Organisms

All the organisms utilized in the Haynes Lab comply with biosafety level 1 (BSL1) and do not possess the potential to cause disease in individuals. All organisms, despite having no association with disease, are treated as potential pathogens, thus personal protective equipment such as gloves, laboratory coats, and protective eyewear/goggles are used to prevent contact with bacteria and yeast samples in the lab, to prevent splashes, and to avoid sources of ultraviolet radiation.

Standard protocols were followed for all genetic manipulation, including PCR, plasmid assembly (restriction, ligation, and transformation), and DNA extraction. These protocols standardize specific safety procedures encountered in day to day labwork.

The following organisms are present in the lab and directly utilized in our project:

• Escherichia coli BL21(DE3)^{[http://www.ncbi.nlm.nih.gov/bioproject/30681]}
• Escherichia coli NEB-10 Beta^{[http://www.neb.com/nebecomm/products/productc3020.asp]}

Biosafety training

All members of our team were required to attend biosafety and bloodborne pathogen training according to Arizona State policy before working in the lab. This course satisfies the OSHA Bloodborne Pathogens training requirement as well as the Biosafety requirements for working with recombinant DNA. The Laboratory-Specific biosafety training checklist was followed^{[http://www.asu.edu/uagc/EHS/forms/asu_lab_specific_biosafety_training.pdf]} to ensure all team members were adequately trained.

Biological Reagents

Reagents and equipment including solid and liquid bacterial growth media, and yeast culture media, micropipette, volumetric pipette tips and centrifuge tubes are autoclave sterilized (heated to 121 degrees celsius at 15 PSI) both preceding and following use, ensuring there is no threat of waste contamination of outside the lab. This is in accordance with Arizona State University's biological waste procedures^{[http://cfo.asu.edu/ehs-biowaste-compliance-guideline]}.

Flammable Reagent Safety

All team members were required to complete fire safety training prior to working in the lab. Reagents that are potentially flammable (e.g. ethanol and isopropyl alcohol) are stored in a flame protective cabinet.

Corrosive and Noxious Reagent Safety

Reagents that have corrosive and/or noxious fumes (e.g. bleach solutions and phenol-chloroform) are kept within a chemical fume hood to prevent inhalation and physical contact.

Device Testing

To be applicable to the field, our biosensor project design requires testing with pathogenic organisms. To resolve this issue, the team developed solutions to determine the accuracy and precision of each type of biosensor without endangering the research team:

• The DNA biosensor, instead of targeting a DNA sequence of pathogenic bacteria, is targeting a sequence of green fluorescent protein as a proof-of-concept.
• The cell surface biosensor, which is specific to a pathogenic strain of E. coli (O157:H7), was tested using heat-killed E. coli O157:H7 cells, supported by previous research as an effective target to determine biosensor accuracy^{[http://www.pnas.org/content/early/2010/10/11/1008768107.full.pdf]}. Originally, expressing the O157:H7 antigen on nonpathogenic strains of bacteria was considered, but the pathogenic nature of the antigen restricted that method of testing.

In the future, to test the biosensor's accuracy and precision in detecting real pathogens, Biosafety Level 2 safety rules will need to be implemented in the laboratory as the research group will be working with pathogenic bacteria.

None of the BioBricks produced by the 2012 Arizona State iGEM team raise any safety concerns.

Public Safety

There is minimal risk associated with the release of the aforementioned organisms. The recombinant DNA (rDNA) experiments conducted in the laboratory provide ampicillin, kanamycin and/or chloramphenicol resistance to E.coli BL21(DE3) and E.coli NEB-10 Beta to select for plasmids. Under the circumstances that any of these genetically altered organisms were released they would have minimal potential for pathogenesis. In accordance with Arizona State University’s Environmental Health & Safety policy, “Nothing in the trash, nothing down the drain”, we autoclave and bleach sterilize all waste from recombinant DNA experiments. This reduces the likelihood of accidental release.

Under the circumstances of “designed” release, the organisms and all rDNA products present no possibility of harming the outside population and ecosystem. Our iGEM team, faculty mentors, and department lab safety manager, find no foreseeable public health threat associated with the organisms and recombinant DNA project utilized by our team. Malicious misuse of the rDNA or organisms used in the lab would be minimal because the DNA biosensors contain sequences that target a benign target, green fluorescent protein, and the cell surface biosensor contains a protein with antimicrobial, rather than pathogenic, properties.

Environmental Safety

The safety of the environment was considered for real-world use of all biosensor designs. The designs for each biosensor was developed so that bacteria containing recombinant DNA would not be exposed to public water supplies in application. The end goal of the biosensor would be application in rural areas with public water supplies in danger of bacterial contamination. After the fusion protein biosensors are isolated in a lab setting, the fusion proteins used as each biosensor would test water samples in a cell-free environment and could subsequently be denatured via heat, reducing any potential contamination of the water or safety concerns by the biosensor protein itself.

Biosafety Regulations and Provisions

The local biosafety group at Arizona State University is the Institutional Biosafety Committee and the Environmental Health and Safety Biosafety Program. The Institutional Biosafety Committee sees no foreseeable public health threat associated with the organisms and recombinant DNA project utilized by our team.