Team:Grenoble/Safety

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<h1>Safety</h1>
 
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<li><a href="#lab">Lab work safety</a></li>
 
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<li><a href="#general">General considerations</a></li>
 
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<li><a href="#instru">Instruments</a></li>
 
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<li><a href="#chemical">Chemical risk-assessment</a></li>
 
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<li><a href="#bio">Biological risks, biosafety rules</a></li>
 
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<li><a href="#microorg">Microorganisms chassis</a></li>
 
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<li><a href="#bioparts">Biobricks parts used</a></li>
 
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<li><a href="#toggle">Toggle switch</a></li>
 
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<li><a href="#qs">Quorum sensing</a></li>
 
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<li><a href="#tomato">Pigment</a></li>
 
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<li><a href="#rsma">RsmA</a></li>
 
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<li><a href="#outlab">A bacteria went out of the lab, another cell was killed, what happened and how likely would that happen ?</a></li>
 
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<li><a href="#robustus">Robustness of our device</a></li>
 
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<li><a href="optional">Optional question</a></li>
 
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<h1>Safety issues</h1>
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In general, the work in a laboratory requires the use of complex equipment or it implies performing delicate operations. The material, that could be a machine, chemicals or biological material involves the existence of risks. Risks for the goods and for people in the room, but also risks for the environment and people outside the lab. The safety rules and procedures as well as the personal and collective protective equipment are made to minimize the risks by decreasing the probability of  an incident to happen.
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<li>Would the materials used in your project and/or your final product pose:</li>
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<li>Risks to the safety and health of team members or others in the lab?</li>
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<li>Risks to the safety and health of the general public if released by design or accident?</li>
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<li>Risks to environmental quality if released by design or accident?</li>
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<li>Risks to security through malicious misuse by individuals, groups or states?</li>
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<li>If your response to any of the questions above is yes:</li>
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<li>Explain how you addressed these issues in project design and while conducting laboratory work.</li>
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<li>Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.</li>
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<li>Under what biosafety provisions will / do you operate?</li>
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<li>Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.</li>
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<li>Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them?</li>
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<li>Describe any concerns or changes that were made based on this review.</li>
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<li>Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.</li>
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<li>Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.</li>
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<li>OPTIONAL QUESTION: Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</li>
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<h2 id="lab">Lab work safety</h2>
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<h3 id="general">General considerations</h3>
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In general considerations, the work in a laboratory requires the use of complex equipment or performing delicate operations. The material, that could be a machine, chemicals or biological material implies the existence of risks. Risks for the goods and for people in the room, but also risks for the environment and people outside the lab. The safety rules and procedures as well as the personal and collective protective equipment are made to minimize the risks by decreasing the probability of  an incident to happen.
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Half of our team made an internship at the CEA Grenoble. The CEA has a specific department working on safety issues. There is also a special team in charge of security and safety called FLS: Formation locale de sécurité, we may translate: Local Group of Security and Safety. They ensure the safety of the people who are working in the center and the visitors and also of the goods and the material. The members of our team who made their internship in CEA have attended a compulsory safety training organised by FLS.  
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During our project, we have seek much information about products and materiel employed in our experiments and the risks associated with these latter. The litterature, the material safety datasheet and moreover the safety engineers of our labs were important sources of knowledge for us.
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<h3 id="general">General considerations</h3>
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Half of our team made an internship at the CEA Grenoble. The CEA have a whole department working on safety issues. There is also a special section of people in charge of the security and the safety. It is called FLS: Formation locale de sécurité, we may translate: Local Group of Security and Safety. They ensure the safety of the people who are working in the center and the visitors and also of the goods and the material. The members of our team who made their internship in CEA have assists to safety conferences organised by FLS.  
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</p>
</p>
<p>
<p>
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The whole team work now together in another lab, the CIME (centre inter-universitaire de microélectronique), next to the CEA labs and the Phelma school buildings. All team members have met the safety engineer of the labs where we conduct the experiments. He explained us the safety rules to be followed.
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The whole team is now working together in another lab, the CIME (centre inter-universitaire de microélectronique), next to the CEA labs and the Phelma school buildings. All team members have met the safety engineer of the labs where we conduct the experiments. He explained us the safety rules to be followed.
</p>
</p>
<p>
<p>
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At CEA some researchers worked on microsystems devices to detect and quantify these pollutants. They will share their experience and knowledge with us about the way to conduct safe experiments with very toxic chemicals like mercury and also about technical aspects of existing measurement device. We would like to compare our work, our biosystem to “technological only” system that already exist, in terms of precision, sensitivity, reliability, speed and costs.
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At CEA some researchers work on microsystems to detect and quantify pollutants like heavy metals. They shared their experience and knowledge with us about the way to conduct safe experiments with very toxic chemicals like mercury.  
</p>
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<p>
 
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We plan to present our work and the synthetic biology to a larger public: companies which fund us, school in our villages and town, a conference at “Midi Minatec”, ... 
 
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<h3 id="instru">Instruments</h3>
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<h3 id="instru">Instruments</h3>
<p>
<p>
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The experiments we made in our project did not require the use of sophisticated equipment. In fact, we only performed very usual operations of microbiology with a commonly used laboratory strains of E.Coli. In terms of experiment the only exotic and hazardous step is the use of mercury. We have used basic devices that we find in every molecular biological laboratory:
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During our experiments, we only performed commonly used protocols and instrumentation for microbiology and common laboratory strains of E.Coli. We have used basic devices that we find in every molecular biological laboratory:
<dl>
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<dt>Ultra violet lamp: </dt>
<dt>Ultra violet lamp: </dt>
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<dd>
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There is a risk for the eyes and in case of long exposure for the skin, but the UV lamp are only used to take a picture of our gel after electrophoresis, so we are never directly exposed, and it is always for very short period.
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There is a risk for the eyes and the skin, but the UV lamp is only used to take a picture of our gel after electrophoresis, so we are never directly exposed because there is a protective cover and we wear a mask that shields from UV.
</dd>
</dd>
<dt>Centrifuge:</dt>
<dt>Centrifuge:</dt>
<dd>
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The main risk is for the material. The centrifuge have to be perfectly balance otherwise the rotor could break. The majority of the centrifuge in our lab have detector that warns the operator in case of bad balance.  
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The centrifuges have to be perfectly balanced. All the centrifuges in our lab have detectors that warn the operator in case of imbalance.  
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<dt>Autoclave:</dt>
<dt>Autoclave:</dt>
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<dd>
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The operation of the autoclave require a specific training  
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The operation of the autoclave require a specific training and has only be performed by trained people.
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<dt>Water bath</dt>
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<dd>
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The risks might be contamination if bacterial suspension is spillt, a burn risks might exists in some case (high temperature of water)
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</dd>
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<h3 id="chemical">Chemical risk-assessment</h3>
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<h3 id="chemical">Chemical risk-assessment</h3>
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BET et Mercure, préciser que les gel sont fait sans BET, qu'il sont juste trempés dedans pour la photo dans une hotte, par conséquent rien de ce qui touche le BET ne s'éloigne de ce poste de travail avec la hotte, un pareil poste est utilisé pour le mercure faire le dessin avec autocad au Open office Draw
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A toxic chemical, the EtBr (ethidium bromide) is commonly used to stain DNA. We do not use EtBr solution while making our gel but we dip the gel in an EtBr bath after the electrophoresis. Due to the hazardous nature of this product, a hood is specially dedicated to its usage. The EtBr and all material that got in contact with it is stored in a special trash in the hood.
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We aim to design a detector and measurement device for a pollutant in water, like heavy metals. We are actualy working on two versions of this quantification device. One of them involves the use of the MerR sensor for the mercury, and the second one, TetR for the tetracycline. We therefore need to use mercury to test this system. That raises questions about security for the researcher but also for the public and the environment.
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We design a biosensor to measure a pollutant (like heavy metals) concentration in water. We are actually working on two versions of this biosensor. One of them involves the use of the MerR sensor for mercury, and the second alternative one, TetR for tetracycline. We therefore need to use mercury to test this system. This raises questions about safety for the researcher but also for the public and the environment. The teracycline is a safer alternative to test our system.
</p>
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<p>
<p>
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We also use for our experiments another toxic chemical, the EtBr (ethidium bromide). However this chemical is commonly used for microbiology. We employ the EtBr to make the DNA visible in a gel under U.V exposure after electrophoresis. We do not use EtBr solution while making our gel but we dip the gel in an EtBr bath after the electrophoresis. Due to the hazardous nature of this product, a hood is specially dedicated to its usage. The EtBr and all material that got in contact with it is store in a special trash in the hood.  
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For the tests we will have to use mercury in a water solution. It is the ionic form Hg2+ that will be used. The mercury is very toxic and mutagenic. To limit the risks in terms of probability of incident and in terms of hazards, we will use a stock solution. We will only have to dilute it to the wanted concentrations. Moreover a chemical hood will be dedicated to the preparation of these solutions and for the test of our system. A specific trash will also be dedicated to store all the wastes in contact with mercury.
</p>
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<p>
<p>
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In order to test our biosensor, mercury should be use in a water solution. It is the form Hg2+ that will be used. To limit the risks we will use a solution already prepared that we will just have to dilute to the wanted concentrations.
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Concerning the toxic waste management, a firm specialized in the processing of hazardous wastes recovers the barrels of toxic chemicals. A tracking sheet is associated to each toxic barrel. The lab receive then a document that certifies the appropriate waste treatment.
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<h3 id="bio">Biological risks, biosafety rules</h3>
<p>
<p>
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the environmental issue of the toxic waste management. Liquid having Mercury or tips and dishes that are in contact with this toxic are kept in specials bins. This rubbish bin is then given to a society specialized into toxic waste treatment. A slip monitoring is sign up by every organism that is involved into the production, transportation and treatment of the toxic waste. When the later is cremated, the producer of the waste receive and attestation that must be kept as a proof of the appropriate treatment.
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However we performed common experiments of molecular biology and biochemistry for which the risks are well known nowadays. The most uncertain part of our project is the genetic modification of living organism. Hence we are presenting the different Biobrick and confronting them to their related safety issues as well as a scenario where we discuss the different hazards and their possibility.
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In our bio-safety analysis, we try to take into consideration :
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<ul>
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<li>The risk of the chassis bacteria</li>
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<li>The one of each biobrick as well as their combinations in the whole device</li>
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<li>The robustness of the whole device</li>
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</ul>
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</p>
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Our project is based on the utilization of mercury, which raises  Mercury is an element that has toxic effects on brain and renal function. The other source of important chemical risks is BET. To avoid these latters, experiments are performed under chemical hoods and used contaminated materials are sterilized.
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<h4 id="microorg">Microorganism chassis</h4>
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After each experiment, all biological wastes are collected in a special bin and autoclaved before leaving the lab, to prevent environmental contamination.
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<p>
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During our project, mercury is conserved in the laboratory and is subjected to special treatment for elimination of heavy metals.  
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We work with a strain of E.Coli designed for lab work : BW25113. This strain is commonly used by students and researchers. It has no virulence genes, and is therefore a riskless chassis. Furthermore, it has got several genetic modifications that will limit its development if ever it was to make it out of the lab. Those modifications are :
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<ul>
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<li>An inactivated lacZ, ara and rha genes : the bacteria can use neither lactose, arabinose or rhamnose as sources of energy.</li>
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<li>A deletion into a gene coding for an enzyme (pyr E) involved in the synthesis of Thymine and Cytosine nucleotides. This deletion however does not make the strain auxotroph for theses bases.</li>
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</ul>
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These mutations are a disadvantage and limit the development of this strain on a minimal medium.
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<h4 id="bioparts">Biobricks parts used</h4>
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<p>
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The genetic device we develop is a toggle switch which includes two quorum sensing biobricks: cin I and cinR genes. We also use a reporter gene coding for a pigment, the lycopen as an output. We include a post transcriptional regulation mechanism extracted from Pseudomonas aeruginosa, rsma.  We present here some details about safet of these biobricks. Moreover we made an event tree to analyse a scenario where one of our bacteria went out of the lab.
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<h5 id="toggle">Toggle switch</h5>
<p>
<p>
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Bio-safety is not only about the nature of the basic parts, but also about their combinations. The toggle switch mechanism is a “man-made” specific combination of inoffensive genetic sequences. It has no hazard on its own but activates the transcription of downstream genes, in our case cinI or cinR (see here after). In an uncontaminated environment (no mercury) only cinR is transcribed and no quorum sensing molecule is synthesized.
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<h5 id="qs">Quorum sensing</h5>
<p>
<p>
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Concerning the reprocessing of toxic chemicals or biological wastes, we follow the standard protocols of our lab. The main  toxic wastes we have to deal with in our experiments is BET, the other very dangerous chemical is the mercury. Each one of this product is collected in a special barrel (one for each dangerous chemical) that are recovered by a company specialized in the reprocessing of hazardous wastes. The biological wastes are sterilized in an autoclave by heat and pressure before reprocessing by another company.
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We used CinI as quorum sensing, share by many species of legume-nodulating rhizobia <a href="#1">(1)</a>, a genus of soil bacteria that fix nitrogen. The cin quorum sensing molecule regulates growth inhibition, expression of nodulation genes, but no harmful response has been noticed so far. It is a Rhizobium-specific communication system. These bacteria colonise plant cells within root nodules and have never shown any pathogenicity towards humans and their environment.
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<h3 id="bio">Biological risks, biosafety rules.</h3>
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However we performed usual operations of biology and chemistry for which the risks are well known nowadays, our experiments aim to genetically modify a living organisms. And this aspects of the project safety issue is the most important in terms of information sought because it is the less known. Here we are dealing with probability, scenario that may happened and where consequences are mostly uncertain."
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<h4 id="microorg">Microorganisms.</h4>
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<h5 id="tomato">The lycopen</h5>
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<p>
<p>
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We work with a strain of E.Coli designed for lab works : BW25113. This strain is commonly used by students and researchers. It has no virulence gene, and is therefore a riskless chassis. Furthermore, it has got several genetic modifications that avoid its development if ever it was to make it out of the lab. Those modifications are :
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We use a combination of three genes that codes for lycopen. This pigment is naturally found in tomato and has no toxicity.
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<h5 id="rsma">The rsma regulation system</h5>
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<p>
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The Rsma translational regulation system we extracted from an opportunistic bacterium called Pseudomonas aeruginosa <a href="#2">(2)</a>⁠. It is very similar to others regulation systems like “Csra” that can be found in many eubacteria <a href="#3">(3)</a>. The Rsma and csra systems both control a large variety of physiological processes such as central carbon metabolism, motility, biofilm formation, virulence, pathogenesis <a href="#4">(4)</a> ⁠and many more <a href="#5">(5)</a>⁠...
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Basically, when CsrA or RsmA proteins are expressed, they bind to a mRNA leader sequence and act as translational repressors by inducing their degradation. Alternatively, when a small RNA molecule is expressed (called rsmy in the Pseudomonas system) it titres and sequesters the protein, allowing the expression of targeted genes <a href="#5">(5)</a>. In most systems, the binding site on the RNA leader sequence is a stem-loop containing repeats of GGA nucleotides <a href="#4">(4)</a>.
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In Pseudomonas, the protein RsmA negatively regulates the type VI secretion system, which has been implicated in the P.aeruginosa chronic infections <a href="#5">(5)</a>⁠. In specific environment conditions, rsmY/rsmZ are transcribed, which produces a syringe base plate <a href="#4">(4)</a>⁠. That is used to inject proteins into a target cell.
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<h5 id="outlab">Analysis of a catastrophic scenario</h5>
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<p>
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Being originally implicated in the activation of virulence genes, this rsma regulation system implies safety issues. For instance, the RsmA system could somehow interfere with the CsrA system of E. coli, which is highly homologous.  
<ul>
<ul>
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<li>An inactivated LacZ and rha genes : the bacteria can use neither the lactose or the rhamnose as a source of energy.</li>
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<li>So what would happen if our strain was to make it out of the lab ?</li>
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<li>A deletion into a gene coding for an enzyme (pyr E) that produce a matrice required for DNA fabrication (Thymine and Cytosine bases).</li>
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<li>Could our genetic device activate genes in a wild type E.coli strains, or in any other bacteria ?</li>
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<li>A deletion into the gene that code for an enzyme involved in the fabrication of arabinose, an amino acid component of most protein.</li>
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<li>Would it, then, become harmful to human or any other organism in the environment ?</li>
</ul>
</ul>
</p>
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<p>
<p>
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So this bacteria has limited source of energy, needs to be supplied into DNA and protein constituents. Those are such disadvantages that this strain can only grow on highly supplemented medium. We also apply the national lab biohazard policy to each experiment : all the biological waste are collected on a special bin and autoclaved before leaving the lab.
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The worst situations we could imagine with an organism containing these bricks are:
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<ul>
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<li>an over development of bacteria in any specific environment</li>
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<li>a health threat to human or any other organism</li>
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</ul>
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</p>
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<p>
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We tried to think about what would be the series of event to cause such a disaster. For each of them we tried to think of how probable it is to occur, and what is know about the possible hazard.
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</p>
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<p>
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In order to illustrate our vision of the risk, we made an event tree analysis composed of three colours representing the level of probability (yellow - orange - red).
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<h4 id="bioparts">Biobricks parts used.</h4>
 
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<h5 id="toggle">Toggle switch</h5>
 
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<h5 id="qs">Quorum sensing</h5>
 
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<h5 id="tomato">Pigment</h5>
 
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<h5 id="rsma">RsmA</h5>
 
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<h5 id="outlab">Scenario, what would happen if bacteria went out of the lab ?</h5>
 
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<a href="https://static.igem.org/mediawiki/2011/a/a8/Event_tree.png"><img src="https://static.igem.org/mediawiki/2011/a/a8/Event_tree.png" alt="Event tree, bacteria out of the lab" title="Event Tree, bacteria are out of the lab ! What would happen ?" width="620"/></a>
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<center>
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<a href="https://static.igem.org/mediawiki/2011/f/fe/Safety_schematic.png"><img src="https://static.igem.org/mediawiki/2011/f/fe/Safety_schematic.png" alt="Event tree, bacteria out of the lab" title="Event Tree, bacteria are out of the lab ! What would happen ?" width="610" class="bordure"></a>
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<table>
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  <th>Hazard</th>
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  <th>Likelyhood of Hazard to occur</th>
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</tr>
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<tr>
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  <td>1. Some bacteria containing our genetic circuit get out of the lab.</td>
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  <td>
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<ul>
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<li>So far the project is experimental : all components are confined to  the laboratory. </li>
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<li>All the biological waste are collected in a special bin and autoclaved before leaving the lab.</li>
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</ul>
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</td>
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</tr>
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<tr>
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  <td>2. These bacteria grow in the environment</td>
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  <td>Our strain has  several mutations that limit its development outside the favourable conditions of the lab</td>
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</tr>
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<tr>
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  <td>3. A living E.coli from the lab tranfers genes to a wild type E.coli by conjugation</td>
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  <td>This strain is F- so it cannot build up any pili that are necessary for gene transfer. It could however become competent if it would acquire the necessary genes in contact with  F+ bacteria.</td>
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</tr>
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<tr>
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  <td>4. Wild-type bacteria of any species incorporate genetic material from a lysed cell released from the laboratory</td>
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  <td>The sequence we use comes originally from Pseudomonas. The risk to transfer this DNA sequence therefore already exists potentially in nature.</td>
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</tr>
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<tr>
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  <td>5. The transferred RsmA protein or rsmy RNA interact with the host regulation system</td>
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  <td>This is likely to occur since homologs exist in many species.</td>
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</tr>
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<tr>
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  <td>6. The transferred DNA can activate the transcription of virulence genes</td>
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  <td>Several check points exist in a cell to control such a global mechanism. A response is triggered only if all checkpoints are coherently functioning.</td>
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</tr>
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<tr>
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  <td>7. Virulence factors are expressed in the bacteria which  has incoporated the brick</td>
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  <td>In a normal condition a virulent cell would express its virulence only when a targeted host is close and triggers it. This might not be the case. Then the expression of virulence factors would be useless, or even a disadvantage.</td>
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</tr>
 +
<tr>
 +
  <td>8. Virulence factors are expressed and threat a specific target </td>
 +
  <td>As far as we know about this system, it  is used by opportunistic bacteria that can become pathogenic only to immune-depressive individuals. </td>
 +
</tr>  
 +
 +
</table>
 +
 +
<h4 id="robustus">Robustness of the device</h4>
 +
<p>
<p>
-
The system we develop needs to be kept off until we want to induce it. In order to achieve that, we extract a post-transcriptional switch mechanism. This system comes from Pseudomonas aeruginosa, a similar one exists in E. coli.
+
<center>
 +
<a href="https://2011.igem.org/Team:Grenoble/Projet/Results/Sensitivity" title="Click here"><img src="https://static.igem.org/mediawiki/2011/f/fd/Bouton_model_sensitivity.png"/></a>
 +
<div class="legend">
 +
Click on the icon above to see the result of mathematical study of the robustness of the device.
 +
</div>
 +
</center>
</p>
</p>
<p>
<p>
-
The rsma system of P.aeruginosa controls numerous genes including some virulence factors expressed in some oportunist condition. When activated, it allows the transcription of some genes involved in the creation of a syringe. The later is used to inject some proteins into a targeted cell. E.Coli has not this kind of pathogenic mechanism at all.
+
In this section, we consider how the device would behave if some of the components stop working properly. Mutations can prevent the functionning of parts of the genetic circuit, with predictible consequences.
</p>
</p>
<p>
<p>
-
The rsma system could somehow interfere with the Csra system of E. coli, which is highly similar and also involved in global regulation mechanism. This has not been tested so far. So what would happen if our strain was to make it out of the lab ?  Could our genetic device activate some gene of a wild E.coli strain, or other bacteria ? We have no experimental data to argue for any response. Considering the worst case - the mechanism we use can be effective in wild bacteria - this event would require several extremely low probability events to occur :
+
In our project, we can divide the system into four components:
<ul>
<ul>
-
<li>Harmless lab bacteria containing the device should be out of the lab. So far the project is experimental and no bacteria should get out, as describe above.</li>
+
<li>the toggle switch component</li>
-
<li>The strain we use can not leave on other medium than the one used in a lab (LB agar) so if spread out, it should quickly die.</li>
+
<li>the communication component based on quorum sensing genes</li>
-
<li>Our strain has no competence to build up a pili and share genetic information. So for any of its DNA to be incorporated, it should be from lysed cell, which decrease the probability of transfer.</li>
+
<li>the output signaling component</li>
 +
<li>a translational regulation component based on rsma</li>
</ul>
</ul>
</p>
</p>
<p>
<p>
-
If incorporated into a wild strain, the risk would be to activate some virulence gene.
+
Several situations were considered and tested using the numerical model we developped. Here are a few examples of unwanted behaviours of components and their expected consequences.
 +
</p>
 +
<p>
<ul>
<ul>
-
<li>First, it implies that the wild strain has a similar mechanism of global gene regulation.</li>
+
<li>If the degradation tags of the repressors in the toggle switch do not work, the toggle switch would become very slow in switching and therefore unable to sense external molecule concentration. The response of the device will be very slow.</li>
-
<li>Even if there is a similar mechanism, the wild one and the one extracted from pseudomonas would need to be made of similar DNA sequence to interact. A  good compatibility of two mechanisms is quite unlikely.</li>
+
<li>If any branch of the toggle switch is not functional, then no activation of the output signaling component can occur. The plate would remain white. </li>
-
<li>To become pathogenic, the wild bacteria should also have virulence genes to human, which is not that common. Otherwise its metabolism would be modified without any consequences to human.</li>
+
<li>If the cinI promotor leaks or is not sensitive enough to AHL, the output signal will be produced whatever the external molecule concentrations. The entire device will be red. </li>
-
<li>The system we use is originally activated in some specific conditions : when pseudomonas is in contact with a target cell. In which environment would be the lucky bacteria that could have got the sequence ? A rubbish bin, a dump, a river ?... In most of the situations, it would not be an environment on that gives an advantage to the cell that produce virulence factor.</li>
+
<li>If the post-transcriptional system doesn't work, the toggle switch would be less sensitive but still working. The red band will be larger.</li>
-
<li>Given that it is a global mechanism control system, there are always numerous other check point into the cell before modifying its expression. So even if integrated, the rsma system would certainly be either a disadvantage or would get inhibited.</li>
+
</ul>
</ul>
</p>
</p>
<p>
<p>
-
An other system used in our device can modify expression of numerous genes : the CinI quorum sensing. The later is shared by many species of legume-nodulating rhizobia : a genus of soil bacteria that fix nitrogen. The cin quorum regulates growth inhibition, expression of some genes that influence nodulation, but no harmful response have been notice so far. It is quite a Rhizobium specific communication system. This bacteria colonises plant cells within root nodules and has never shown any pathogenic sign to human.
+
This last result suggests that the RsmA/rsmY regulation system is not critical but modelling show it improves dramatically the sensitivity of our device. Given that it is a global regulation system, it might interfere with cinI (see section biobrick safety). Before integration into our final genetic network we will have to test this possibility.
-
</p>
+
</p>
 +
 +
<center>
 +
<a href="https://2011.igem.org/Team:Grenoble/Projet/Results/rmsA" title="Click here"><img src="https://static.igem.org/mediawiki/2011/9/97/Bouton_regulation.png"/></a>
 +
<div class="legend">
 +
Click on the icon above to know why RsmA is important.
 +
</div>
 +
</center>
</div>
</div>
-
 
-
 
-
<h4 id="patho">Pathogenicity, Infectivity and Toxicity.</h4>
+
-
+
<div class="blocbackground">
<div class="blocbackground">
 +
 +
<h2 id="bibli">References</h2>
<p>
<p>
-
Manipulation of living organism allows producing artificial form of life and metabolism. These modifications, although well controlled, require application of the precautionary principle.  
+
<div id="1">1.</div> Wisniewski-dy, F. and Downie, J.A. Quorum-sensing in Rhizobium. Antonie van Leeuwenhoek 397-407(2002).
-
</p>
+
-
<p>
+
-
Even if in our project we don't plan to take our work out of the lab, so this can not be a cause of safety issue, engineered bacteria might be accidentally or on purpose released in the environment. So, caution involves the implementation of different blocking to limit the propagation of these organisms in the nature:
+
</p>
</p>
-
 
<p>
<p>
-
<dl>
+
<div id="2">2.</div> Brencic, A. and Lory, S. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Molecular Microbiology 72, 612-632(2009).
-
<dt>Nutritional blocking:</dt>  
+
</p>
-
<dd>Organisms could survive only with artificial substances. In this way, in case of release into the nature such organisms would die.</dd>
+
<p>
-
<dt>Evolutionary blocking:</dt>
+
<div id="3">3.</div> Timmermans, J. and Melderen, L.V. Post-transcriptional global regulation by CsrA in bacteria. Cellular and Molecular Life Sciences 2897-2908(2010).doi:10.1007/s00018-010-0381-z
-
<dd>Organisms couldn’t adapt themselves and evolve alone in the nature. This blocking prevents mutations of the organisms that allow them to survive.</dd>
+
</p>
-
<dt>Preprogrammed cellular death:</dt>  
+
<p>
-
<dd>implementation of a suicide gene which is inhibited during wet work. In this way, organisms couldn’t survive outside the laboratory.</dd>
+
<div id="4">4.</div> Mercante, J. et al. Molecular Geometry of CsrA ( RsmA ) Binding to RNA and Its Implications for Regulated Expression. Journal of Molecular Biology 392, 511-528(2009).
-
</dl>
+
</p>
-
</p>
+
<p>
 +
<div id="5">5.</div> Bernard, C.S. et al. MINIREVIEW Nooks and Crannies in Type VI Secretion Regulation . Society 192, 3850-3860(2010).
 +
</p>
</div>
</div>
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<strong>Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</strong>
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<h2 id="bio">Biological risks, biosafety rule</h2>
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<h2 id="optional">OPTIONAL QUESTION:</h2>
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<strong>Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</strong>
+
-
+
-
<div class="blocbackground">
+
-
+
<p>
<p>
-
In our project, and we think it is the case of many other, the major problem we want to avoid is
+
Manipulation of living organism allows producing artificial form of life and metabolism. These modifications, although well controlled, require application of the precautionary principle. Even if in our project we do not plan to take our work out of the lab, engineered bacteria might be accidentally or on purpose released in the environment.
-
In case of a bacteria would go outside the lab, efficient methods can be used to limit or prevent their development. Synthetic biology project should employed living organisms mutated to become enable to survive outside the lab. For exmple bacteria that must use amino acids which do not exists in the nature. It is also possible to force the microorganisms to use rare carbon sources. An other possibility is the use of a suicide gene repress by an artificial molecule that can not be found out of a laboratory. Another ways is to make bacteria weak face to the micro-organisms natural selection.  
+
</p>
 +
<p>
 +
In many projects, the main issue is that bacteria may leave the lab. In such a case, efficient methods should be used to limit or prevent their development ouside. Synthetic biology project should employ living organisms unnable to survive outside the lab. For exemple microorganisms forced to use rare carbon sources. Another possibility is to have a suicide gene repressed by an artificial molecule no tpresent in nature. Another way is to make bacteria weaker so that they cannot face natural selection.  
</p>
</p>
<p>
<p>
-
+
To increase the safety of iGEM competition, we propose to use auxotrophic bacteria that cannot grow in the absence of a given amino acid, for instance.
 +
</p>
 +
<p>
 +
A team of researcher from CEA (IG/Genoscope – Évry), Institut für Biologie (Freie Universität, Berlin), CNRS, University of Evry, Katholieke Universiteit (Leuven) and Heurisko company (USA) worked on <a href="http://www.cea.fr/le_cea/actualites/evolution_d_un_genome_bacterien-60034"" title="chemical evolution of a bacterial genome">Chemical evolution of a bacterial genome.</a> They forced the bacteria to use a synthetic chemical instead of Thymine.
</p>
</p>
-
<p>
+
</div>
-
For the researcher’s safety in lab, the work in sterile middle, overall and gloves wearing and all other standard protections things are evidently recommended.
+
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</p>
+
-
<p>
+
-
To increase the safety off iGEM competition, we think about bacteria which have an inducible essential gene for binary division by a chemical not existing or rare in nature, by this way the bacteria can’t be divide itself so it will be not selected and going to disappear nearly.
+
-
</p>
+
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http://www.cea.fr/le_cea/actualites/evolution_d_un_genome_bacterien-60034
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document.getElementById('submenu').innerHTML = '<h3><span class="vert">Safety</span> Issues</h3><ul><li><a href="#lab">Lab work safety</a></li><ol><li><a href="#general">General considerations</a></li><li><a href="#instru">Instruments</a></li><li><a href="#chemical">Chemical risk-assessment</a></li></ol><li><a href="#bio">Biological risks, biosafety rules</a></li><ol><li><a href="#microorg">Microorganism chassis</a></li><li><a href="#bioparts">Biobrick parts used</a></li><ol><li><a href="#toggle">Toggle switch</a></li><li><a href="#qs">Quorum sensing</a></li><li><a href="#tomato">The lycopen</a></li><li><a href="#rsma">The rsma regulation system</a></li><li><a href="#outlab">Analysis of a catastrophic scenario</a></li></ol><li><a href="#robustus">Robustness of the device</a></li></ol><li><a href="#bibli">References</a></li><li><a href="#optional">Optional question</a></li></ul>'
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{{:Team:Grenoble/Design/pied}}

Latest revision as of 20:50, 28 October 2011

Grenoble 2011, Mercuro-Coli iGEM


Safety issues

In general, the work in a laboratory requires the use of complex equipment or it implies performing delicate operations. The material, that could be a machine, chemicals or biological material involves the existence of risks. Risks for the goods and for people in the room, but also risks for the environment and people outside the lab. The safety rules and procedures as well as the personal and collective protective equipment are made to minimize the risks by decreasing the probability of an incident to happen.

General considerations

Half of our team made an internship at the CEA Grenoble. The CEA has a specific department working on safety issues. There is also a special team in charge of security and safety called FLS: Formation locale de sécurité, we may translate: Local Group of Security and Safety. They ensure the safety of the people who are working in the center and the visitors and also of the goods and the material. The members of our team who made their internship in CEA have attended a compulsory safety training organised by FLS.

The whole team is now working together in another lab, the CIME (centre inter-universitaire de microélectronique), next to the CEA labs and the Phelma school buildings. All team members have met the safety engineer of the labs where we conduct the experiments. He explained us the safety rules to be followed.

At CEA some researchers work on microsystems to detect and quantify pollutants like heavy metals. They shared their experience and knowledge with us about the way to conduct safe experiments with very toxic chemicals like mercury.

Instruments

During our experiments, we only performed commonly used protocols and instrumentation for microbiology and common laboratory strains of E.Coli. We have used basic devices that we find in every molecular biological laboratory:

Ultra violet lamp:
There is a risk for the eyes and the skin, but the UV lamp is only used to take a picture of our gel after electrophoresis, so we are never directly exposed because there is a protective cover and we wear a mask that shields from UV.
Centrifuge:
The centrifuges have to be perfectly balanced. All the centrifuges in our lab have detectors that warn the operator in case of imbalance.
Autoclave:
The operation of the autoclave require a specific training and has only be performed by trained people.

Chemical risk-assessment

A toxic chemical, the EtBr (ethidium bromide) is commonly used to stain DNA. We do not use EtBr solution while making our gel but we dip the gel in an EtBr bath after the electrophoresis. Due to the hazardous nature of this product, a hood is specially dedicated to its usage. The EtBr and all material that got in contact with it is stored in a special trash in the hood.

We design a biosensor to measure a pollutant (like heavy metals) concentration in water. We are actually working on two versions of this biosensor. One of them involves the use of the MerR sensor for mercury, and the second alternative one, TetR for tetracycline. We therefore need to use mercury to test this system. This raises questions about safety for the researcher but also for the public and the environment. The teracycline is a safer alternative to test our system.

For the tests we will have to use mercury in a water solution. It is the ionic form Hg2+ that will be used. The mercury is very toxic and mutagenic. To limit the risks in terms of probability of incident and in terms of hazards, we will use a stock solution. We will only have to dilute it to the wanted concentrations. Moreover a chemical hood will be dedicated to the preparation of these solutions and for the test of our system. A specific trash will also be dedicated to store all the wastes in contact with mercury.

Concerning the toxic waste management, a firm specialized in the processing of hazardous wastes recovers the barrels of toxic chemicals. A tracking sheet is associated to each toxic barrel. The lab receive then a document that certifies the appropriate waste treatment.

Biological risks, biosafety rules

However we performed common experiments of molecular biology and biochemistry for which the risks are well known nowadays. The most uncertain part of our project is the genetic modification of living organism. Hence we are presenting the different Biobrick and confronting them to their related safety issues as well as a scenario where we discuss the different hazards and their possibility.

In our bio-safety analysis, we try to take into consideration :

  • The risk of the chassis bacteria
  • The one of each biobrick as well as their combinations in the whole device
  • The robustness of the whole device

Microorganism chassis

After each experiment, all biological wastes are collected in a special bin and autoclaved before leaving the lab, to prevent environmental contamination.

We work with a strain of E.Coli designed for lab work : BW25113. This strain is commonly used by students and researchers. It has no virulence genes, and is therefore a riskless chassis. Furthermore, it has got several genetic modifications that will limit its development if ever it was to make it out of the lab. Those modifications are :

  • An inactivated lacZ, ara and rha genes : the bacteria can use neither lactose, arabinose or rhamnose as sources of energy.
  • A deletion into a gene coding for an enzyme (pyr E) involved in the synthesis of Thymine and Cytosine nucleotides. This deletion however does not make the strain auxotroph for theses bases.

These mutations are a disadvantage and limit the development of this strain on a minimal medium.

Biobricks parts used

The genetic device we develop is a toggle switch which includes two quorum sensing biobricks: cin I and cinR genes. We also use a reporter gene coding for a pigment, the lycopen as an output. We include a post transcriptional regulation mechanism extracted from Pseudomonas aeruginosa, rsma. We present here some details about safet of these biobricks. Moreover we made an event tree to analyse a scenario where one of our bacteria went out of the lab.

Toggle switch

Bio-safety is not only about the nature of the basic parts, but also about their combinations. The toggle switch mechanism is a “man-made” specific combination of inoffensive genetic sequences. It has no hazard on its own but activates the transcription of downstream genes, in our case cinI or cinR (see here after). In an uncontaminated environment (no mercury) only cinR is transcribed and no quorum sensing molecule is synthesized.

Quorum sensing

We used CinI as quorum sensing, share by many species of legume-nodulating rhizobia (1)⁠, a genus of soil bacteria that fix nitrogen. The cin quorum sensing molecule regulates growth inhibition, expression of nodulation genes, but no harmful response has been noticed so far. It is a Rhizobium-specific communication system. These bacteria colonise plant cells within root nodules and have never shown any pathogenicity towards humans and their environment.

The lycopen

We use a combination of three genes that codes for lycopen. This pigment is naturally found in tomato and has no toxicity.

The rsma regulation system

The Rsma translational regulation system we extracted from an opportunistic bacterium called Pseudomonas aeruginosa (2)⁠. It is very similar to others regulation systems like “Csra” that can be found in many eubacteria (3). The Rsma and csra systems both control a large variety of physiological processes such as central carbon metabolism, motility, biofilm formation, virulence, pathogenesis (4) ⁠and many more (5)⁠...

Basically, when CsrA or RsmA proteins are expressed, they bind to a mRNA leader sequence and act as translational repressors by inducing their degradation. Alternatively, when a small RNA molecule is expressed (called rsmy in the Pseudomonas system) it titres and sequesters the protein, allowing the expression of targeted genes (5). In most systems, the binding site on the RNA leader sequence is a stem-loop containing repeats of GGA nucleotides (4).

In Pseudomonas, the protein RsmA negatively regulates the type VI secretion system, which has been implicated in the P.aeruginosa chronic infections (5)⁠. In specific environment conditions, rsmY/rsmZ are transcribed, which produces a syringe base plate (4)⁠. That is used to inject proteins into a target cell.

Analysis of a catastrophic scenario

Being originally implicated in the activation of virulence genes, this rsma regulation system implies safety issues. For instance, the RsmA system could somehow interfere with the CsrA system of E. coli, which is highly homologous.

  • So what would happen if our strain was to make it out of the lab ?
  • Could our genetic device activate genes in a wild type E.coli strains, or in any other bacteria ?
  • Would it, then, become harmful to human or any other organism in the environment ?

The worst situations we could imagine with an organism containing these bricks are:

  • an over development of bacteria in any specific environment
  • a health threat to human or any other organism

We tried to think about what would be the series of event to cause such a disaster. For each of them we tried to think of how probable it is to occur, and what is know about the possible hazard.

In order to illustrate our vision of the risk, we made an event tree analysis composed of three colours representing the level of probability (yellow - orange - red).


Event tree, bacteria out of the lab


Hazard Likelyhood of Hazard to occur
1. Some bacteria containing our genetic circuit get out of the lab.
  • So far the project is experimental : all components are confined to the laboratory.
  • All the biological waste are collected in a special bin and autoclaved before leaving the lab.
2. These bacteria grow in the environment Our strain has several mutations that limit its development outside the favourable conditions of the lab
3. A living E.coli from the lab tranfers genes to a wild type E.coli by conjugation This strain is F- so it cannot build up any pili that are necessary for gene transfer. It could however become competent if it would acquire the necessary genes in contact with F+ bacteria.
4. Wild-type bacteria of any species incorporate genetic material from a lysed cell released from the laboratory The sequence we use comes originally from Pseudomonas. The risk to transfer this DNA sequence therefore already exists potentially in nature.
5. The transferred RsmA protein or rsmy RNA interact with the host regulation system This is likely to occur since homologs exist in many species.
6. The transferred DNA can activate the transcription of virulence genes Several check points exist in a cell to control such a global mechanism. A response is triggered only if all checkpoints are coherently functioning.
7. Virulence factors are expressed in the bacteria which has incoporated the brick In a normal condition a virulent cell would express its virulence only when a targeted host is close and triggers it. This might not be the case. Then the expression of virulence factors would be useless, or even a disadvantage.
8. Virulence factors are expressed and threat a specific target As far as we know about this system, it is used by opportunistic bacteria that can become pathogenic only to immune-depressive individuals.

Robustness of the device

Click on the icon above to see the result of mathematical study of the robustness of the device.

In this section, we consider how the device would behave if some of the components stop working properly. Mutations can prevent the functionning of parts of the genetic circuit, with predictible consequences.

In our project, we can divide the system into four components:

  • the toggle switch component
  • the communication component based on quorum sensing genes
  • the output signaling component
  • a translational regulation component based on rsma

Several situations were considered and tested using the numerical model we developped. Here are a few examples of unwanted behaviours of components and their expected consequences.

  • If the degradation tags of the repressors in the toggle switch do not work, the toggle switch would become very slow in switching and therefore unable to sense external molecule concentration. The response of the device will be very slow.
  • If any branch of the toggle switch is not functional, then no activation of the output signaling component can occur. The plate would remain white.
  • If the cinI promotor leaks or is not sensitive enough to AHL, the output signal will be produced whatever the external molecule concentrations. The entire device will be red.
  • If the post-transcriptional system doesn't work, the toggle switch would be less sensitive but still working. The red band will be larger.

This last result suggests that the RsmA/rsmY regulation system is not critical but modelling show it improves dramatically the sensitivity of our device. Given that it is a global regulation system, it might interfere with cinI (see section biobrick safety). Before integration into our final genetic network we will have to test this possibility.

Click on the icon above to know why RsmA is important.

References

1.
Wisniewski-dy, F. and Downie, J.A. Quorum-sensing in Rhizobium. Antonie van Leeuwenhoek 397-407(2002).

2.
Brencic, A. and Lory, S. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Molecular Microbiology 72, 612-632(2009).

3.
Timmermans, J. and Melderen, L.V. Post-transcriptional global regulation by CsrA in bacteria. Cellular and Molecular Life Sciences 2897-2908(2010).doi:10.1007/s00018-010-0381-z

4.
Mercante, J. et al. Molecular Geometry of CsrA ( RsmA ) Binding to RNA and Its Implications for Regulated Expression. Journal of Molecular Biology 392, 511-528(2009).

5.
Bernard, C.S. et al. MINIREVIEW Nooks and Crannies in Type VI Secretion Regulation . Society 192, 3850-3860(2010).

OPTIONAL QUESTION:

Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Manipulation of living organism allows producing artificial form of life and metabolism. These modifications, although well controlled, require application of the precautionary principle. Even if in our project we do not plan to take our work out of the lab, engineered bacteria might be accidentally or on purpose released in the environment.

In many projects, the main issue is that bacteria may leave the lab. In such a case, efficient methods should be used to limit or prevent their development ouside. Synthetic biology project should employ living organisms unnable to survive outside the lab. For exemple microorganisms forced to use rare carbon sources. Another possibility is to have a suicide gene repressed by an artificial molecule no tpresent in nature. Another way is to make bacteria weaker so that they cannot face natural selection.

To increase the safety of iGEM competition, we propose to use auxotrophic bacteria that cannot grow in the absence of a given amino acid, for instance.

A team of researcher from CEA (IG/Genoscope – Évry), Institut für Biologie (Freie Universität, Berlin), CNRS, University of Evry, Katholieke Universiteit (Leuven) and Heurisko company (USA) worked on Chemical evolution of a bacterial genome. They forced the bacteria to use a synthetic chemical instead of Thymine.