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From 2012.igem.org

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Home / Team / Official Team Profile / Project / Parts Submitted to the Registry / Notebook / Safety / Attributions / Talk / Human Practices

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Researcher Safety

All researchers on our iGEM team are undergraduate students guided by the professors at WLC. Our primary faculty advisor, Dr. Balza, oversees our entire project which includes safety precautions. Furthermore, all of our student researchers have laboratory experience from past biology classes where we learned biological safety procedures. Our basic researcher knowledge includes appropriate clothing, protective gloves and eyewear, appropriate waste disposal, and care of flammable substances, among others.

The Biochemistry laboratory available to the WLC iGEM team is Biosafety Level 1. The NFPA (National Fire Protection Association) warning diamond further identifies possible dangers present in our lab. They are a health hazard of 3, flammability rating of 3, and a reactivity rating of 2. Furthermore, we also have an oxidizer warning.

Hazardous Reagents, Tools, and Equipment

GelGreen (Biotium)

GelGreen is a nucleic acid gel stain engineered as an alternative to the toxic ethidium bromide. It is still a suspected mutagen in the levels used for our experiments. By wearing gloves when forming and moving agarose gel, the danger is nearly nonexistent.

DAPI

DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is a nucleic acid stain used for visualization of the nucleus under a fluorescent microscope. The MSDS states that the potential hazards are skin irritation, serious eye irritation, respiratory irritation, or an allergic skin reaction.

Antibiotics

The antibiotics used (ampicillin, kanamycin, puromycin, neomycin) are not commonly used in clinical settings and all the bacterial waste is put in a biohazard bin for autoclaving. This prevents environmental exposure.

Ampicillin

Ampicillin is an antibiotic we used in the selection process. The MSDS states that ampicillin may cause an allergic skin reaction, and allergy symptoms, asthma symptoms, or breathing difficulties if inhaled. However, the danger of inhalation is greatly reduced once the stock solution is diluted from the ampicillin salt.

Kanamycin

Kanamycin is an antibiotic that is used in our project to select for e. coli that have received the cardiac promoter-reporter gene construct. The MSDS states that kanamycin is a teratogen and may damage fertility. Kanamycin is used in solution, made from the kanamycin salt.

Puromycin

Puromycin is an antibiotic used in our project to select for mouse stem cells that have received the complete ANF-pEGFP-pcDNA3.1+puro plasmid. The MSDS states that puromycin may cause respiratory tract irritation if inhaled, skin irritation if absorbed through skin, eye irritation, and as most laboratory chemicals, should not be eaten. Puromycin is used in solution, made from the puromycin salt. This greatly reduces the likelihood that puromycin will be inhaled.

Neomycin

Neomycin is an antibiotic used in our project to select for mouse stem cells that have received the complete MLC2v-pECFP-pcDNA3.1+neo plasmid. The MSDS states that neomycin may cause an allergic skin reaction, and allergy symptoms, asthma symptoms, or breathing difficulties if inhaled. However, the danger of inhalation is greatly reduced once the stock solution is made from the neomycin salt.

UV Light

Ultraviolet light is used in our laboratory a) in a biosafety cabinet for aseptic procedures, and b) for visualizing DNA in agarose gels after electrophoresis. Ultraviolet light exposure is hazardous to skin and eyes. Even a few seconds of exposure can cause skin cancer. The cornea can also be injured by UV exposure, causing inflammation and eventually cataracts. In our lab, a protective shield is in place when agarose gels are viewed under UV light.

Bacteria-Contaminated Items

The bacteria used for research purposes in our experiment are TOP10 cells, which are non-pathogenic and would not survive outside of laboratory conditions. All disposable objects that have been in contact with bacteria are to be placed in a special red biohazard bin, which is autoclaved before disposal. Small objects such as glass rods that are reused are dipped in alcohol and flame sterilized in the lab. Large objects are autoclaved by stockroom staff and returned.

Biosafety of Organisms and Biobricks

TOP 10 Cells (Invitrogen)

TOP10 cells are a strain of e. coli specially designed for laboratory use. The TOP10 cells are chemically competent, have a high transformation efficiency, and are non-pathogenic. In an uncontrolled environment outside of the laboratory, this strain of e. coli would soon die.

Murine Stem Cells (WLC cultures)

The WLC iGEM team uses murine, induced pluripotent stem cells. No embryos are destroyed in the process, and murine cells are less objectable for scientific research than human cells. These stem cells require growth media to feed, and would not survive outside of the laboratory. Furthermore, these stem cells present no danger to humans or the environment.

pcDNA3.1+neo (Part: BBa_J176122)

The pcDNA3.1+neo construct was sent to us by Dr. Haynes as a biobrick. There is no promoter in the plasmid, but we plan to add a constitutive promoter to regulate the neomycin production as well as the pECFP-MLC2v sequence.

pcDNA3.1+puro (Part: BBa_J176128)

The pcDNA3.1+puro construct was sent to us by Dr. Haynes as a biobrick. There is no promoter in the plasmid, but we plan to add a constitutive promoter to regulate the puromycin production as well as the pEGFP-MLC2v sequence.

pEGFP-ANF

The pEGFP-ANF construct was generously given to us by Dr. Paul Krieg at Arizona State University. The ANF (Atrial Natiuretic Factor) promoter sequence was isolated from Xenopus, a genus of frog. This promoter was inserted into pEGFP-1 (clontech). The ANF promoter inserted near the EGFP gene uses cis-regulatory action to promote EGFP. EGFP (Enhanced Green Fluorescent Protein) is a red-shifted variant that provides greater fluorescence than the natural GFP gene.

pECFP-MLC2v

The pECFP-MLC2v construct was generously sent to us by Dr. Puceat at Inserm. The MLC2v (Myosin Light Chain, Ventricular) promoter sequence was isolated from Mus musculus. This promoter was inserted into the promoterless pECFP vector (clontech).

Safety to the Public and Environment

Our stem cells are designed for use in research labs by knowledgeable personnel; they would not be available for use by the general public. The murine stem cells we are using require special cell media to live. These engineered stem cells would not be competitive or even viable outside of a laboratory. Furthermore, the fluorescent genes are not harmful to animals or plants. Antibiotic resistant genes will do no good to stem cells that do not have food to survive. It could be said with confidence that our engineered murine stem cells pose no threat.

For any major hazard, our organism would need to be purposefully engineered to cause harm, greatly changing the intended structure and function. The murine stem cells we are engineering will be non-pathogenic, will produce no toxic product to humans, and will not interact with the human body or organisms found in nature.

The worst hazard that could be imagined would be the constitutive promoter and antibiotic resistance taken up by a pathogenic bacterium. Fortunately, however, it would only be resistant to that one antibiotic. Another antibiotic, as well as intentional physical or chemical stress (such as heat, acids, alcohol, uv light), could easily destroy the resistant bacteria.

As of now, antibiotic resistance is a simple means to select for certain cells in the laboratory. A more dangerous alternative would be transmission of the desired sequence through a virus. The widespread use of antibiotic resistance in genetic engineering makes it both safer and more dangerous. On one hand, there are more knowledge and resources for use. Dangers are much more manageable than other techniques for DNA transmission. However, more people have access to possibly dangerous genes, increasing the chance for misuse. There is also a higher probability for a rare occurrence such as a mutation. Finally, less qualified or knowledgeable people such as students are directly involved. Antibiotic resistance genes do pose a danger to the general public. MRSA, for example, is already prevalent in hospitals and public areas. It threatens the health and life of those who are infected by it.

Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?

Our bio bricks pose no serious threats. All parts were made using non-pathogenic strains of bacteria and the parts themselves focus more on mammalian cells than bacterial cells. Basic BSL1 protocol was used when conducting our experiments.

Is there a local biosafety group, committee, or review board at your institution?

Due to the non-pathogenic nature of the research at WLC, our school does not have a biological safety committee. All of our projects fall within BSL 1 containment arrangement protocols.

How do you think synthetic biology, and especially the iGEM competition, is perceived by the public? What could be done to influence that?

Public perception of the iGEM competition is impacted by many factors, first and foremost is the content of each year’s entries in the competition. Some years, there will be fewer projects that have negative connotations to the public (if they are knowledgeable enough to understand what the projects consist of), and other years there will be more controversial projects that may impact the image of the entire program. However, it is likely that the projects within the iGEM program may encounter some public resistance regardless of the content. This is because, for the most part, changing the genomics of anything is viewed negatively by modern society.

In a culture that is developing a “Green” revolution, altering that which makes an organism into what it naturally would become (changing genetics and altering bio-mechanisms), is frowned upon. This is due in part to an over publicity of information regarding organic foods and invasive species. Unfortunately, a lot of this publicity is government mandated. Anything that touches a GMO (Genetically Modified Organism), must be labeled as such. This impacts the societal perception of genetic engineering and bio-mechanical alteration. The fears associated with these two practices are mainly harm to humanity and harm to the environment. To combat these fears, the iGEM program can begin a regulatory measure insuring that testing is done on any products that are associated with the competition or its competitors to ensure that these products will not be harmful to either of the above.

Publicity and public outreach programs to educate about genetically modified organisms and the risks and benefits that they could potentially yield would create a sense of security and reduce irrational fear on the public’s behalf. As always, education is the key to creating public acceptance of any new idea.

Bio-safety Engineering

Bacteria can become pathogenic or harmful to the environment. Because of this it is important that with any engineered organism we must be able to quickly and safely eradicate it. Antibiotics are a great way of killing bacteria, but there is always the possibility that the bacteria will form antibiotic resistance. This makes antibiotics not the best choice. One solution is inserting a "suicide gene" into engineered strains of bacteria. A suicide gene will allow us to induce bacterial cell death exactly when and if we need it. The key to having this work is that the induction agent must be safe for the environment and for those working with it. That is why every researcher working on a project must first understand the primary environment in which the bacteria will be used. After careful analysis of that environment, a suitable inducer can be identified to safely bind to the modifiable promoter region of the suicide gene. The environment will be left intact, but the modified bacteria will be killed.