With our project, there are several levels of safety precautions and concerns. We have been mindful of these concerns as we developed and executed our experiments.
General Laboratory Safety
The RAIN laboratory spaces are equipped with standard laboratory safety equipment, including a fume hood, two biosafety cabinets, fire extinguishers, spill kits, sharps containers, flammable cabinets, broken glass boxes, first aid kits, flammables storage cabinets, eyewash stations, etc. Personal protective equipment, including lab coats, eye protection, and gloves, is available for everyone working in the lab.
All members of the iGEM team received a comprehensive lab safety orientation to include accident prevention, fires, building evacuation, handling and disposal of biohazards, chemical storage, etc. A checklist of topics was used to ensure that each team member received the same information, and at the end of the orientation, each member signed their sheet to acknowledge that the information had been covered to their satisfaction. Safety orientation was done by Co-PI Jennifer McKee-Johnson, PhD, who is the Laboratory Manager and Safety Officer for a biotechnology company in Seattle.
Widely accepted laboratory safety rules and guidelines, such as no food, gum, or drink in the lab and wearing laboratory-appropriate clothing (closed-toe shoes, no loose or drapey clothing, etc.) are enforced.
Additionally, our laboratory seeks to follow all regulatory laws and guidelines set out by the Occupational Safety and Health Administration (OSHA) and by the Washington (state) Administrative Code (WAC).
Because our project centers around the use of molecular techniques to create a genetically-modified organism (GMO), there are a variety of safety concerns. Here we address both known physical hazards for these types of experiments and the potential concerns.
Molecular biology experiments
Our project relied on the use of gel electrophoresis and polymerase chain reaction (PCR) in addition to other basic molecular biology techniques. The most significant hazards included razor blades for excising gel fragments, hot surfaces (PCR machine), and shock from powered gel boxes.
The otsA-expression vector was assembled and propagated in E. coli DH5α cells. DH5α cells are considered to be non-pathogenic and to pose minimal risk of infection in humans (Chart et al., 2000). Because E. coli can act as an opportunistic pathogen and cause a variety of symptoms, care was used when handling bacteria to prevent auto-inoculation (use of PPE) and contamination of workspaces (good “housekeeping”). All liquid E. coli cultures were treated with 10% bleach prior to disposal; all solid cultures (i.e., agar plates) were autoclaved prior to disposal.
The rhizobia strain, R. leguminosarum, is non-pathogenic to humans. We purchased it from Carolina Biological supply as this strain is approved for use by high school science classes in the United States. However, we continued to use best practices with respect to handling microorganisms when working with this strain.
An additional concern would be inadvertent “escape” of this strain from the lab after transfer of the expression vector. Over-expression of otsA and resulting increased production of trehalose could confer a selective advantage to the modified strain over wild-type strains of R. leguminosarum. To prevent this, we have worked to minimize exit of this bacterium from our facility. As the modified R. leguminosarum will be used to inoculate bean seeds for growth in the laboratory, all soil, irrigation flow through, and dead plant matter will be autoclaved prior to disposal. Plants will be grown in “greenhouses” consisting of large plastic storage containers modified to accommodate the plants. This growth paradigm should minimize any accidental spread via dirt spillage or air dispersion.
Chart, H; Smith, HR; La Ragioni, RM; and Woodward, MJ. (2000) An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5a and EQ1. J Appl Micro 89: 1048 – 1058.