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| − | <title>iGEM Toulouse 2015</title>
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| − | <center> <h3>Safety</h3> </center>
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| − | <center><img src="https://static.igem.org/mediawiki/2015/6/67/TLSE_BG.png"></center>
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| − | <a href="#main1"><h3>Content</h3></a>
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| − | <center>
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| − | <div id="breadcrumb" class="clear" style="float: center;" >
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| − | <ul>
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| − | <li><a href="#part1">- Team safety and training</a></li>
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| − | <li><a href="#part2">- Safety in the lab</a></li>
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| − | <li><a href="#part3">- Safety of our project</a></li>
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| − | </ul>
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| − | <center><div class="title" id="part1">
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| − | <h3>Team safety and training</h3>
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| − | <div class="subtitle" >
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| − | <h3>INSA safety training</h3>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | During the summer, our lab was in the engineering school INSA wich possesses a safety department with one prevention advisor and several prevention assistants assigned to the laboratories. Their goal is to ensure the well being of the employees regarding safety rules and risk prevention. In our laboratory, the LISBP, safety is supervised by Nathalie Doubrovine who was the one instructing us regarding safety procedures.
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| − | <br>
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| − | As new interns we had to take part in a general training session to learn how to identify the risks and prevent
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| − | ourselves and our colleagues from harm. We also followed several other trainings that allowed us to use technical apparatus such as autoclave or Nuclear Magnetic Resonance (NMR).<br>
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| − | <i>The laboratory safety training requirements of the LISBP are detailed in the Rules and Procedures of the LISBP.</i>
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| − | </p>
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| − | <div class="subsubtitle" >
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| − | <h3>New employees safety training</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | Every new LISBP employee has to attend this training session regardless of its status (researcher, PhD student, intern...).
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| − | The training is divided into two parts. The first one is a training concerning general prevention in research laboratories. This one is taken individually with the NEO software and the explanations of the prevention assistant. It also informs the employee about the emergency numbers.<br>
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| − | The second one is a training about the techniques used during our stay concerning microbiological, chemical and incendiary risks.
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| − | <br>
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| − | We then have a test to make sure that we were understood and aware of the previous presentations.
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| − | </p>
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| − | </div>
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| − | <div class="subsubtitle" >
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| − | <h3>Autoclave</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <div class="one_half first">
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| − | <p align="justify" style="font-size:15px;">
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| − | The whole team went through an autoclave training, detailing the explosive/implosive danger surrounding work with an under pressure apparel.<br>
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| − | A lab coat, heat resistant gloves and glasses must be worn when manipulating the autoclave.
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| − | </p>
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| − | </div>
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| − | <div class="one_half">
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| − | <img src="https://static.igem.org/mediawiki/2015/a/a8/TLSE_safety_autcl.png" />
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| − | </div>
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| − | <div class="subsubtitle" >
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| − | <h3>Liquid nitrogen</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <div class="one_half first">
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| − | <p align="justify" style="font-size:15px;">
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| − | We also had a training explaining the risks faced when manipulating liquid nitrogen, and how to manage incidents.
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| − | Isothermic gloves, glasses and of course a lab coat must be worn when working with it.
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| − | </p>
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| − | </div>
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| − | <div class="one_half">
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| − | <img src="https://static.igem.org/mediawiki/2015/c/c8/TLSE_safety_nitro.png" />
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| − | </div>
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| − | <div class="subsubtitle" >
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| − | <h3>NMR</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | We used the NMR machine kindly made available to us by the MetaToul platform to analyse our culture supernatant. This is a powerful but very expensive equipment, based on the magnetic resonance of the atoms. Due to this, specific rules have to be respected when working with it. <br>
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| − | In order to protect the user, access to the NMR is not allowed for people wearing pacemakers, drug pump systems, staples on soft or hard tissue, and pregnant women. <br>
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| − | Secondly, in order to protect the technological material, when entering the room the user should not wear a lab coat, nor have anything in his pockets, especially anything made of metal.
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| − | </p>
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| − | </div>
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| − | <div class="subsubtitle" >
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| − | <h3>Legislation and French Labor Law</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | INSA Toulouse is a public school for engineers thus the biosafety guidelines are not specific to our institution; the French national regulations about working conditions and the manipulation of genetically modified organisms are applied.<br>
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| − | The regulation on workers' protection against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994) includes microorganisms, cell cultures and human endoparasites which may cause infections, allergies or toxicity. <br>
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| − | 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.)<br>
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| − | The <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000000465273&dateTexte=&categorieLien=id">Decree of the 16th July 2007</a> describes the technical preventive measures that are to be set up in research laboratories (including containment), education, analysis, anatomy and surgical pathology, autopsy rooms, and industrial and agricultural facilities where workers are likely to be exposed to biological pathogens.<br>
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| − | The rules of health, safety, and preventive medicine applied in public services in France (and thus in all public facilities working in scientific and technological domains) are set out in the <a href="http://www.legifrance.gouv.fr/affichTexte.do?cidTexte=LEGITEXT000006063791">Decree No. 82-453</a>. This decree refers to the Labour Code, Public Health Code and Environmental Code.<br>
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| − | The <a href="http://legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000024584737&categorieLien=id">Decree No. 2011-1177</a> is related to the use of genetically modified organisms.<br>
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| − | <br>
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| − | </p>
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| − | </div>
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| − | <center><div class="title" id="part2">
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| − | <h3>Safety in the lab</h3></center>
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| − | <div class="subtitle" >
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| − | <h3>Equipment</h3>
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| − | <div class="group center">
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| − | <ul align="left" style="font-size:15px;">
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| − | <li>A conventional lab coat, closed with long sleeves</li>
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| − | <li>Closed shoes</li>
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| − | <li>Gloves</li>
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| − | <li>Glasses if needed (UV exposure, hot water or chemical handling)</li>
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| − | </ul>
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| − | </div>
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| − | <div class="subtitle" >
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| − | <h3>Waste</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <table style="font-size:15px;" align='left'>
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| − | <th colspan=2><p>Different trash containers are available in the lab:</p></th>
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| − | </tr>
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| − | <td><p align="left">One for biological waste (yellow). <br>This waste will be autoclaved before being thrown out.</p></td>
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| − | <td><img src="https://static.igem.org/mediawiki/2015/6/6e/TLSE_safety_fig1.png" style="padding:20px;width:50%;"/></td>
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| − | </tr>
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| − | <td><p align="left">One for common waste (green or orange).</p></td>
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| − | <td><img src="https://static.igem.org/mediawiki/2015/7/77/TLSE_safety_commonWaste.png" style="padding:20px;width:70%;"/></td>
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| − | </tr>
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| − | </table>
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| − | </div>
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| − | <div class="subtitle" >
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| − | <h3>Storage</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <table style="font-size:15px;" align="left">
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| − | <tr>
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| − | <th colspan=2><p>We have three cupboards, each one dedicated to a different kind of chemical product we use:</p></th>
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| − | </tr>
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| − | <td><p align="center">Flammable products</p></td>
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| − | <td><img src="https://static.igem.org/mediawiki/2015/6/64/TLSE_safety_flam.png" style="padding:20px;width:50%;"/></td>
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| − | </tr>
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| − | <td><p align="center">Acids</p></td>
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| − | <td><img src="https://static.igem.org/mediawiki/2015/2/2f/TLSE_safety_acid.png" style="padding:20px;width:50%;"/></td>
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| − | </tr>
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| − | <td><p align="center">Bases</p></td>
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| − | <td><img src="https://static.igem.org/mediawiki/2015/4/44/TLSE_safety_bases.png" style="padding:20px;width:50%;"/></td>
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| − | </tr>
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| − | </table>
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| − | </table>
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| − | </div>
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| − | <div class="subtitle" >
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| − | <h3>Rules</h3>
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| − | </div>
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| − | <p align="justify" style="font-size:15px;">
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| − | Our workspace is divided between two rooms. We have a break room where no biological material is brought. In this room, we are able to eat and drink (a fridge, a kettle and a coffeemaker are available)
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| − | but it is also the space where we hold our meetings and work on our computers.
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| − | On the contrary, in the lab, we have to wear protective equipment and respect basic rules:
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| − | </p>
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| − | <li>No smoking (all rooms)</li>
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| − | <li>No drinks or food</li>
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| − | <li>Obligation to wear a closed cotton lab coat</li>
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| − | <li>Oblligation to wear closed shoes</li>
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| − | <li>Long hair must be tied up</li>
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| − | <li>Oral pipetting of any substance is prohibited in any laboratory</li>
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| − | <li>Regular handwashing</li>
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| − | <li>In some cases (UV light, projection risk), obligation to wear protection glasses</li>
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| − | </ul>
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| − | </div>
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| − | </div>
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| − | <div class="subtitle" >
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| − | <h3>Apparatus</h3>
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| − | </div>
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| − | <div class="subsubtitle">
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| − | <h3>Chemical hood</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | We used the chemical hood when we had to manipulate dangerous and volatile chemicals. For example, all manipulations dealing with formic and butyric acid were done under such a hood.
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| − | </p>
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| − | </div>
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| − | <div class="subsubtitle" >
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| − | <h3>Water-bathes</h3>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | The water-bathes were used extensively (transformation, digestion, etc.). However, they can be dangerous because of the exposition to hot or even boiling water. To prevent the burning risk associated with heat, steam and projections, we used special gloves. We made sure to always check that the water-bathes were turned off at the end of the day.
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| − | </p>
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| − | </div>
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| − | <div class="subsubtitle" >
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| − | <h3>Biological safety cabinet</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | To manipulate into a sterile area and thus avoid external contamination by unwanted microorganisms we used a Biological safety cabinet (FASTER – Ultrasafe). The bench of the BSC was cleaned with ethanol before and after each manipulation. We also cleaned the BSC completely every two weeks.
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| − | </p>
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| − | </div>
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| − | <div class="subtitle">
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| − | <h3>Ethidium Bromide</h3>
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| − | </div>
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| − | <h3>Dark room</h3>
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| − | </div>
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| − | </center>
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| − | <p align="justify" style="font-size:15px;">We have a dark room dedicated to the use of EtBr and UV. This room is key-closed and everyone entering the room has to wear gloves, glasses and a lab coat. Everything in direct contact with something in this room has to stay there.
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| − | </p>
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| − | <div class="one_half">
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| − | <h3>Waste</h3>
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| − | </div>
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| − | </center>
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| − | <p align="justify" style="font-size:15px;">Two specific trash cans are dedicated to the gloves or paper and the contaminated agarose gels.</p>
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| − | </div>
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| − | <center>
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| − | <img src="https://static.igem.org/mediawiki/2015/f/f1/TLSE_safety_BET.png" style="width:50%;"/></center>
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| − | </div>
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| − | <div class="one_half">
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| − | <img src="https://static.igem.org/mediawiki/2015/d/d2/TLSE_safety_BETwaste1.png" style="width:50%;"/></center>
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| − | </div>
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| − | </div>
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| − | <center><div class="title" id="part3">
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| − | <h3>Safety of our project</h3>
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| − | </div>
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| − | <div class="group center">
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| − | <p align="justify" style="font-size:15px;">
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| − | We have described above the relevant security measures taken all through the summer to minimize the risks of incident in the lab. But 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 general public and the environment. <br> <br>
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| − | First of all, we decided to work with the E. Coli chassis, since its genome is very well characterized and it was well adapted to the molecules we wanted to produce. Of course the strain we chose to work with was non-pathogenic. Moreover the used parts are also non-pathogenic (the acids doses produced are harmless for humans). <br>
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| − | The main threat is thus the dissemination of our bacteria in the external environment where its DNA or RNA would be in contact with microorganisms present in the external environment. <br> <br>
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| − | To minimize this risk, all our waste is autoclaved before being specifically disposed of and the French regulation does not allow any genetically modified microorganisms to be taken outside the lab. For example, if we would like to test our device on an actual hive, it would be by using the chemicals alone, and not the strain that produces them. <br> <br>
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| − | But since regulations are different around the world, and to have a complete overview of our project, we decided to reflect on the biosafety issue, were our device to ever be put in place on hives. <br> <br>
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| − | We first thought about addressing the containment issue through physical means. In fact, the trap we designed is in itself a first barrier, preventing the engineered bacteria from being in contact with their environment (and particularly the bees). But that was not enough, and the second barrier was developed relying on Groeningen’s team work in 2012. <br>
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| − | In fact, we reproduced their idea of packing our bacteria in a small bag made of a special plastic called TPX®, by Mitsui Chemicals. This special material is actually microporous, which means it will let gases such as oxygen or formic and butyric acid pass through while the bacteria stay inside the bag. You can see <a href="https://2015.igem.org/Team:Toulouse/Results">here</a> the sterility tests we realized. You can learn more about this in the video below. <br> <br> <br>
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| − | <i>VIDEO DE SACLAY</i><br> <br> <br>
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| − | Apart from this physical containment, we were also thinking of adding to our system an auxotrophy to a specific nutriment not found in the natural environment. In this case, bacteria directly died if they are in contact with the external environment. <br> <br>
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| − | Finally, one issue specific to our project is the fact that the small bag containing bacteria would probably have to be manipulated by beekeepers. Indeed our system would be optimized to last at most 2 to 3 weeks, after which the bag would have to be replaced. That would designing a specific training for the beekeepers, and a safe way to get rid of the used bags.
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| − | References
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