Team:Lyon-INSA/safety

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<h1>Safety</h1>
<h1>Safety</h1>
<div class="contenuTexte introduction" style="margin:20px">
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We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified <i>Bacillus subtilis</i> strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked also with an <i>Escherichia coli</i> strain and as a biofilm model with <i>Staphylococcus epidermidis</i>.  
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We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified <i>Bacillus subtilis</i> strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked with an <i>Escherichia coli</i> strain. <i>Staphylococcus epidermidis</i>,  <i>S. aureus</i> and  adherent <i>E. coli</i> strains were used as biofilm models.  
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<h2>Researcher/Public/Environmental Safety</h2>
<h2>Researcher/Public/Environmental Safety</h2>
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Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their toxicity for example. For these reasons: the access of the laboratory was limited to those involved in the project and all work benches were cleaned every day and the whole lab cleaned every week.
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<strong>Health and safety training</strong><br> <br>
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Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider that having a proper training in safety and security at the beginning of our experimental work prepared us to be more organized, responsible for our actions and respectful for those of others.  
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CLEAN AREAS TO ENCOURAGE GOOD PRACTICES
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We have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. No one was allowed to work alone in the laboratory. In addition, the French system provides all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life-saving diploma and are also trained for firefighting. A specific formation for handling chemicals and modified bacteria carrying antibiotic resistance genes was given to each student before starting the bench work.
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Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider the fact of having a proper training in safety and security at the beginning of our experimental work prepared us to be more organised, responsible for our actions and respectful for those of others. To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.
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<strong>Global hazards and risks :</strong> Electricity and gas: we have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. Personal protective equipement, including appropriate labcoats, gloves and safety glasses were worn for lab experiments. No one was allowed to work alone in the laboratory. In addition, the French system provide all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life saving diploma and also have trained for firefighting.
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<strong>Main chemical hazards and risks :</strong> Most of the reagents used are irritant, toxic and can be potential carcinogens (agarose, polyacrylamid, methanol, Ethidium bromide…)
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To minimize the impact of their use, they are manipulated following the supplier’s instructions, wearing appropriate personnal safety equipement, i.e. gloves, safety glasses,  labcoats and under extractor hood when necessary. All samples, tubes, vials are clearly identified/labelled to avoid inappropriate mix between two non compatible solvents. All reagents are eliminated in the appropriate waste recovered barrels.
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We took specific safety measure for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates whithin the double strand DNA helix. Many biological processes can thus be affected such as transcription and replication. To avoid the dissemination of EtBr in the lab, it is stored and used in the same room where the electrophoresis gels are revealed. EtBr is NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. This room is locked by a key. The access to this room is forbidden to any person who does not carry a lab coat. Dedicated gloves are used for EtBr manipulation and must not leave the room. They are discarded in a specific trash barrel for genotoxines contaminated material to gather with stained gels. EtBr-contaminated material is further processed and decontaminated by an external service.
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Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.
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<strong>Main biological hazards and risks:</strong> All Bacillus subtilis, Echerichia coli and Staphylococcus epidermidis strains we used have a biosafety level of 1, which means they are not known to cause disease and have minimal environmental hazards.
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At this level the precautions concerning the lab-material are minimal: wearing gloves and face protection when needed. Any contaminated material is discarded in a trash can to be autoclaveded within 2 days. The decontamination procedure is similar to any other applied to frequently encountered virus or bacteria (ex: to wash hands with antibacterial soap, disinfect any surface in the laboratory that has been exposed). Our work benches are decontaminated with ethanol 70% after each manipulation. And contaminated media and materials are autoclaved at least every 2 days to avoid any release in environment.
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All the students must have their vaccinations up to date. In case a student harbors an injury, it must be covered so it is not exposed to the bacteria. All rooms are equiped with with a first aid kit.
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In the building where the labwork was performed, no laboratory is manipulating pathogenic microorganisms, which limits the risks to students, and the recombination possibility between a pathogenic bacteria and the lab strains.
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For better protection the following basic safety rules applied for all students during lab work:<br/>
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<strong>Restricted access</strong><br/><br/>  
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Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their harmfulness. For these reasons, the access of the laboratory was limited to those involved in the project only.
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Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.
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<li>Wear a buttoned up lab coat</li>
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<strong>Waste management</strong><br/><br/>
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Chemical wastes. All reagents were eliminated in the appropriate waste recovery barrels.
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Biological wastes. We used the autoclave of the microbiology teaching platform to decontaminate our biological solid and liquid wastes : no bacteria, modified or not, are released in the environment.
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<li>Contact lenses are forbidden except if people wear a face protection or safety glasses</li>
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<li>Spillage need to be cleaned up  immediately</li>
 
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<strong>Cleaning</strong><br/><br/>
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<li>Technical and safety files are available neat  all the lab equipments</li>
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All work benches were cleaned every day. The whole lab was cleaned every week.
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<li>No chemical touching, sniffing or tasting</li>
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CLEAN AREAS TO ENCOURAGE GOOD PRACTICES!
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<li>No mouth-pipeting</li>
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<strong>Protection</strong><br/><br/>
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Some of the reagents used are irritant, toxic and can be potential carcinogens (polyacrylamid, Ethidium bromide…). To minimize the impact of their use, they were manipulated following the supplier’s instructions, wearing appropriate personal safety equipment, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials were clearly identified/labelled to avoid inappropriate mix between two non-compatible solvents.
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<li>No returning unused chemicals to their containers</li>
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<li>No hazardous material disposal down the drain</li>
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This specific localisation in a public research laboratory building allows us to benefit from researcher and student law safety (or under application in France) which are mandatory in each lab in France. For example, we have specific place for each kind of experimentation:
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We took specific safety measures for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates within the double strand DNA helix. To avoid the dissemination of EtBr in the lab, it was stored and used in the same room where the electrophoresis gels were revealed. EtBr was NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. Specific trash barrel for genotoxic contaminated material were used for EtBr-contaminated material.
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Rooms dedicated for:
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To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.
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We use our personal autoclave in the lab to decontaminate and sterilize our materials.
 
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<h2>BioBrick parts safety issues</h2>
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<h2>Specific biological hazards and risks linked to the “Biofilm Killer” project</h2>
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A GMO may contain harmful parts and be dangerous for the environment. The potential danger of a GMO is also due to its potential interactions with the environment. When an iGEM project is conceived, the team has to think about the possible interactions of their parts with the environment and design tests to document the effects of their parts on the environment. Synthetic biology is a science which worries people, so we have to provide them the insurance of the complete safety of our experiments.<br/>
 
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<strong>Our molecules : Lysostaphin, Dispersin, Surfactin</strong>
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<strong>Bacterial strains</strong>
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None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|Lysostaphin, an endopeptidase specific to the cell wall peptidoglycan of <i>Staphylococcus</i>, is an extremely potent antistaphylococcal agent. Lysostaphin has been used in animals and topically in human against certain infections. Concerning the safety precautions it is indicated to not breathe gas/fumes/vapor/spray, to avoid contact with skin and eyes.">lysostaphin</span>, <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|Surfactin is a lipopeptide antibiotic and is used as a surfactant to mediate flux of mono- and divalent cations, such as calcium, across lipid bilayer membranes. It is a powerful biosurfactant which can cause lysis of erythrocytes and bacteria. It is also known as a clotting inhibitor. However, considering the concentrations in which it is produced by bacterial cultures, the risk is minimal. In addition, the “Biofilm Killer” GMO will produce the molecule only locally, further reducing the risks.">surfactin</span> and <span class="tippable" style="color:red;font-style:italic;cursor:pointer" title="|DispersinB is commercialized by Kane Biotech. It should be used in conjunction with an antibiotic or an anti-microbial, such as silver. Last year, Kane Biotech provided a brief document on DispersinB wound spray to the FDA, in preparation for the submission of an Investigational New Drug Application. They have started clinical trials in order to test the effect of this molecule on infected people. To date,  there is no evidence for a negative effect of this molecule on human health.">dispersin</span>. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here.  
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All <i>Bacillus subtilis</i>, <i>E. coli</i> and <i>S. epidermidis</i> strains we used at INSA-Lyon have a biosafety level of 1, which means that they are not known to cause disease and have minimal environmental hazards. <i>Staphylococcus aureus</i> has a biosafety level of 2 and requires P2 equipment. Manipulations with this organism were conducted in the Bioadhesion/Biofilm lab in Massy (Paris) under Romain Briandet's supervision.
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<i>B. subtilis</i> is the chassis chosen for “Biofilm Killer”. <i>B. subtilis</i> is widely used in the field as a biocontrol agent against plant pathogens and food industry processes. It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA).  However, if modified by biobricks, <i>B. subtilis</i> has to be physically contained (double filter 0.45/0.2 µm)(<a href="https://2012.igem.org/Team:Lyon-INSA/BiofilmK#solution"> See process in "Our solution"</a>)<br/><br/>. <strong>Enhanced safety can be achieved by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"</strong>
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<i> "The Bacillus subtilis species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1]</i>
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<strong>Our chassis</strong>
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<i>B. subtilis</i> is known to produce endospores [2] that are resistant to different treatments (heat, UV irradiation…) [3]. Spores of a variety of species are of major concern for the food industry, and even if <i>B. subtilis</i> is non-pathogenic, use of a sporulating strain can be a problem in food industry because spores are resistant to most cleaning procedure and thus can get out of usual control.  
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Therefore, we plan to introduce a mutation in the <i>B. subtilis</i> strain 168 to disrupt the sporulation process and offer an asporulating version of Biofilm Killer for food application.
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In our Biofilm Killer project, we aim to construct a bacterium able to destroy biofilms and to synthesize biocide molecules or a biosurfactant in order to develop a novel method to remove biofilms and clean surfaces in several industrial processes. So, we chose a strain that would have a minimum impact on the process itself, the quality of the product and biological treatment that could exists on site (waste water treatment plant).
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Our genetic constructions have been transferred into a strain of <i>Bacillus subtilis</i>. This species are well known by researchers but also by many industries such as the food industry. <i>Bacillus subtilis</i> is also widely used in the field as a biocontrol agent against plant pathogens.
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<strong>“Biofilm Killer” power: Lysostaphin, Dispersin, Surfactin</strong><br/><br/>
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It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA)
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None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: <i>lysostaphin, surfactin and dispersin</i>. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here.
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<div style="margin:30px;font-style:italic;">"The <i>Bacillus subtilis</i> species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA)[10] and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1]</div>
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But <i>Bacillus subtilis</i> is also known to produce endospores that are resistant to different treatments (heat, UV irradiation…). A possible impact on human health cannot be excluded. Indeed, it has been shown that the ingestion of endospores of non-modified <i>B. subtilis</i> does not prevent their germination into the gastrointestinal tract (GIT) [4]. The bacteria are even capable of growth in the lower part of the GIT. But, in this particular case, the consequence seem to be beneficial on health. The presence of <i>B. subtilis</i> in the GIT seems to be linked to the development of the gut-associated lymphoid tissue (GALT). This is one of the reason <i>B. subtilis</i> can be used as a probiotic.
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When the environment becomes unadapted to its growth, the bacterium induces the formation of an endospore in its intracellular compartment [2].  The core of the endospore contains the bacterial chromosome. The core is protected by a double layer membrane, called the forespore. This membrane is itself protected by the coat, composed by multilayered proteins. In <i>B. subtilis</i>, the Cot family proteins is widely involved in the formation of the coat. The resistance of the spore is dependant on the state of hydratation of the core. Spores can be dispersed in the environment and turn into germination processus leading to the contamination of new areas previously devoid of this micro-organism. In our case, we work with <i>B. subtilis</i> strain 168, which is the most widely used, and capable of sporulation, <i>B. subtilis</i> strain in research labs.
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Sporulation can be hazardous to the environment and humans because spores are resistant to most cleaning procedure and thus can survive for a very long time, even in extreme conditions [3]. Spores can be dispersed in the environment and turn into germination processus leading to the contamination of new areas previously devoid of this micro-organism.
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In our case, we work with <i>B. subtilis</i> strain 168, which is the most widely used, and capable of sporulation, <i>B. subtilis</i> strain in research labs.
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The high spore resistance to adverse conditions imply that avoiding the induction of sporulation is one task that we need to consider in our project if we use a sporulant <i>B. subtilis</i>.
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If the cells were released in the environment after the germination of spores, we estimate the chance of survival to be minimal, because strain 168 has been modified and habituated to laboratory conditions. Even so, as mentioned below, <i>B. subtilis</i> is not a pathogenic bacterium.
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<strong>For all these reasons, and in the aim of an industrial application, we propose to introduce our genetic constructions in an NON-sporulant <i>B. subtilis</i> strain, such as QB1133.</strong>
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The use of a non sporulant <i>B. subtilis</i> to host our genetic construction would not significantly impact neither the environment nor the human health. The odds of the bacteria surviving to the low pH of the stomach, and the very hard life conditions (anaerobia, pH, competition with the intestinal flora) are very low in the vegetative state.
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In addition to the use of non sporulant mutant of <i>B. subtilis</i>, we could enhance safety by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"
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<strong>References</strong>
<strong>References</strong>
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1 European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944.
 
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2 Morphogenesis of <i>Bacillus</i> Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21)
 
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3 Role of the Spore Coat Layers in <i>Bacillus subtilis</i> Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626
 
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4 The Intestinal Life Cycle of <i>Bacillus subtilis</i> and Close Relatives, Duc, Tran T. Hoa, Claudia R. Serra, Adriano O. Henriques Nguyen K. M. Tam, Nguyen Q. Uyen, Huynh A. Hong, Le H.and Simon M. Cutting, J. Bacteriol. 2006, 188(7)
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<br>1. European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944.  
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<br>2. Morphogenesis of Bacillus Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21)  
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<br>3. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626
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Our institution (INSA Lyon) has a biosafety group. However, we have a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students follow a 4 hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training all along the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies to all the french biosafety regulations.
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Our institution (INSA Lyon) has, as every public institute, a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students have followed a 4-hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training though the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies with all French biosafety regulations.
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As far as the legal aspect is concerned, these is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is quite restrictive, based on the precautionary principle. Even though synthetic biology doesn’t yet have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.
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<center><a href="https://static.igem.org/mediawiki/2012/f/ff/Deathm.jpg" class="fancyable"><img src="https://static.igem.org/mediawiki/2012/f/ff/Deathm.jpg" style="border:5px solid black;border-radius:5px" width="500"/></a></center>
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As far as the legal aspect is concerned, there is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is based on the precautionary principle. Even though synthetic biology doesn’t have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.
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<h2>New ideas for safety in iGEM</h2>
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<h2>After standard parts, why not a standard chassis ?</h2>
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After standard parts, with not standard chassis ?
 
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The INSA de Lyon team propose that a “safety kit” should be provided to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.
 
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This kit should contain:
 
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<li>a collection of chassis that could be used to receive the DNA constructions. The strains’ genome would be the result of the gene knockout method which is used to inactivate a specific gene. With the new genetic technologies that evolve each day, now it is even possible to purchase a commercial Gene Knockout System which can be designed to knock-out the gene of interest. For exemple, an applications of this genetical instrument is the construction of auxotrophic strains (or nutrient-deficient) which are mutant strains of bacteria that are unable to grow on minimal media. The proliferation of these organisms outside the laboratory is limited because the lab chemicals are not found in nature.</li>
 
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<li>a toxin/antitoxin system coupled with a lysing agent/cytotoxic compount/degradation agent which is provided on a separate backbone (it would let the choice to the participants whether or not they want to use it). If the strain containing this system is released, horizontal gene transfer between a non-natural bacteria and a natural one is prohibited. Thus, with the anti-toxin inside the chassis’ genome, and the toxin and cytotoxic compound in the plasmid DNA, the horizontal gene transfer will induce the death of the recipient.
 
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<li>a strain suicide option represented by a gene-killer (also found on a backbone) which is activated in the absence/presence of a certain substrate. For example, Contreras <i>et al.</i> (1991) [1], proposed the construction of a confined strain capable of digesting polluted substance. In the absence of pollutant in the environment, a suicide gene is expressed and the cell is destructed.
 
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We are aware that engineering these DNA sequences is not easy. This is why we suggest that a new section or a new reward should be proposed : “ Best Safety Device”. For teams it would be a real challenge and their investment for creating new safety devices would be rewarded.
 
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In synthetic biology the safety and security measures are of primary importance. Every team should have a proper formation in safety and security issues. INSA de Lyon team suggests that one of the competition requirements should be a diploma validating a proper safety formation.
 
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Beyond traditional <strong>physical containment</strong> (by restricting organisms to a physical space), disabling of the organisms in some way so as to ensure they cannot survive if accidentally or incidentally introduced into the environment. Such disabling leads to <strong>chemical containment</strong> (based on engineering "kill switches" or reliance on nutrients not found in the wild) or <strong>informational containment</strong> (by making genetic information incompatible between natural and synthetic species).
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The Lyon-INSA team proposes to provide a “safety kit” to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.
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This kit would contain a collection of “disabled chassis” for the most popular chassis (<i>E. coli, B. subtilis</i>…) that could be used to receive the DNA constructions.
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As engineering these chassis is not easy, we suggest to create a <strong>new “ Best Safety Device” reward</strong> to motivate iGEM teams.
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<strong>References</strong>
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Latest revision as of 12:03, 8 June 2013

Safety

We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified Bacillus subtilis strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked with an Escherichia coli strain. Staphylococcus epidermidis, S. aureus and adherent E. coli strains were used as biofilm models.

Click on the title to show/hide the text.

Researcher/Public/Environmental Safety

Health and safety training

Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider that having a proper training in safety and security at the beginning of our experimental work prepared us to be more organized, responsible for our actions and respectful for those of others.
We have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. No one was allowed to work alone in the laboratory. In addition, the French system provides all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life-saving diploma and are also trained for firefighting. A specific formation for handling chemicals and modified bacteria carrying antibiotic resistance genes was given to each student before starting the bench work.

Restricted access

Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their harmfulness. For these reasons, the access of the laboratory was limited to those involved in the project only.
Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.


Waste management

Chemical wastes. All reagents were eliminated in the appropriate waste recovery barrels.



Biological wastes. We used the autoclave of the microbiology teaching platform to decontaminate our biological solid and liquid wastes : no bacteria, modified or not, are released in the environment.

Cleaning

All work benches were cleaned every day. The whole lab was cleaned every week.
CLEAN AREAS TO ENCOURAGE GOOD PRACTICES!

Protection

Some of the reagents used are irritant, toxic and can be potential carcinogens (polyacrylamid, Ethidium bromide…). To minimize the impact of their use, they were manipulated following the supplier’s instructions, wearing appropriate personal safety equipment, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials were clearly identified/labelled to avoid inappropriate mix between two non-compatible solvents.


We took specific safety measures for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates within the double strand DNA helix. To avoid the dissemination of EtBr in the lab, it was stored and used in the same room where the electrophoresis gels were revealed. EtBr was NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. Specific trash barrel for genotoxic contaminated material were used for EtBr-contaminated material.

To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.

Specific biological hazards and risks linked to the “Biofilm Killer” project



Bacterial strains

All Bacillus subtilis, E. coli and S. epidermidis strains we used at INSA-Lyon have a biosafety level of 1, which means that they are not known to cause disease and have minimal environmental hazards. Staphylococcus aureus has a biosafety level of 2 and requires P2 equipment. Manipulations with this organism were conducted in the Bioadhesion/Biofilm lab in Massy (Paris) under Romain Briandet's supervision.

B. subtilis is the chassis chosen for “Biofilm Killer”. B. subtilis is widely used in the field as a biocontrol agent against plant pathogens and food industry processes. It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA). However, if modified by biobricks, B. subtilis has to be physically contained (double filter 0.45/0.2 µm)( See process in "Our solution")

. Enhanced safety can be achieved by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"

"The Bacillus subtilis species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1] B. subtilis is known to produce endospores [2] that are resistant to different treatments (heat, UV irradiation…) [3]. Spores of a variety of species are of major concern for the food industry, and even if B. subtilis is non-pathogenic, use of a sporulating strain can be a problem in food industry because spores are resistant to most cleaning procedure and thus can get out of usual control. Therefore, we plan to introduce a mutation in the B. subtilis strain 168 to disrupt the sporulation process and offer an asporulating version of Biofilm Killer for food application.

“Biofilm Killer” power: Lysostaphin, Dispersin, Surfactin

None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: lysostaphin, surfactin and dispersin. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here.

References

1. European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944.
2. Morphogenesis of Bacillus Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21)
3. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626

Biosafety group

Our institution (INSA Lyon) has, as every public institute, a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students have followed a 4-hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training though the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies with all French biosafety regulations.



As far as the legal aspect is concerned, there is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is based on the precautionary principle. Even though synthetic biology doesn’t have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.

After standard parts, why not a standard chassis ?

Beyond traditional physical containment (by restricting organisms to a physical space), disabling of the organisms in some way so as to ensure they cannot survive if accidentally or incidentally introduced into the environment. Such disabling leads to chemical containment (based on engineering "kill switches" or reliance on nutrients not found in the wild) or informational containment (by making genetic information incompatible between natural and synthetic species).
The Lyon-INSA team proposes to provide a “safety kit” to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.
This kit would contain a collection of “disabled chassis” for the most popular chassis (E. coli, B. subtilis…) that could be used to receive the DNA constructions.
As engineering these chassis is not easy, we suggest to create a new “ Best Safety Device” reward to motivate iGEM teams.

References

1 Contreras A., Molin S, Ramos JL. 1991. Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics. Applied and Environmental Microbiology.

Retrieved from "http://2012.igem.org/Team:Lyon-INSA/safety"