Team:Cornell/project/safety

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<h6 style="margin-top:32px;">Human Practices</h6>
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<a href="https://2012.igem.org/Team:Cornell/project/hprac">Overview</a>
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/CEA">Comprehensive Environmental Assessment</a>
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/bioethics">Bioethics</a>
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/cayuga_watershed">Cayuga Watershed</a>
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<a href="https://2012.igem.org/Team:Cornell/project/hprac/safety">Safety</a>
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<h2 class="centered">Safety Overview</h2>
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<h6> View our <a href="http://www.dropbox.com/s/grdh8fyd6bmnpzk/Cornell_iGEM_2013_Basic_Safety_Form%20%282%29.pdf" target="_blank">safety form</a>.</h6>
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<h3>Safe Practices in the Lab</h3>
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Our team conducted our work on the Cornell University campus, in the Biomedical Engineering Instructional Lab in Weill Hall, as well as in the <a href="http://angenent.bee.cornell.edu/molecularlab.html" target="_blank">Angenent Lab</a> in the Department of Biological &amp; Environmental Engineering in Riley-Robb Hall. All work was conducted in a biosafety level (BSL) 1 laboratory: this means that the strains we are using (<i>Escherichia coli</i> DH5a and WM3064, <i>Shewanella oneidensis</i> MR-1 and JG700, and <i>Pseudomonas putida</i>) are non-pathogenic and well-characterized. Before gaining access to the lab space, team members were required to:
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<h3>Specific Safety Concerns</h3>
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We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.
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<h5>Laboratory Safety</h5>
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Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.
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<h5>Chassis Organisms</h5>
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<img style="max-height:1000px;margin:10px" src="https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png"/><br />
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The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities.
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<h5>Novel Coding Regions</h5>
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None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.
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<img style="max-height:1000px;margin:10px" src="https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png"/>
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<h3>Safety Protocol</h3>
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<h5>Wet Lab</h5>
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In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.
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There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.
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<li>complete chemical waste disposal training,</li>
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<li>attend an orientation with Todd Pfeiffer, the Weill Hall Facilities Director, regarding the Cornell Environmental Health &amp; Safety guidelines, and </li>
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<li>receive specific training from <a href="http://www.bme.cornell.edu/people/adj-profile.cfm?netid=sda4"> Dr. Shivaun Archer</a>, the manager of the BME Instructional Lab, for the equipment in the lab.</li>
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Our standard lab practices were in compliance with the World Health Organization’s Biosafety Level 1 <a href="http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf" target="_blank">guidelines</a>. Many of our basic lab practices are described below.  
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All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS.  
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<li>Researchers were required to wear gloves while in the lab space, and to remove one glove when going into lab common areas. </li>
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<li>No gloves were allowed to leave the lab space, and no food or drink was allowed into the lab space. </li>
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<li>Within Weill Hall, microbiological samples were transported only via the internal service elevator, to avoid contamination of public areas.</li>
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<li>Any materials transported between the two on-campus lab spaces were held in secondary containment during transport. </li>
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<li>All flammable liquids were kept in a flammable storage  cabinet.</li>
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<li>The lab contained distinct waste containers for general waste, biohazard waste, biohazard sharps, and non-biohazard broken glassware.</li>
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<li>Liquid bacterial waste was treated with bleach before being discharged into sanitary sewage.</li>
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<li>Benchtops were decontaminated with ethanol before and after lab work was conducted. Tools that came into contact with bacteria were soaked in ethanol and flame-treated before and after use.</li>
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<li>An autoclave was used to decontaminate growth media, glassware, tubes, pipette tips, etc. All lab members were trained in proper autoclave use by the Weill Hall Facilities Director in order to avoid dangers to researcher safety.</li>
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<li>An emergency shower, eyewash, and first aid kit were available withing the lab space in case of emergency.</li>
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<li>Lab notebooks were maintained in a taped-off no-glove area.</li>
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Safety procedures specific to our project, in addition to best microbial practices, were followed during testing with arsenic and naphthalene. We modified EH&amp;S standard operating procedures for <a href="https://static.igem.org/mediawiki/2012/d/d7/Sodium_Arsenite_SOP.pdf" target="_blank">arsenites</a>, <a href="https://static.igem.org/mediawiki/2012/3/37/Sodium_hydrogen_arsenate_heptahydrate_SOP.pdf" target="_blank">arsenates</a>, and <a href="https://static.igem.org/mediawiki/2012/c/ca/Naphthalene_SOP.pdf" target="_blank">naphthalene</a> for our specific experimental needs. Most importantly, all work with arsenic and naphthalene was conducted by team members wearing appropriate PPE in a taped-off, designated area, and with equipment labeled as arsenic- or naphthalene-contaminated. Waste was collected separately, labeled appropriately as a cancer-hazard and disposed of through the Cornell EH&amp;S.
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Similar precautions were taken in working with Ethidium Bromide - all EtBr-staining was done in designated boxes, and in taped off areas used only for running and staining gels. Gels were visualized on a UV light box protected from EtBr contamination using saran wrap, which could be easily disposed of in the biohazard bin rather than risking ineffective decontamination of the UV light box. Team members were protected from exposure to UV rays with a UV shield. Facial shields were also available for use when needed.
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We maintain two copies of MSDS for every chemical stock that we maintain in our lab: one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.
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<h5>Dry Lab</h5>
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We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely.  Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe.
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Material Safety Data Sheets for these three hazardous compounds, as well as all other hazardous compounds used in the lab, were readily available in a binder in the lab space, as well as in the team’s shared online file folder.
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All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate.  Closed toe shoes and long pants were also worn when working in the machine shop.  While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times.  When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects.
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<h3>Training and Enforcement</h3>
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<h5>Required Safety Training</h5>
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All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.
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<h5>Safety Officers</h5>
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The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/" target="_blank">Institutional Biosafety Committee</a>, to ensure proper equipment usage.
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<h5>Is there a local biosafety group, committee, or review board at your institution?</h5>
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These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx" target="_blank">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.
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Yes, <a href="http://sp.ehs.cornell.edu/Pages/Home.aspx" target="_blank">Cornell University Environmental Health &amp; Safety</a> oversees safe procedures in on-campus laboratory research. The Cornell EH&amp;S mandates training required of researchers before they can gain access to lab spaces. Through the EH&amp;S, all team members took a required course on Chemical Waste Disposal.
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<h5>If yes, what does your local biosafety group think about your project?</h5>
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Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.
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We have been in contact with the EH&amp;S at our university about best practices for testing with arsenic. We have consulted them about disposal of carcinogenic wastes, as well as about how to keep researchers safe by using secondary containment and designated equipment. The EH&amp;S has expressed satisfaction that our standard operating protocols and waste disposal plan will ensure researcher safety, as well as public safety.  
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<h3>References</h3>
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<h3>Safety Concerns for Our Project</h3>
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We are working with biosafety level 1 organisms - <i>Escherichia coli</i> DH5α and WM3064 (a conjugation-enabled DAP auxotroph), <i>Shewanella oneidensis</i> MR-1 and JG700 (an mtrB knockout strain), and <i>Pseudomonas putida</i>. None of these strains pose a threat to researcher or public health before modification. The pathways that we are modifying in <i>S. oneidensis</i> JG700 are natural pathways already found in <i>Shewanella</i> and <i>Pseudomonas</i>, so they do not raise any safety concerns. As our project aims to detect the unwanted presence of toxic compounds in the environment, our device could not easily be used to negatively impact the public or the environment, and thus does not pose any foreseeable biosecurity risk.
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<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: <a href="http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml" target="_blank">http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</a>
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<h5>Would any of your project ideas raise safety issues in terms of:</h5>
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<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013)  Retrieved from: <a href="http://www.atcc.org/~/ps/32471.ashx" target="_blank">http://www.atcc.org/~/ps/32471.ashx</a>
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<h6>researcher safety,</h6>
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As we were working at BSL 1, the primary concern in terms of researcher safety was testing with our toxins of interest. Please see safe lab practices and standard operating procedures for arsenic and naphthalene, above.
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<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: <a href="http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm" target="_blank">http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</a></li><br>
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<h6>public safety,</h6>
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Again, our project itself could not be easily used to negatively impact the public. However, even when a research project does not pose a security threat, the conducting of research too must be done in a way that protects public interests and safety. All bacteria and waste were disposed of properly, in biohazard bags and through the Cornell EH&amp;S, rather than in common garbage cans, or decontaminated with bleach before being poured down the drain.
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<br>Additionally, in order to make our biosensor field-deployable, i.e. suitable for placement in water that will be drinking water, our device includes a thorough effluent filtration system to prevent the escape of our modified strains into the environment. We’re using a safe species and safe modifications that would not pose any foreseeable threat to native species or biodiversity. Finally, though our current selective pressure on our engineered strains is antibiotic resistance, the final device would use strains that were auxotrophs for some essential nutrient or that have our genetic modifications integrated into the bacterial chromosome; this would avoid the possibility of conferring antibiotic resistance to harmful bacterial strains in water. In the case of auxotrophic selection, our strain would be mutated such that it is an auxotroph for two essential nutrients: one of these nutrients would be provided in the media, so that the strain could only survive inside our bioreactors. Selective pressure for the plasmid would then be maintained through the second essential nutrient, which the plasmid would enable the bacteria to synthesize.
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<h6>or environmental safety?</h6>
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We do not foresee any adverse effects of deploying our device in natural streams and lakes. As detailed above, we have thought carefully about how to prevent our modified strains from getting into the environment. However, because we are knocking out mtrB - an essential component of <i>S. oneidensis</i>’s respiration pathway - and putting its production under the control of an inducible promotor, it is likely that our modified strains would be less able to compete than the wild type organisms already in the lake. If our parts were to lose their function, through mutation or other means, the most likely outcome would be that our strains would lose their capacity for facultative anaerobic respiration entirely, again leaving them less able to compete with wild-type <i>Shewanella</i>. There is the concern that antibiotic resistance could be horizontally transferred to other organisms in the wild, if our strain were to be released into the wild. This could again be addressed by selecting for strains auxotrophically.
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<h5>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</h5>
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The only safety issue associated with our new BioBrick parts is researcher safety in testing the parts. We have documented our standard operating procedures above, which other teams can reference if they want to use our parts, or test other parts using arsenic or naphthalene.
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We are also conducting extensive tests to assess the functionality of our BioBrick parts, to ensure that they behave as expected under different conditions.
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<h3>Safety Concerns for iGEM Competition</h3>
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<h5>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</h5>
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While our team is fortunate enough to have a biosafety group readily available for on-campus consultation, we recognize that many other teams may not have comparable biosafety resources available to them. In addition to the documentation by teams of their own safe practices, which can be used by other teams as references, more explicit safety guidelines, a “safety checklist” for instance, could be provided by the iGEM competition itself. One possibility is a checklist that rates a given lab from “unsafe” to “very safe” based on the number of safety provisions satisfied by the user filling out the checklist - rather like the Internet sites which assess the strength of inputted passwords.
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Many of the safety concerns we expressed above are the same concerns that many other iGEM teams also face. Mainly, the prominent use of antibiotic resistance in the construction and retention, via selective pressure, of novel genetic circuits poses a continued risk to public health due to the possibility of conferred antibiotic resistance. While the genes of antibiotic resistance can be carefully controlled in the lab setting, for any device to be used in a real-world setting, this possibility of horizontally transferring antibiotic resistance must be addressed. With this in mind, future iGEM teams should make an effort, where possible, to transition to other forms of selective pressures, such as limited growth media.
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Latest revision as of 00:57, 29 October 2013

Cornell University Genetically Engineered Machines

Safety

View our safety form.

Specific Safety Concerns

We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of biological safety mechanisms to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.
Laboratory Safety
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.
Chassis Organisms

The wet lab subteam regularly works with a non-pathogenic strain of E. coli; as part of our project this year, we are also working with two fungal species, Ganoderma lucidum and Cochliobolus heterostrophus, as well as Agrobacterium tumefaciens and Aspergillus niger, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities.
Novel Coding Regions
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.

Safety Protocol

Wet Lab
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.

There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.

All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS.

We maintain two copies of MSDS for every chemical stock that we maintain in our lab: one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.
Dry Lab
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe.

All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects.

Training and Enforcement

Required Safety Training
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.
Safety Officers
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the Institutional Biosafety Committee, to ensure proper equipment usage.

These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the Environmental Health & Safety guidelines. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.

Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.

References

  1. Lawrence Berkeley National Lab (2011) Appendix B Pathogen and Toxin Lists Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml

  2. Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx

  3. University of Pittsburgh (2013) Risk Group and Biosafety Chart Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm