, with integrity and full conscience, and to evaluate the risks linked to using GMOs.

Ethics of our Project

The Lascaux cave protection

The Lascaux caves since 1979 belongs to the World Heritage of UNESCO. In 1946, representatives of 37 countries met in London to sign and enforce the Constitution of UNESCO (The United Nations Educational, Scientific and Cultural Organization), defining the concept of world heritage and establishing an initial list of this heritage. This concept is unique by its universal application. Indeed, the World Heritage sites belong to everyone, irrespective of the territory in which they are located. Places as amazing and diverse as the Serra da Capivara National Park in Brazil, the Pyramids of Egypt, or the historic town of Trogir in Croatia are the heritage of our world.

[Sphinx and Chephren-Pyramid, Serra da Capivara National Park (Brazil), Historic city of Trogir]

Among the 1052 world treasures that need to be protected, the Lascaux cave and its frescoes, impressive by their number and their aesthetic quality, are listed since October 1979 (ICOMOS Recommendation). This prehistoric site is located in the French countryside. It includes 147 deposits dating from the Palaeolithic era and 25 decorated caves. There is no parietal cave in the world that matches it in terms of the quality and variety of the findings (skeletons, objects, flint, various utensils). The painted hunting scenes include more than a hundred animal figures with amazing accuracy of observation, rich colors and life-like quality. The site is particularly interesting from ethnological, anthropological and aesthetic point of views, marking a milestone in the history of prehistoric art and in our human culture. Like the links of a long human chain, the Lascaux cave shows to future generations a work executed by ancient men. In order to preserve this chain, it is our duty to protect this legacy. Indeed, the walls of the cave are contaminated by black and white fungi that threaten to hide the frescoes.

[Localisation of the Lascaux cave, The lascaux cave]

Why save art?

Art is a paradox in itself. Indeed, at the time of the Renaissance, an interesting definition of art appeared: it is a disinterested and free activity, pleasant in itself, having no utility for production. But if art is aimless, why of all times, men made art and attempted to protect it?
In other words, is it really necessary to save art? Indeed, one may think that science is useful only when it is in the service of health or the environment. But why limit oneself? Combining science and disciplines based on beauty and aesthetics for example is an interesting combination. Art has existed for as long as we did, and has always seemed to be associated with what makes us humans.
However, isn’t a work of art destined to disappear? In the Tibetan Buddhist tradition, Buddhist conceived mandalas (support for meditation and visualization, symbol of the universe) in the sand (very sensitive material overtime). These disappear with the tide and weather. For Buddhists, a work of art made of sand, then destroyed, allows the artist to understand and accept the transience of material things and to detach oneself. Even today, ephemeral art is practiced for example in the Landart. This is a trend of contemporary art that uses the framework and materials of nature (wood, earth, stones, sand, water, rock, etc.). Thus, at any time, ephemeral art persists. Does it really make sense to save these frescoes? Moreover, despite all our efforts, the world tends toward entropy (second law of thermodynamics), which means that the universe tends toward disorder and disintegration. Will we actually counteract this law of nature by saving the frescoes in the cave? Or maybe, we could be satisfied with the survival to transmit this work as long as possible to the future generations? Humans have used art to fight oblivion and leave a trace of their passages on earth. Therefore, Art is an important part of our humanity. It is a proof of our intelligence and our necessity of expression. It is through the power and intelligence of men that we can organize the formless and chaos of the real world in a style, an expression. Thus, Nietzsche promotes art as the purest of spiritual entertainment. Although it is not directly useful, art can be engaged, be a witness and memory. Some works also have a decorative goal. They exist for their beauty, even though beauty is subjective. For the artist, art is also used to escape and to express themselves. Generally, art is a means of timeless communication. Thus, the Lascaux cave is a work of art, it deserves to exist, to be maintained and protected. It carries a message and a memory of the Paleolithic man.

Some philosophers of aesthetics, defined art as "sensitive knowledge": an autonomous knowledge which is opposed to knowledge through concepts. In this case, art is not for art, it has no goals but is a means of knowledge, and is necessarily a role in men’s evolution; it goes beyond the individual. It belongs to no one and is not the object of desire: it is there for contemplation of a generation, and then transmitted to the next.

These frescoes show the representation that men had of their world 18,000 years ago. Notwithstanding, wildlife depicted on walls of the cave does not match the species hunted and consumed at the time. It includes mainly horses, buffalos and goats, and rare and often dangerous animals, such as bears, rhinos and big felines. A single reindeer engraved was found when it seems that it was very consumed at the time. This art does not represent the time of the hunting scenes as one might imagine. Still, the frescoes are extremely realistic in terms of morphology and attitudes of animals. For many prehistorians, the cave is actually a sanctuary, a sort of religious monument. Other theories circulated on the significance of the frescoes compared with the celestial constellations, shamanic worship, magic destruction of these terrifying animals, etc. Anyway, these frescoes carry the message of men 18,000 years ago, and they show us how they lived and what they believed in. They carry a page of humanity. Is it not necessary to keep this message alive as long as possible?

Ethical fundaments of the Paleolitis project

Why using synthetic biology to save art? Let’s not forget that the Lascaux cave is not a usual work of art, not only by age but especially by the presence of life. In fact, the cave has a very complex ecosystem, diverse and constantly interacting. It contains its own microflora as well as other living beings like insects. Since the opening of the cave by humans caused an imbalance in the cave ecosystem, one would think it’s only coherent that men attempt to repair their mistakes. As biologists, we offer to solve this biological problem, in this fragile ecosystem, with a biological solution. Physicochemical approaches have been partially successful but the threat is still there. Our conviction is that no mechanical or chemical solution could efficiently and sustainably restore a biological balance. We therefore decided to act with the most modern means from this emerging science that is synthetic biology to build a biological solution, and so contribute to the preservation of an ancient heritage and ensure its transmission to future generations.

However, our project is merely a proof of concept. We are aware that it won’t be possible to right away test our genetically modified bacteria in real conditions. Indeed, the ecosystem of the cave is very delicate and vital for the preservation of the frescoes. Thus, it is necessary to know what might be the impact of our modified microorganism on the environment before using it to treat the caves. We need to remove the black and white fungi while preserving the balance of the ecosystem. However, after getting in touch with scientists and people working in the Lascaux cave, it turned out that we will be able to test our genetically modified bacteria in a "cave laboratories". Among others, we contacted Yvan Moënne-Loccoz, part of the scientific council of the Lascaux cave and Jean-Jacques Cleyet-Merle Director of the National Museum of Prehistory.

It is obvious that any project involving the release of genetically modified bacteria in an ecosystem, needs an in-depth studies to evaluate the evolution of the ecosystem following the addition of a genetically modified native bacterium. Such studies have been already done before. For example, a Canadian study (1) analyzed changes in microbiota of rhizosphere (soil area directly formed and influenced by roots and microorganisms) and endorhiza (internal root tissue) cucumbers after adding a genetically modified native bacterium. This allowed the estimate of the risks associated with the future use of genetically modified microorganisms (GMMs) in crops. This study shows that the ecosystem’s modification in this project could be mastered. It gives an encouraging result for the use of synthetic biology. It may be possible in the near future to get the same results for our project and why not imagine being able to reopen the cave to the public in the future?

1. Mahaffee, W. F. & Kloepper, J. W. Bacterial communities of the rhizosphere and endorhiza associated with field-grown cucumber plants inoculated with a plant growth-promoting rhizobacterium or its genetically modified derivative. Can. J. Microbiol. 43, 344–353 (1997).

SAFETY

Even if the danger of GMOs for the environment and the health have not been proved, it is still necessary to take precautionary measures. As consequence, we have set up maximum actions in the lab to avoid the biodissemination of our genetically modified bacterium. These actions had to ensure our well-being, regarding safety rules and risk prevention.

Safety in our school department

INSA safetyDuring summer, our team worked in the engineering school INSA, which has a safety department. In our laboratory, the LISBP, safety is supervised by Nathalie Doubrovine who is the person in charge to instruct us safety procedures. Thanks to her, we have completed a formation (with the NEO software) to learn the different risks related to our work in the laboratory and how to react in case of danger. The formation contained four parts: prevention, fire risk, biological risk and chemical risk. At the end, we had a test to validate our formation.

Autoclave training: our team has undertaken an autoclave training, to explain to us the explosive and implosive dangers of the dispositive and the security measures to take in order to protect ourself. A lab coat, heat resistant gloves and glasses were required for the manipulation of the autoclave.

Legislation and French Labor Law: We work in a public school for engineers (INSA Toulouse), thus we have to respect the French national regulations about working conditions and the manipulation of genetically modified organisms. As we work with microorganisms and cell cultures, we are concerned by the regulation on workers’ protection against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994). It also includes human endoparasites which may cause infections, allergies or toxicity. This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5). This Decree of the 16th July 2007 describes the technical preventive measures that are to be followed in research laboratories, where workers are likely to be exposed to biological pathogens. We have to obey to the rules of health, safety, and preventive medicine applied in public services in France (Decree No. 82-453). This decree refers to the Labour Code, Public Health Code and Environmental Code.

Basic rules in a lab

We have to apply the basic safety principles in all laboratory rooms:

It is forbidden to smoke in all rooms.
It is forbidden to drink and eat in the laboratory rooms.
It is compulsory to wear a closed lab coat in cotton.
It is compulsory to wear closed shoes.
Long hair must be tied back.
Oral pipetting of any substance is prohibited in any laboratory.

There are also others precautions when working with biological organisms:

We need to wash our hands regularly.
It is compulsory to wear gloves except for the use of an electric burner.
In some cases (UV light, projection risk), it is compulsory to wear protection glasses.
Equipments: As soon as we manipulate in the lab, we have to wear the following personal protective equipments (a conventional lab coat, closed with long sleeves, closed shoes, gloves, glasses if needed).
Storage: we used three dedicated cup-boards for different kinds of chemical products: flammable, acids, bases.
Waste: Different trash containers are available in the lab: one for biological waste (these wastes will have to be autoclaved before being thrown out), one for common waste, one for special chemical wastes, and another for wastes soiled with ethidium bromide.
Biological safety cabinet: To work into a sterile area and thus avoid external contamination by unwanted microorganisms, we have used a biological safety cabinet (FASTER – Ultrasafe). This BSC was cleaned with ethanol before and after each manipulation. A control of maintenance is done each year. The last control was executed on October 25th 2015.
Water bathes: We often used the water-bathes for transformation or digestion, etc. They can be dangerous because of the exposition to hot or even boiling water. To protect us, wearing a lab coat and special gloves was indispensable. The same equipment is necessary for the microwave.
Ethidium Bromide: For some manipulations (to reveal gel electrophoresis), we have used ethidium bromide and UV light. Thus, a dark room is dedicated for this usage. This room is key-closed and the wearing of a lab coat, gloves and glasses is mandatory. Everything in direct contact with something in this room has to stay there. Wastes are treated specifically. A specific trash can is dedicated to gloves or paper and another is dedicated to the agarose gels that were contaminated.
Chemical hood: We used the chemical hood when we had to manipulate dangerous and volatile chemicals (for example: the usage of alcohol 96°)

Safety for our project

We have described above the relevant security measures taken all along summer to minimize the risk of incidents in the lab. However, when working in the field of synthetic biology, one of the main concerns is the dissemination of our engineered strain, which could be a threat to the public and the environment.

We have used E. coli and B. subtilis chassis, since their genomes are very well characterized and, of course, since these bacteria are non-pathogenic. E. coli was used for genetic manipulations, and the genes that were inserted (BioBricks) into this bacterium, did not increase its pathogenicity. B. subtilis was the final chassis because of the natural presence of Bacilluss species in the cave’s microbiote. Therefore, the main threat is the dissemination of our bacteria in the external environment where gene transfer could occur between microorganisms.

This is the reason why, to prevent any risk of dissemination, all our wastes were autoclaved before being appropriately disposed. Moreover, the French regulation does not allow any genetically modified microorganisms to be taken outside the lab. We are therefore aware that it won’t be possible to test our genetically modified bacteria in real conditions. In fact, it is unthinkable to release our bacteria in the environment without being confident enough that there won’t be any disturbances of the ecosystems of the Lascaux caves and the surrounding environment. So our effort should be regarded as a proof of concept.

To minimize both the risk of dissemination and of gene transfer, several solutions were planned. First, we have designed a physical device that would confine our engineered bacteria to the area to be treated (see the Modeling part). Then we also designed the use of a double toxin/anti-toxin systems to prevent gene transfer (see project design section). We also thought of including a biological clock that would limit the lifespan of our therapeutic bacterium. Moreover, we planned to use a spo0A- B. subtilis strain to prevent the bacterium to form spores that would allow it to resist to any competitors and to poor nutriment conditions.

Chassis organisms

Bacteria	Risk group	Risk Group Source	Disease risk for humans ?
Escherichia coli DH5 alpha	1	DSMZ	No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)
Bacillus subtilis 168	1	DSMZ	No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)

Fungi	Risk group	Risk Group Source	Disease risk for humans ?
Aspergillus niger 246.65 CBS	1	DSMZ	No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)
Talaromyces funiculosum CBS 235.94	1	DSMZ	No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)
Chaetomium globosum CBS 148.51	1	DSMZ	No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)

WOMEN IN SCIENCES

This year, chance has brought together 8 girls and only girls to create the 2016 IGEM Toulouse team. This peculiar and quite funny situation has somehow triggered our thinking about the actual position of women in Science, especially taking into account that our principal supervisors are mostly men…

Results of a vast survey on the role of women in scientific research conducted by the Boston Consulting Group were published in March 2014 in the European Union, showed that only 11% of the highest academic posts are occupied by women in science subjects. The percentage of women heads of scientific institutions varies from one country to another: only 6% in Japan, 27% in the US, 29% in France or 34% in Spain. Clearly, women are underrepresented in scientific professions.

Nevertheless, these percentages are slowly increasing, mainly because women are fighting for to reach the goal that the parity would be respected in all levels of society. Regarding specifically the scientific area, many women have raised the flag of parity such as Caroline Herschel, the first woman recognized as scientist but also the first one to publish a scientific paper in 1786 about the discovery of a comet, or such as Marie Curie who obtained two Nobel prizes in physics and chemistry. However, efforts are still needed for a much fair treatment of women in order to grant full equality in all countries around the world.

[Caroline Herschel , Marie Curie]

Thus today, 8 young women were involved in the iGEM adventure in Toulouse. We wondered if this is something we should mention. In one hand, we do not have to highlight that we are only women in this project. Actually the selection to be part of the team was made based on the skill and motivation of the student and not on his gender. At the end of the selection process, it turned out that the most appropriate candidates were all women! On the other hand, we realized, from our media interviews and discussion with the public (see Engagement), that clichés about science being a man field are still present in the nowadays society and we felt it was our duty to promote the place of women in science.

We are of course aware of the statistical study from the 2013 iGEM Paris-Bettencourt team showing that parity inside a team positively influences its success. Against this statistic and for modestly contribute to the fight for men-women parity, we would like to demonstrate that only complementary skills are the secret ingredient to the success in this competition, beyond the gender.

CRITERIA FOR SILVER MEDAL

To fullfil the requirements for the silver medal, we completed the three silver criteria:
1- Validated Part: 8 parts have been validante during this project (see Parts)
2- Collaboration: We initiated and piloted a collaborative project including 7 iGEM teams(see Collaboration)
3- Human Practices: Ethics, safety and the place of women in today sciences have been investigated in the présent page.

Note: to fullfil the requirements for the gold medal, we completed two gold criteria (integrated human practice and improvement of a previous part)

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