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Team:Arizona State/Safety
From 2013.igem.org
Researcher and Lab Safety
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]
- Human Dendritic Cells
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.
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.
Listeriolysin O Safety Analysis
Toxicity
There is a risk of toxicity from one of the genes used in the system, listeriolysin O, via human cell damage. Listeriolysin O (LLO) is a virulence factor protein naturally found in Listeria monocytogenes. The pathogenic function of LLO occurs when Listeria enters the host’s macrophage immune system cells, and then enters the phagolysosome organelle. Listeria secretes LLO protein, the protein ruptures the phagolysosome, and live Listeria escapes the macrophage cell. In combination with other expressed toxins such as invasin or internalin, this process allows listeria to escape macrophages and continue infecting the host. LLO could potentially lyse human blood cells if unintentionally ingested by team members, other people at ASU, or the public.
The LLO in our E. coli chassis is expressed without the secretion tag found naturally preceding the LLO coding sequence in Listeria monocytogenes. Once our bacteria is engulfed by a macrophage, an acidic phagolysosomes must first degrade the membrane of the E. coli before LLO can be exposed and subsequently break open the phagolysosome for proper antigen presentation. This prevents the bacteria from continuously escaping macrophages inside the body and spreading unintentionally. The ASU team is conducting assays of this process on blood agar plates to confirm that LLO is not secreted from E. coli. The below images indicate correct functionality of the LLO protein. We have also included the following warnings on the Registry page: (1) Do not add a secretion tag to LLO, (2) Do not express LLO along with BioBricks or in chassis that enable active cell invasion (e.g., BBa_BBa_I10001).
LLO Biosafety Engineering
The LLO-based BioBrick does not contain the secretion tag. Therefore, we believe that LLO functions only after the chassis (E. coli) is degraded by the phagolysosome. LLO should result in the release of the therapeutic payload (anti-cancer antigens), and not live E. coli cells. We are performing assays to confirm that E. coli will not secrete LLO prior to deeming that there is no safety risk to this part.
If the LLO protein were to escape the E. coli chassis in the bloodstream before the bacteria was engulfed by a macrophage, then LLO, in the absence of the Listeria internalin cofactors, would be disrupted by the neutral pH of blood[http://www.ncbi.nlm.nih.gov/pubmed/16105950][http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002356 ]. We believe that if an unintended subject ingested the E. coli, their macrophages and dendritic cells would engulf the bacteria and LLO would break open the phagolysosomes of those cells, resulting in unintended immunization against cancer. Although the system is designed as a prophylactic vaccine and we envision seeing no immediate negative consequence of unintended vaccination, it may lead to a dangerous autoimmune response against self-antigens in at-risk subjects that are unintentionally vaccinated.
Dual-Use Potential
There is a potential for malicious misuse of the LLO toxin as a biological weapon. However, the construct that we have built in the iGEM lab is not functional as a stand alone biological weapon because it lacks invasive proteins, such as invasin or internalin, is only active in acidic phagosomal conditions, and prematurely folds the proteins needed for pore-forming activity in neutral pH conditions[http://www.ncbi.nlm.nih.gov/pubmed/16105950][http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002356 ].
LLO Necessity and Alternatives
Phagolysosome lysis, induced by LLO after the E. coli membrane is degraded inside a dendritic cell, is necessary for our cancer antigens can be released from the E. coli chassis into the macrophage cytoplasm, which results in presentation on MHC Class I complexes and subsequent cytotoxic T cell activation, needed for destroy tumor cells. Without LLO, the antigens would be confined to MHC Class II presentation, which would only activate B cells. Because cells in the body, such as tumor cells, only contain MHC Class I proteins on their surface, B cell activation is not sufficient to trigger an anti-tumor response because tumor cells would still be indistinguishable from healthy cells. B cells produce antibodies, but they will not be able to recruit cytotoxic T cells to tumor cells without the previous existence of T cells that recognize tumor antigens. MHC Class I activation is the only method to activate cytotoxic T cells to recognize antigens on the surface of tumor cells. We are, however, looking into safer alternatives to LLO, such as ubiquitin, to replace LLO in future iterations of the vaccine system.
Environmental Safety
Our project involves developing a modular bacterial lab-strain, and eventually probiotic bacteria currently being consumed by humans, cancer vaccine platform. None of the genes themselves have had any previous literature detailing detrimental environmental effects. Because the introduced genes themselves are either from human tumor cells or are the LLO gene, we do not believe they pose any significant environmental hazarsds. In addition, because we are attempting to develop a bacterial-based vaccine, we envision this system to be used only in basic and clinical laboratories.
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.
National Guidelines:
- National Institutes of Health[http://www.nih.gov/]
- NIH Guidelines for Research Involving Recombinant DNA Molecules[http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm]
- NIH Risk Group Classifications[http://rpi.edu/research/office/ibc/riskgroupclassifications.html]
- Center for Disease Control[http://www.cdc.gov/]
- CDC Biosafety[http://www.cdc.gov/biosafety/]
- American Biological Safety Association[http://www.absa.org/]
- Environmental Protection Agency[http://www.epa.gov/]
- Toxic Substances Control Act (TSCA) Biotechnology Program[http://www.epa.gov/opptintr/biotech/index.htm]
- Occupational Safety and Health Administration[http://www.osha.gov/]
- Toxic and Hazardous Substances: Blood borne pathogens[http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=standards&p_id=10051]
Arizona State Environmental Health and Safety Program[http://cfo.asu.edu/ehs-biosafety]
- Biosafety manual[http://www.asu.edu/uagc/EHS/documents/biosafetymanual.pdf]
- Biowaste compliance guidelines[http://cfo.asu.edu/ehs-biowaste-compliance-guideline]
More biosafety and human practices analysis conducted on our project can be found here.
