Safety Information

Safety at the University of Minnesota

Biosafety rules for the University of Minnesota are described in the Biosafety Manual, which is produced by the Department of Environmental Health and Safety (DEHS). The Biosafety Manual is available online here: http://www.dehs.umn.edu/bio_pracprin.htm

The University of Minnesota Institutional Biosafety Committee (IBC) oversees any research involving the use or manipulation of recombinant DNA. Information about the University of Minnesota IBC can be found online here: http://www.research.umn.edu/ibc/#.UElFPo60zQc

The United States of America also has safety regulations and guidelines maintained by the Center for Disease Control (CDC). Information about Biosafety in Microbiological and Biomedical Laboratories (BMBL) are found in a publication made available by the CDC. The BMBL 5th Edition can be found online here: http://www.cdc.gov/biosafety/publications/bmbl5/index.htm

In addition to previous laboratory safety training obtained in classes, all iGEM participants are required to undergo safety training modules appropriate for his or her research as well as the research of others in shared lab space. Training modules for completion can be found online here: http://www.dehs.umn.edu/training_newlabsafety.htm

Additionally, safety information and orientation within the lab space are given by instructors on the first day in laboratory. Lab-specific SOPs were described and provided by instructors in case of biological contamination and spill. Waste disposal techniques for different types of waste were described and a template was provided for student reference.

Researcher Safety

E. coli strains used in our project are non-pathogenic strains that pose minimal to no health risk to researchers handling them. Saccharomyces cerevisiae and closely related strains have not been associated with pathogenicity towards humans. Neither organism poses risk towards team members or others who enter our laboratory and are considered Biosafety Level 1 (BSL-1) materials. As such, BSL-1 standard practices, safety equipment, and laboratory access standards are always employed by our team and others working in our labs.

Genes and gene constructs in our project include:

• yeast-e coli shuttle vector (constructed through Gibson of pSB1C3 with minor point mutations, yeast 2u origin, and industrial G418 resistance gene driven by pADH1)
• pGPD driven yeast-optimized DXMT1 with tADH1 (obtained as gBlock fragments)
• pCyc driven yeast-optimized XMT1 with tCycE1 (obtained as gBlock fragments)
• pUCBB
• DHQS (from Anabaena variabilis)
• NRPS (from A. variabilis)
• ATP-Grasp (from A. variabilis)
• O-Methyltransferase (from A. variabilis)
• ScyA (from Nostoc punctiform)
• ScyB (from N. punctiform)
• ScyC (from N. punctiform)

None of these genes have the capacity to confer pathogenic or toxic character onto constructs in either host organism or any other organisms in the lab. Enzyme activities of XMT1 and DXMT1 produce caffeine, which is toxic in extremely high doses (150-200 mg/kg body mass), but is neither present nor available at such doses in the laboratory. All DNA constructs (circularized or linear) are handled with care to ensure minimal contact with surfaces and organisms other than those for which they are intended.

In addition to iGEM Team Minnesota specific safety concerns, common substances used in the laboratory were stored and used as indicated by standards given by their manufacturers. Flasks and culture tubes have secondary containment during use and transfer to the autoclave. Wastes also have secondary containment during autoclaving. Aborted autoclave cycles are repeated.

Equipment that could also pose potential risks to researchers was identified and handled with caution. Standard handling and disposal protocol for broken glass and sharps was followed to ensure safe use of hazardous equipment. Centrifuging is done in closed tubes in a closed, lidded centrifuges.

Substances that pose risk to health and safety were identified and treated with special care. These substances include:

Ethidium bromide: ethidium bromide is a DNA intercalating agent with probable carcinogenic properties. Ethidium bromide is stored in a sealed container in a cool, dry, well ventilated location. Latex or nitrile gloves and other appropriate personal protective equipment are worn when handling ethidium bromide. Ethidium bromide is diluted to less than 0.1% weight by volume and disposed of in the laboratory drain if the material is liquid or in the regular refuse container if the material is contained within a gel. In addition to manufacturer guidelines, labs clearly marked “Ethidium Bromide Zones” as the only place where the use of ethidium bromide and gloves that have come in contact with ethidium bromide is allowed.

Public Safety

Our biological systems do not contain parts which pose a significant threat to the health of the public should they be released accidentally. As previously stated, our team is working with non-pathogenic laboratory strains of E. coli and S. cerevisiae is not known to be pathogenic to humans. Recombinant proteins introduced into bacteria by our team produce products which are not known to be harmful or are only harmful in extremely large doses (as with caffeine).

Plasmids used in our research contain antibiotic selective markers which have been designated for use in research. As such, use of these antibiotics does not pose a epidemiological threat.

Environmental Safety

E. coli and the recombinant strains produced are not likely to have adverse effects on the environment. Similarly S. cerevisiae and closely related strains have not been associated with adverse effects on the environment.