Team:Alberta/Humanpractices

This year, for the first time, we carried out a bit of a social experiment: we invited a substantial number of local high school students to join the team. In the end, the team is composed of five high school students, in grades ranging from 10 to 12, and four undergraduates, one from each academic year and none with previous iGEM experience. The idea is two-fold: firstly, to extend the reach of the iGEM experience to a different audience of students, effectively expanding the outreach of synthetic biology; and secondly, to see if the different background and perspectives of younger students would have an impact on the nature of the science done.

Although we expected that the HS students would have formal coursework background, there were several unanticipated obstacles. One was school schedules. High school classes run until nearly the end of June, limiting the participation of the HS students during the initial phase of the project. Another was logistics and transportation. Generally the HS students neither lived on campus nor drove a vehicle, complicating their availability especially for after-hours lab sessions. However there were also unanticipated benefits. The younger students brought a level of enthusiasm which readily overcame any differences in background. Moreover, the undergraduates and HS students bonded during lab hours and discussions of topics ranging from religion (adherents of most of the world’s major groups were present) to card games and beyond.

Overall, the effort was demanding but positive, and something we will recommend U of A iGEM teams consider in future years.

Members of the iGEM team presented to WISEST Teacher Appreciation Day, Aug 14, 2012, discussing the iGEM program and our project. WISEST is a U of A training program aimed at encouraging the attraction, retention and advancement for women of all ages in science, engineering and technology.

We also presented at the Alberta Genetically Engineered Machines competition on Sep 15-16, 2012. aGEM, currently in its fourth year, is a western Canada gathering of iGEM teams, where they present and receive feedback from a star-studded panel of judges. Although patterned after iGEM, aGEM attracts its own audience, including more than 100 largely local luminaries who came to hear about advancements in synthetic biology at the hands of Canadian students. Although this year Calgary won aGEM (boo! hiss! the University of Calgary is a traditional rival to the University of Alberta), we received encouragement and a slate of useful suggestions for the remainder of our project.

High school students who took part in our team will be an important component of our outreach. Their experience, paired with the fact that they are still active in the local high school community will allow them to be powerful proponents of high school iGEM, and will hopefully allow them to have a part in setting up future high school iGEM teams in Edmonton.

We would first like to thank Uppsala 2011 iGEM team, for introducing amilGFP to the iGEM universe. Although we did not need to contact them directly, the data and sequences they contributed to the parts registry formed the core of the colors we used this summer.

We have supplied strains and plasmids to a number of other iGEM teams. Both the University of British Columbia and University of Calgary iGEM teams relied on E. coli knockout strains we had access to and provided to them. Additionally, the CINVESTAV-IPN-UNAM_MX iGEM team used a number of BioBricks first made by the 2007 and 2008 University of Alberta iGEM teams, which we provided to them along with technical suggestions. (Parenthetically, we note that the border issues faced by some teams pales in comparison to those faced by Canadians.)

      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 ([http://ntp.niehs.nih.gov/?objectid=6F5F63F6-F1F6-975E-79965F7EE68AE7C0 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.

      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.

      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.

      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.

Project main members:

• Dr. Douglas Ridgway & Professor Mike Ellison: Concept, instruction, oversight, and fundraising
• Ryan Mercer: Instruction, technical assistance
• Peter West: Gradient plates, wiki writer, presentation of materials, handbook writer
• Abdullah Farooq: PCR, plasmid manipulation, presentation of materials, handbook writer
• Torrin Lemire: Transformations, plating, gradient plate master, handbook writer/editor
• Easwar Chidambaravasi: PCR expert, plasmid manipulation, plasmid cloning
• Rick (Nien-Tsu) Tseng: PCR expert, plasmid manipulation and cloning, wiki writer
• Tom Patrick: Plasmid manipulation, plasmid loss expert, plasmid cloning
• Spencer Short: Plasmid manipulation, sponsor liaison
• Aidan Burke: Gradient plates, gel electrophoresis expert, handbook writer
• Sarah Haeubl: LB agar/broth technician, handbook writer

Additional thanks to:

• Michael Feist: web programming
• Justin Fedor: assistance in fluorescence measurements
• Dept Biochemistry: providing space
• Uppsala 2011: for introducing amilCP to the iGEM universe

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