Team:Grenoble/Safety

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<h1>Safety</h1>
 
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<h1>Safety issues</h1>
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<li><a href="#risk">Risk-assessment of our project</a></li>
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<li><a href="#instru">Instrumentation.</a></li>
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<li><a href="#chemical">Chemical risk-assessment.</a></li>
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<li><a href="#biological">Biological risk-assessment.</a></li>
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<li><a href="#microorg">Microorganisms.</a></li>
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<li><a href="#bioparts">Used biobriks Parts.</a></li>
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<li><a href="#eval">Evaluation methods of the biobricks parts.</a></li>
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<li><a href="#patho">Pathogenicity, Infectivity and Toxicity.</a></li>
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<li><a href="#environment">Environmental impact.</a></li>
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<li><a href="#safety">Biosafety provisions</a></li>
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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="risk">Risk-assessment of our project</h2>
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<h3 id="general">General considerations</h3>
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Laboratory work requires the using of complex equipment or performing delicate operations, it also involves the use of toxic, flammable or explosive. The execution of this work may cause accidents or serious poisoning; the effects can be immediate or insidious. For this all reasons there are safety rules to follow.
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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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<p>
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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 datsheet and moreover the safety engineers of our labs.
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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.
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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.  
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<h3 id="instru">Instrumentation</h3>
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<h3 id="instru">Instruments</h3>
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The experiences of our project did not require the use of sophisticated equipment. We have used basic devices that we find in 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:
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<dt>Ultra violet lamp: </dt>
<dt>Ultra violet lamp: </dt>
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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.
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<dt>Centrifuge:</dt>
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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>
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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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<h3 id="chemical">Chemical risk-assessment.</h3>
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<h3 id="chemical">Chemical risk-assessment</h3>
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Our project is based on the utilization of mercury, which raises questions about security for the researcher but also for the public and the environment. 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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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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During our project, mercury is conserved in the laboratory and is subjected to special treatment for elimination of heavy metals. About searchers, protections are simpler: it is necessary to work without contaminating the material. If it is contaminated, it shall not be touched with bare hands. Hence the use of protective equipment.
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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.
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<p>
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We are making a device that can quantify a component in water, such as heavy metals pollutants. Two models are being developed. One of them involves the use of the Mer sensor. We therefore need to use mercury to test this system. These raise 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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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.
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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="biological">Biological risk-assessment.</h3>
 
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<h4 id="microorg">Microorganisms</h4>
 
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<h3 id="bio">Biological risks, biosafety rules</h3>
<p>
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We work with a strain of E.Coli designed for the lab works. It is commonly used by the students and the researcher.  
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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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<h4 id="bioparts">Used biobriks Parts.</h4>
 
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The system we develop needs to be kept off until we want to induce it. In order to achieve that, we develop a post-transcriptional switch mechanism. This system is extracted from Pseudomonas aeruginosa, a highly similar system exists in E. coli.  
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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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<h4 id="microorg">Microorganism chassis</h4>
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<p>
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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.
</p>
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<p>
<p>
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The system of P.aeruginosa controls numerous genes including virulence factors, a syringe mechanism to inject toxic compounds to a targeted cell, but the bacteria E.Coli we work with does not have this kind of system. We have chosen the system from P.aeruginosa to avoid interferences between our genetic circuit and the metabolism of E.Coli.
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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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<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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<h5 id="eval">Evaluation methods of the biobricks parts.</h5>
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Manipulation of living organism allows producing artificial form of life and metabolism. These modifications, although well controlled, require application of the precautionary principle.
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These mutations are a disadvantage and limit the development of this strain on a minimal medium.
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<h5 id="patho">Pathogenicity, Infectivity and Toxicity.</h5>
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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:
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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>
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<p>
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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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<h5 id="tomato">The lycopen</h5>
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<p>
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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> Nutritional blocking: organisms could survive only with artificial substances. In this way, in case of release into the nature such organisms would die. </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> Evolutionary blocking: organisms couldn’t adapt themselves and evolve alone in the nature. This blocking prevents mutations of the organisms that allow them to survive. </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> Preprogrammed cellular death: implementation of a suicide gene which is inhibited during wet work. In this way, organisms couldn’t survive outside the laboratory. </li>
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<li>Would it, then, become harmful to human or any other organism in the environment ?</li>
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<h3 id="environment">Environmental impact.</h3>
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The toxic wastes like BET that we use during our experiments are collected in special barrels that are recovered by the reprocessing hazardous wastes company.
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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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<h2 id="safety">Safety provisions</h2>
 
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We have FLS (Formation locale de sécurité) that regulates what is coming in the laboratory where we are working; they ensure the safety of the researchers, public, and the environment. Six member of our team have had safety training, and all team members have met the safety engineer, who explained the safety rules to be followed.
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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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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 these chemicals and also about technical aspect 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 and costs.
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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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<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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</center>
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<br/>
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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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  <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>
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<tr>
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  <td>8. Virulence factors are expressed and threat a specific target </td>
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  <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>
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</tr>  
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</table>
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<h4 id="robustus">Robustness of the device</h4>
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<p>
<p>
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So our work for the safety section of our wiki is not over, it will continue all over our project and after the jamborees. Indeed, 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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<center>
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<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>
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<div class="legend">
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Click on the icon above to see the result of mathematical study of the robustness of the device.
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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.
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</p>
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<p>
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In our project, we can divide the system into four components:
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<ul>
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<li>the toggle switch component</li>
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<li>the communication component based on quorum sensing genes</li>
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<li>the output signaling component</li>
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<li>a translational regulation component based on rsma</li>
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</ul>
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</p>
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<p>
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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.
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</p>
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<p>
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<ul>
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<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>
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<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>
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<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>
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<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>
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</ul>
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</p>
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<p>
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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.
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<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>
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<div class="legend">
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Click on the icon above to know why RsmA is important.
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</center>
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<strong>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?</strong>
 
<div class="blocbackground">
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<h2 id="bibli">References</h2>
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From the environmental point of view, simple and efficiency methods can be used like bacteria not able to survive outside by use Amino Acids which not existing in nature. It’s too possible to use rare carbon source for the bacteria. We can also use a suicide gene repress by a chemical molecule not found out of a laboratory. Another ways is to make bacteria weak face to the micro-organisms natural selection. 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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<div id="1">1.</div> Wisniewski-dy, F. and Downie, J.A. Quorum-sensing in Rhizobium. Antonie van Leeuwenhoek 397-407(2002).
</p>
</p>
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<p>
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<p>
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<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).
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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>
</p>
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+
<p>
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<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
 +
</p>
 +
<p>
 +
<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).
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</p>
 +
<p>
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<div id="5">5.</div> Bernard, C.S. et al. MINIREVIEW Nooks and Crannies in Type VI Secretion Regulation . Society 192, 3850-3860(2010).
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<div class="blocbackground">
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ARTICLE DU CEA SUR Base artificielle
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<h2>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>
 +
<p>
 +
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.
 +
</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>
 +
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.
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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.