Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?

      Biosafety regulations are national, international and local measures installed to minimize any risks involved in biological research. When determining the dangers that one’s research poses to their fellow researchers, the public at large, and the surrounding environment, we must first refer to the World Health Organization (WHO) biosafety manual to determine the risk level of the microorganisms involved. According to the WHO manual, a risk level one microorganism is one “that is unlikely to cause human or animal disease.” The University of Alberta team is using two non-virulent, relatively harmless strains of Escherichia coli: TG1 and TOP10. Consequently, these microorganisms are classified under risk level one.

      If our project goes completely according to plan this year, there will be no major safety concerns. Our microorganisms, biological parts, laboratory equipment and chemicals are all relatively harmless. The biological parts we are using simply consist of color, antibiotic-resistance (Chloramphenicol, Kanamycin, Ampicillin) and repressor genes. In terms of chemicals, other than the myriad of flammable solvents in the lab, the only other regularly used chemical that poses a possible health risk is ethidium bromide (EtBr). When exposed to extremely high concentrations of EtBr in experiments, human cell lines have received damage to their DNA. The concentration of EtBr used in our lab (10 mg/mL) is much higher than these levels which have shown toxic effects. To mitigate such risks, good microbiological techniques are consistently used in the lab, so gloves are always worn. The most dangerous laboratory equipments we use are a transilluminator, gel apparatus, and a Bunsen burner. The risks posed by exposure to UV radiation, electric shocks and open flames can be easily controlled with proper laboratory safety training. At the University of Alberta, the safety of our researchers is always our top priority. When any student first steps in the lab, they must go through the necessary laboratory safety training. Students are taught how to properly use equipment, dispose of wastes, autoclave used glassware and biologically-contaminated waste, disinfect counters and when to wear safety protection. Counters are always clean and uncluttered, and proper pipetting protocol is always used.

      Proper laboratory safety training is practiced by all undergraduate and high school students to ensure cross-contamination does not occur, thus keeping results consistent from trial to trial. Proper technique is also practiced to prevent microorganisms from adhering to our clothes, and infecting others, including family members.

      Our project not going according to plan would pose as little risk as us succeeding. The standard and well characterized biobytes we are using do not have the potential to combine in dangerous ways. As with many other synthesized DNA constructs, we have found that our plasmids are lost from the bacteria over time (if plated without antibiotic present), due to the expression of colors being an unproductive strain on the cell’s resources. Consequently, there is little risk of our microorganisms spreading. Even if our E. coli strains survived and replicated in the wild, they are highly unlikely to cause harm to any humans or animals as risk level one microorganisms. The microorganisms we are creating in the lab would have less potential for malicious misuse than any bacteria normally found in the environment.

Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?

      The new BioBricks created by the University of Alberta this year do not raise any safety concerns. The BioBricks are novel circuits made from common genes coding for color proteins and repressors. The three color proteins being produced are red fluorescent protein (RFP), blue pigment protein (BPP), and green fluorescent protein (GFP), and the two inducible repressors are LacI, and TetR. All of the BioBricks parts produced this year are safe, non-virulent, and are consequently categorized under a Bio Safety Level One (BSL1). According to the WHO manual, BSL1 microrganisms require no safety equipment and allow for open bench work.

Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?

      On most university campuses, there is a local Biosafety Committee that ensures that research labs are following safety protocols put in place by the national government. At the University of Alberta, the Biosafety Committee is a division of the Environmental, Health and Safety Faculty. The Biosafety Committee is mainly concerned with the proper handling of biohazardous materials. Biohazardous materials are defined as “materials of biological origin or synthetic material which mimic biological entities and may induce adverse conditions to humans, other animals, plants, or the environment”. Under the national and local regulations, our non-virulent strains of E. coli are considered Biosafety level 1 and are of minimal potential hazard. Standard practices, such as wearing gloves when handling microorganisms, as well as using autoclave and bleach to decontaminate materials are stringently followed. Therefore, our iGEM project is of very little concern to the biosafety committee. The Biosafety committee only has to be contacted when organisms of risk level two or higher are used in research.

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?

      The use of antibiotics to select for microorganisms of interest has become prevalent in research and industry. In large scale ethanol production, Saccharomyces cerevisiae is used to break down sugars in a corn-based emulsion and produce ethanol as a major product. Unfortunately, contamination of the emulsion with other bacteria like Lactobacillus spp. is extremely common, so antibiotics are used to control bacterial growth, eliminating competition for yeast growth and maximizing ethanol production. Using antibiotics on such a large scale works wonderfully for industry; it is cheap and effective. However, the wide spread use of antibiotics has caused rampant antibiotic resistance in wild-type strains of bacteria. The leftover antibiotic laden emulsion from ethanol production is often sold to farmers as cattle feed, further spreading antibiotic resistance.

      Antibiotics are also commonly used in synthetic biology to select for bacteria with the plasmids of interest. While this practice works extremely well in the present, it cannot be maintained in the future, as more antibiotic resistance is spread to wild-type strains from iGEM teams and other sources. Hence, alternative selection measures may have to be developed for future iGEM teams to not only prevent the spread of antibiotic resistance, but also to set an example and lead the way for industry. One possibility is to implement a common selection practice used in yeast genetics and integrate it into E. coli. An auxotrophic strain of E. coli with a mutation in Tryptophan (TRP1) could be transformed with a plasmid vector containing a TRP1 gene. The transformation mix would then be plated on an agar plate with growth media lacking Tryptophan. Only auxotrophic E. coli colonies containing the plasmid vector of interest would be present on the plate. By decreasing the dependence of antibiotics in the lab, iGEM can help stop the spread of antibiotic resistance, which is a rising health and safety concern across the world.

      We also note that one of our advances provides a potential new mechanism for preventing the leakage of genetically modified material into the environment, i.e. a safety switch. By placing an essential gene in the origin of replication under inducible control, plasmid replication becomes dependent on the presence of the inducer. In a situation such as a hypothetical accidental environmental release, the plasmid will be rapidly lost, preventing the replication of genetically modified DNA in the environment. This is a safety switch, a genetic mechanism which backstops the usual lab hygiene practices which prevent unintended environmental contamination. Widespread use of conditional replication mechanisms such as this one could help ameliorate public concerns about the presence of genetically modified organisms in the environment.