TEAM ALBERTA

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Team Alberta iGEM Safety Proposal

Team Alberta recognizes the important role that safety plays in the completion of any scientific undertaking. Our team members are fully committed to not only ensuring the safety of those within our lab but also addressing any potential risks that our project may pose to the greater community.

Please find below Team Alberta’s answers to the new, required iGEM safety questions.

1. Would the materials used in your project and/or your final product pose:

a. Risks to the safety and health of team members or others in the lab?

The materials used in our project pose little to no risks to the safety and health of our team members or others in the lab and our final product poses no risk to these individuals.

The organism that our group has genetically manipulated, Neurospora crassa, is well-documented as being safe¹. N. crassa is a biosafety level 1 organism. Since 1941 the species has been used extensively in laboratories resulting in the publication of thousands of research papers. In none of these numerous publications has the Neurospora genus been implicated in causing disease in either animals or plants^1-4.

Neurospora’s minimal threat can largely be attributed to it being an obligate aerobe. Unlike other species such as yeasts, Neurospora is unable to grow in organs such as the gut or bladder, tissues, or systemically within an organism¹. Furthermore, despite Neurospora being regarded by some as an allergen, medical literature generally fails to mention the genus^5-8.

Our team acknowledges that though certain fungal species themselves may not be pathogenic, they may produce dangerous toxins. Throughout our research into Neurospora our group has not found any documentation of the species producing dangerous mycotoxins or any dangerous secondary metabolites¹. Given that Neurospora has had such a long history of living in close association with humans (please see below), it is a generally regarded as being harmless.

It should be noted that there are still materials used in our project that may potentially be hazardous. Our esterification procedure involves the use of strong chemicals and reagents, some of which have the potential to cause harm. These reagents are primarily volatile and require careful handling and storage.

As well, Hygromycin b, an antibiotic and antifungal agent that is used for selection, is toxic if inhaled. Care must be taken around powdered or lyophilized Hygromycin B, and precaution should be taken to cover up respiratory passageways, skin, and the eyes. However, Hygromycin B is most commonly purchased in liquid form, and in liquid carries with it significantly less potential for accidental inhalation.

b. Risks to the safety and health of the general public if released by design or accident?

Our organism poses no threat to the safety and health of the general public. Neurospora has a well-documented history of living in close association with humans. It has historically resided in a variety of businesses including bakeries, lumber yards and plywood factories1;9- 11. Growth has also been observed on the stubble of burnt sugar cane fields and along burnt railway tracks13. Thus, it poses no threat to the health and safety of those in its surroundings.

c. Risks to environmental quality if released by design or accident?

As mentioned, N. crassa has a long history of living in close association with humans; however, these instances are largely documented as having occurred in moist tropical or subtropical environments. Like many fungi, warm and moist is the favored climate for Neurospora growth1. Thus, if our organism, WT or manipulated, were to be released in our dry, temperate environment of Edmonton, Alberta, Canada, with its bitterly harsh winters, we would expect its survival and environmental impact to be minimal. Even if it were to survive, we cannot see it affecting local ecosystems by any appreciable amount.

Nonetheless, our alternative biodiesel has the potential to revolutionize economies throughout the world, as discussed in our human practices component, and we must therefore regard the effects of environmental exposure to Neurospora in other climates. Were Neurospora to be released in other warmer climates it may readily

survive, creating its own niche within the environment. However, the aforementioned nonpathogenicity of the genus results in this not being a source of alarm1-4.

Further, Team Alberta predicts that our manipulated strain of Neurospora crassa would be disadvantaged to survive relative to WT in any environment; our genetic manipulation results in the organism’s energy being diverted away from essential metabolic functions, inhibiting organismal growth and manipulation. This puts it at a disadvantage against natural competing fungi.

d. Risks to security through malicious misuse by individuals, groups or states?

Unforeseen alteration of biopart functions may represent issues of concern. However, in our case if one or several of our bioparts were changed to alter their function or stop working as intended, limited to no safety issues would arise. Our knockout of the FadD gene results in there being limited potential for its alteration of function. Further, our manipulation of thioesterases similarly represents limited safety concerns due to their prevalence in all organisms. If further alterations were to be made to thioesterase genes and resulted in overproduction, our organism would likely die or have a limited existence; if further alterations to thioesterase genes were to prevent their function, then the organism may return to a more WT phenotype12.

The misuse of our bioconverter represents a potential threat to safety. The esterification process requires heat and flammable chemicals contained within a sealed vessel and thus the potential for serious injury exists.

2. If your response to any of the questions above is yes:

a. Explain how you addressed these issues in project design and while conducting laboratory work.

To deal with such issues our team greatly scrutinized all steps within our procedure for potential hazards and subsequently collectively addressed all areas of concern. Please see the outline below.

(esterification diagram goes here)

Our careful consideration of the potential safety concerns associated with the completion of our project and our careful documentation and safety training have allowed us to work both

responsibly and cautiously, limiting the probability of injuries.

All MSDS data sheets were followed in working with strong acids, bases, volatile chemicals, or otherwise potentially harmful reagents. All antibiotics and antifungals were stored cold and in sealed containers, and any reacting chemicals were kept in separate storage areas. All experiments done involving the use of open flame, electrical power sources, or flammable substances were treated with the utmost caution. All experiments with distillation used properly fitted apparatus and were designed to eliminate all possibility of heating closed containers. (For details on the safe handling of Hygromycin b, please see question 3).

Safety issues arising from the completion of our bioreactor were addressed in the stages of its design. Several important safeguards were incorporated. For example, our bioconverter is designed to automatically turn off upon exceeding the recommended temperature and pressure past specific margins.

b. Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.

In submitting parts to the registry, Team Alberta has actively undertaken the practice of describing and documenting any safety, security, health and/or environmental issues of our parts as they are submitted to the registry. None of our parts confer any pathogenicity or otherwise harmful effects upon either Neurospora crassa or Escherichia coli.

One of our parts will confer Hygromycin B resistance to Neurospora crassa, and such strains that contain this part should be noted as being resistant to that particular antifungal agent.

3. Under what biosafety provisions will / do you operate?

a. Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.

Yes, the University of Alberta contains a specified set of Biosafety Rules that are outlined within the Biosafety Manual that is published by the Office of Environmental Health and Safety. A link to this valuable resource can be found here: http://www.ehs.ualberta.ca/en/EHSDivisions/~/media/ 8C72A3F626FB47B6BD70EC79C94CD1CE.ashx

b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them? Describe any concerns or changes that were made based on this review.

Team Alberta consulted the University of Alberta’s Office of Environmental Health and Safety throughout the planning of our project. The Biosafety Division of this office provides both technical expertise and support to labs throughout campus and thus ensures a safe working and learning environment for both staff and students. In addition, the division also monitors compliance with federal and provincial legislation16.

Our team submitted a written overview of the relevant safety considerations associated with our project to this office. Upon perusal of this document, the Biosafety Officer provided us with recommendations to fully ensure that our project met all biosafety requirements. Please see the Biosafety Officer’s recommendations below and how we met each of them:

i) It was recommended that if our lab were to grow N. Crassa beyond a 10L culture volume that we would have to obtain permission from the federal government, as such a culture would be considered large scale production. The Biosafety Officer therefore recommends avoiding production of cultures beyond a 9.5L volume. In addition, it was recommended that our lab prepare a Biological Spill Remediation kit according to the Standard Operating Protocols (SOP) of the university.

To avoid having to proceed with a lengthy governmental registration process in the relatively short timeframe of the iGEM competition, our lab will ensure that no culture we prepare will be larger than 9.5L. To date, the largest culture that we have prepared has been less than 1.5L. As well, the members of our lab assembled the materials required for a Biological Spill Remediation kit, which will allow for the safe and efficient cleanup of any potential spills. ii) The Biosafety officer recommended that our lab ensure all cultures of N. crassa be rendered inert prior to disposal.

The members of our lab have already been adhering to this practice by treating all cultures of N. crassa with a household bleach mixture for a minimum of one hour prior to disposal. Our bleach mixture contains a minimum of one part bleach to three parts culture, which is consistent with the guidelines outlined by the Biosafety Division of the

Environmental Health and Safety Office.

iii) The Biosafety Officer recommended that all individuals working within our lab should wear personal protective equipment (PPE), comprised of fully-fastened laboratory coats or gowns, disposable gloves, safety glasses, closed-toe shoes and floor-length pants.

The members of our lab have been adhering to this common laboratory practice since the very beginning.

iv) Due to the use of the antibiotic and antifungal Hygromycin b, the Biosafety Officer recommended that all work with this antibiotic be carried out in a fume hood. He further recommended that members of our team coming in contact with this antibiotic be fit-tested for N95 respirators in the event that this antibiotic cannot be handled within the fume hood.

The members of our team who are not fit-tested for N95 respirators will not work with Hygromycin b. The two members of our team that will work with this antibiotic have been fit-tested with N95 respirators so that they may safely handle this compound, regardless of its form. All work with Hygromycin b will be carried out within a fumehood.

c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

The members of our lab have all had variable levels of laboratory experience upon beginning the summer. Though most members had some level of biosafety training from previous lab experiences, including WHMIS certification, some members did not have any previous safety training. Because of this uneven experience, all members of our team received a laboratory tour at the beginning of the project. This included an overview of general laboratory safety procedures and protocols. Additionally, all team members were briefed on new safety procedures as our project developed. For example, upon beginning work with our organism, N. crassa, all lab members were briefed on its safe disposal.

d. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.

Yes, the Public Health Agency of Canada outlines national biosafety guidelines. Specifically, the Laboratory Biosafety and Biosecurity Division of this agency is responsible for publishing

biosafety regulations. A quick synopsis of these guidelines may be found here:

http://www.phac-aspc.gc.ca/lab-bio/regul/index-eng.php.

4.Optional Question: Do you have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

In order to motivate future iGEM teams to further emphasize safety in their daily laboratory practices, we propose that each team completes a detailed safety report for a minimal medal requirement. Bearing in mind the increasing importance of safety in the rapidly expanding synthetic biology field, Team Alberta further proposes that future iGEM competitions require teams to engineer their parts and organisms with several safeguards in place in order to achieve a gold medal. If these components were medal requirements, we feel that it would greatly motivate iGEM participants to address potential safety issues, and not just obvious ones, far ahead of time. This attention towards possible problems, and the solutions around them, will pay immense rewards in their future work, iGEM or otherwise. Moreover, this would encourage teams to be more creative with respects to synthetic biology and genetic engineering.

Our team recognizes that biosafety engineering has the potential to make many devices and systems safer. For example, essential gene knockouts can be created through partial or full amino acid deletions instead of single nucleotide alterations that are susceptible to mutation. Auxotrophic (or nutrient-deficient) strains can be created to ensure that an organism has no chance to exist outside of the laboratory environment, ensuring a minimal chance of contaminating the outside world. Organisms can even be designed to positively or negatively respond to varying wavelengths of light and electrical stimuli. All this and more should be taken into account when designing any organic circuit. References

References

1. Perkins DD and Davis RH: Evidence for Safety of Neurospora Species for Academic and Commercial Use. Applied and Environmental Microbiology 2006, 66 (12): 5107-5109.

2. Davis RH: Neurospora: contributions of a model organism. Oxford University Press US; 2000.

3. Perkins DD: Neurospora: the organism behind the molecular revolution. Genetics 1992, 130: 687-701.

4. Perkins DD: Neurospora genetics at the turn of the century. Fungal Genetics Newsletter 2000, 47: 83-88.

5. Frank MM, Austen KF, Claman HN, Unanue ER: Samter’s immunological diseases, 5th ed. Little, Brown & Co US; 1995.

6. Kay AB: Allergy and allergic diseases. Blackwell US; 1997.

7. Middleton E, Reed CE, Ellis EF, Adkinson, NF, Yunginger, Busse, WW: Allergy: principles and practice, 5th ed. Mosby US; 1998.

8. Patterson R, Grammer LC, Greenberger PA: Allergic diseases: diagnosis and management, 5th ed. Lippncott-Raven US; 1997.

9. Shear CL, Dodge BO: Life histories and heterothallism of the red bread-mold fungi of the Monilia sitophila group. Journal Agricultural Research 1927, 34: 1019-1042.

10. Yassin S, Wheals A: Neurospora species in bakeries. Journal Applied Bacteriology 1992, 72: 337-380.

11. Shaw DE: Honeybees collecting Neurospora spores from steam Pinus logs in Queensland. Mycologist 1993, 7: 182-185.

12. Nelson DL, Cox MM: Lehninger Principles of Biochemistry. W.H Freeman US; 2009.

13. iGEM website: https://2011.igem.org/Safety

14. Hygromycin material and safety data sheet:
http://www.invivogen.com/MSDS/HygromycinB_solution_v4_MSDS.pdf

15. Kafer E, Luk D: Sensitivity of bleomycin and hydrogen peroxide of DNA repair-defective mutants in Neurospora crassa. Mutant Research 1989, 217(1): 75-81.

16. University of Alberta Environmental Health and Safety website: http://www.ehs.ualberta.ca/