Team:Nevada/Safety

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iGEM Safety Sheet University of Nevada, Reno (Team Nevada)

Safety Questions

1. Is there a local biosafety group, committee or review board at your institution?

o Yes, The Nevada iGEM team operates under the guidelines established by The University of Nevada, Reno’s Environmental Health & Safety Department (EH&S) and the National Institutes of Health (NIH) guidelines for Research Involving Recombinant DNA.

2. If yes, what does your local biosafety group think about your project?

o We have presented our project proposal to EH&S, and after a review of our materials and procedures, the safety office has approved our project and deemed our practices consistent with The University of Nevada and NIH safety regulations.

3. Would any of your project ideas raise safety issues in term of:

Researcher Safety

o All team members were required to attend two safety workshops. The first course is a basic safety class outlining the proper way to handle and dispose of chemicals and biological hazards. The second safety course is to teach us the ethical and safety issues associated with genetically engineering organisms.
o The Nevada team’s laboratory environment is certified for Biosafety Level 1 (BSL-1) work as listed in the Appendix G-II-A-1 of NIH Guidelines

o Access to the laboratory is limited or restricted at the discretion of the Principal Investigator when experiments are in progress.

o Work surfaces are decontaminated once a day and after any spill of viable material.

o Contaminated liquid or solid wastes are decontaminated before disposal.

o Mechanical pipetting devices are used; mouth pipetting is prohibited.

o Persons wash their hands:
(i) after they handle materials involving organisms containing recombinant DNA molecules and animals, and
(ii) before exiting the laboratory.

o All procedures are performed carefully to minimize the creation of aerosols.

o In the interest of good personal hygiene, facilities (e.g., hand washing sink, shower, changing room) and protective clothing (e.g., laboratory coats, safety glasses and nitrile gloves) shall be provided that are appropriate for the risk of exposure to viable organisms containing recombinant DNA molecules.
o Synthetic biology research does sometimes require interaction with potentially dangerous substances. Listed below are those substances which pose the greatest risk to researcher safety.

o Eithidium Bromide (EtBr) - This substance was used to stain DNA for gel electrophoresis. Even though it is used in highly diluted solutions, it is still a toxic substance and a suspected mutagen. Because of this, everyone who handles EtBr must wear nitrile gloves, a lab coat and safety glasses. The amount of Eithdium Bromide utilized by this lab is small and it poses little environmental danger at its functional concentration. All liquid and solid waste that comes in contact with this substance is stored and disposed of as required by EH&S.

o Ultraviolet Light (UV) – Used for the visualizing of stained DNA, this form of light poses the risk of UV radiation. To avoid exposure when reading gels, UV lights were contained inside of equipments that acted as a UV-blocking shield and when performing gel extractions, exposure was prevented by wearing lab coats, nitrile gloves and using safety glasses and UV-protection screens.

o Bunsen Burners – Used for sterilizing certain laboratory equipment such as inoculation loops or plate spreaders. This equipment can cause burns and fires if neglected or misused.

o Culture Mediums – Solutions and plates containing nutrient rich media were used to grow E. coli and Cyanobacteria. If media, such as Lauria Broth, are released in large quantities into the environment they have the potential to disrupt ecosystems by promoting the growth of abnormal bacteria and fungi. All leftover stocks and plates are autoclaved or combined with a bleach solution before being disposed of as specified by the EH&S.

o All other toxic and hazardous chemicals were handled to avoid direct contact and with observance of proper safety procedures.
Public and Environmental Safety
o There is no concern for the safety of the public or the environment because the bacteria cells we worked with are all non-pathogenic with controlled antibiotic resistance and they would not be able to survive outside of the laboratory setting.

o Escherichia Coli (E. coli) - we are working with the NEB10 Beta strain of E. coli (New England Bio Labs) that is deficient in producing leucine and cannot survive on media that is not supplemented with this amino acid (auxotrophic mutant). NEB Iq strain of E. coli (New England Biolabs) is also currently being used in the lab. It is not currently reported as an auxotroph by New England Bio Labs but upon attempting to grow the strain in the presence of C and N without an amino acid source, we have been unable to demonstrate grow for this cell line.

o Cyanobacteria Synechocystis PCC6803 - We have created our own auxotrophic mutant by using homologous recombination to create an insertional mutation in the thiamine monophosphate pyrophosphorylase synthase (ThiE) gene. In doing so, we will be creating a mutant that requires thiamine to grow and cannot survive outside the laboratory.
o Also, we are currently designing a self-contained apparatus that will be used when co-culturing our E. coli and Cyanobacteria. This apparatus will prevent introduction of organisms into the environment and can be autoclaved.
o In addition to the above mentioned safety precautions, all materials and waste products that come into contact with live bacteria are disinfected with a 10% bleach solution and all used glassware is autoclaved before being disposed of in adhesion to EH&S standards. Gloves are always worn while handling living bacteria and hands are always washed before a student leaves the lab. All of which prevents the public from ever encountering the bacteria used in our research.
o All chemicals and biological agents were disposed of in their designated biohazard receptacles as determined by the EH&S.

4. Do any of the new BioBrick parts (or devices) that you made this year raise any of the above mentioned safety issues?

o The one public safety concern with this year’s project was the BioBrick part that produces ethanol.

o Ethanol – Ethanol will be produced by our E. coli cells and has the potential to cause respiratory irritation, acute toxic effects, severe eye irritation and is highly flammable when stored in high concentrations. Because of its hazardous nature it is deemed a controlled substance. But when ethanol is diluted and unrefined its health risks are greatly decreased.
o To prevent our ethanol production from creating any type of research, environmental or public safety concerns we contacted the University of Nevada’s Enviromental Health and Safety Department. The University of Nevada’s officials granted us permission to execute the project with the following restrictions:

1) Large volumes of ethanol cannot be produced

2) The resulting culture medium will not be distilled to purify the ethanol

3) Consumption of the ethanol containing culture medium or any of its derivatives is strictly prohibited.

o We have also generated new parts related to fatty acid production in E. coli and hexose sugar production in Synechocystis. Based on our search of the literature (view our Reference page) none of these genes or gene products pose any public, environmental or research safety concerns.

5. Do you have any other ideas of 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?

o For any iGEM team working with Cyanobacteria, we would highly recommend that they make their own auxotrophic mutant by undergoing homologous recombination at the ThiE locus or other known location on the chromosome that would result in the production of an auxotroph.

NEW SAFETY AND SECURITY QUESTIONS
1. Would the materials used in your project and/or your final product pose:

Answer to Safety Question 1a:

Risks to the safety and health of team members or others in the lab?
No. None of the materials used in our project pose safety/health risk to team members or other in the lab. The products generated from this project are glucose, ethanol and free fatty acids. Ethanol will be produced by our E. coli cells and has the potential to cause respiratory irritation, acute toxic effects, severe eye irritation and is highly flammable when stored in high concentrations. Because of its hazardous nature it is deemed a controlled substance. But when ethanol is diluted and unrefined its health risks are greatly decreased. We have also generated new parts related to fatty acid production in E. coli and hexose sugar production in Synechocystis. Based on our search of the literature (view our Reference page) none of these genes or gene products pose any public, environmental or research safety concerns.

b. Risks to the safety and health of the general public if released by design or accident?
c. Risks to environmental quality if released by design or accident?
Answer to Safety Questions 1b and 1c
No, there is no risk to the safety/health of the general public if project is released by design or accident. No, there is no risk to the environmental quality if the project is released by accident or design.
o There is no concern for the safety of the public or the environment because the bacteria cells we worked with are all non-pathogenic with controlled antibiotic resistance and they would not be able to survive outside of the laboratory setting.

o Escherichia coli (E. coli) - we are working with the NEB10 Beta strain of E. coli (New England Bio Labs) that is deficient in producing leucine and cannot survive on media that is not supplemented with this amino acid (auxotrophic mutant). NEB Iq strain of E. coli (New England Biolabs) is also currently being used in the lab. It is not currently reported as an auxotroph by New England Bio Labs but upon attempting to grow the strain in the presence of C and N without an amino acid source, we have been unable to demonstrate grow for this cell line.

o Cyanobacteria Synechocystis PCC6803 - We have created our own auxotrophic mutant by using homologous recombination to create an insertional mutation in the thiamine monophosphate pyrophosphorylase synthase (ThiE) gene. In doing so, we will be creating a mutant that requires thiamine to grow and cannot survive outside the laboratory.
o Also, we are currently designing a self-contained apparatus that will be used when co-culturing our E. coli and Cyanobacteria. This apparatus will prevent introduction of organisms into the environment and can be autoclaved.
o In addition to the above mentioned safety precautions, all materials and waste products that come into contact with live bacteria are disinfected with a 10% bleach solution and all used glassware is autoclaved before being disposed of in adhesion to EH&S standards. Gloves are always worn while handling living bacteria and hands are always washed before a student leaves the lab. All of which prevents the public from ever encountering the bacteria used in our research.
o All chemicals and biological agents were disposed of in their designated biohazard receptacles as determined by the EH&S.

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

Answer to Safety Question 1d:

No, there is not risk to security through malicious misuse by individuals, groups or states. All laboratory facilities are limited to Nevada iGEM Team members. Laboratories and building access is secured by key locked doors are electronic card key access. All iGEM Team members have been vetted through the UNR Police Department before being granted access to laboratory facilities. Hazardous materials are maintained in locked cabinets and refrigerators/freezers. Transgenic organism are maintained in growth chambers secured with combination pad locks.
Please explain your responses (whether yes or no) to these questions. Specifically, are any parts or devices in your project associated with (or known to cause): - pathogenicity, infectivity, or toxicity? - threats to environmental quality? - security concerns? 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. b. Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.

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-o	Escherichia coli (E. coli)  - we are working with the NEB10 Beta strain of E. coli (New England Bio Labs) that is deficient in producing leucine and cannot survive on media that is not supplemented with this amino acid (auxotrophic mutant). NEB Iq strain of E. coli (New England Biolabs) is also currently being used in the lab. It is not currently reported as an auxotroph by New England Bio Labs but upon attempting to grow the strain in the presence of C and N without an amino acid source, we have been unable to demonstrate grow for this cell line.
+o	Escherichia Coli (E. coli)  - we are working with the NEB10 Beta strain of E. coli (New England Bio Labs) that is deficient in producing leucine and cannot survive on media that is not supplemented with this amino acid (auxotrophic mutant). NEB Iq strain of E. coli (New England Biolabs) is also currently being used in the lab. It is not currently reported as an auxotroph by New England Bio Labs but upon attempting to grow the strain in the presence of C and N without an amino acid source, we have been unable to demonstrate grow for this cell line.
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