Team:Michigan/Safety

Contents

1. MSBT Safety Affiliation
   1. Standard Microbiological Guidelines for BL-1 (as listed on the Appendix G-II-A-1 of NIH Guidelines)
2. Safety Concerns Regarding Pix Cells
3. Potentially Health-Risk Reagents/Equipment Used in the Lab

MSBT Safety Affiliation

Michigan Synthetic Biology team operates under the guidelines of Operation Safety and Environmental Health (OSEH) here at the University of Michigan. We are overseen by OSEH’s Biological & Laboratory Safety Program Area (B&LS), which are responsible for the biosafety that are required under university standards, federal regulations and sponsored research contracts. Additionally, since our team is working with recombinant DNA, OSEH has a special section regarding rDNA. “OSEH-B&LS provides Biosafety Officer (BSO) services to the Institutional Biosafety Committee (IBC), that oversees this work on the U-M Campuses...including services such as research protocol review and biosafety risk assessment, inspections, reporting, and training of research staff on the unique risks associated with rDNA.” (OSEH-rDNA).

Our team complies to the guidelines as listed in the NIH (National Institutes of Health) guidelines for conducting research specifically involving recombinant DNA molecules (NIH Guidelines for rDNA). We have discusses the status of our lab with OSEH’s IBC group and we have been classified as biosafety level 1 (BL1); defined as the following from the NIH Guidelines for rDNA (Appendix G-III-M) “Biosafety Level 1 is suitable for work involving agents of unknown or minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the general traffic patterns in the building. Work is generally conducted on open bench tops. Special containment equipment is not required or generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science”

Standard Microbiological Guidelines for BL-1 (as listed on the Appendix G-II-A-1 of NIH Guidelines)

• Access to the laboratory is limited or restricted at the discretion of the Principal Investigator when experiments are in progress.
• Work surfaces are decontaminated once a day and after any spill of viable material.
• contaminated liquid or solid wastes are decontaminated before disposal.
• Mechanical pipetting devices are used; mouth pipetting is prohibited.
• Persons wash their hands: (i) after they handle materials involving organisms containing recombinant DNA molecules and animals, and (ii) before exiting the laboratory.
• All procedures are performed carefully to minimize the creation of aerosols.
• In the interest of good personal hygiene, facilities (e.g., hand washing sink, shower, changing room) and protective clothing (e.g., uniforms, laboratory coats) shall be provided that are appropriate for the risk of exposure to viable organisms containing recombinant DNA molecules.
• Special Practices (BL1): Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leak-proof container which is closed before being removed from the laboratory.
• Containment Equipment (BL1): Special containment equipment is generally not required for manipulations of agents assigned to BL1.

Each member on the team is required to go through OSEH Laboratory Safety training prior to working in the lab. Once all members have been trained, the lab director of the building we work in gave us a detailed, highly specific to the building’s harzard waste disposal presentation. We asked questions regarding proper disposal of bioharzard waste and their location within the lab. It was an insightful experience for each member on the team to learn the potential consequences of their actions in the lab to the environment.

Safety Concerns Regarding Pix Cells

Since our project involves the usage of Escherichia coli, we follow closely to the provisions listed in Appendix C-II. Escherichia coli K-12 Host-Vector Systems. However, we are not dealing with E. coli K-12 strain specifically, but BL21 DE3 and DH5-alpha. We assume the protocol and guidelines for E. coli K-12 strains are applicable to BL21 DE3 and DH5-alpha because both are related to the E. coli cloning system.These two strains are auxotrophs and F- (can not transmit its plasmids to others) so their overall cloning mechanism is not completely divergent from K12 strains. Moreover, as cited in Chart et.al 2000, “it was concluded that E. coli strains EQ1 and DH5a, in common with other strains of E. coli K-12 (Anderson 1975; Brenner and Wigby 1982; Smith and Rowe 1982) and BLR and BL21, did not have the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections”. This reassures the E. coli strains we are cloning into are non-pathogenic to the environment.

Again, we would like to emphasis that the two strains of E. coli (BL21 DE3 and DH5-alpha) we use in our project contains F- (F minus). In other words, it is highly unlikely our strains would transmit its plasmids to other strains. This also applies to the unlikely chance of transferring antibiotic resistance plasmids to the environment. In the lab, we commonly use ampicillin and kanamycin, which are often times used in public health settings to treat infections. However, since BL21 DE3 and DH5-alpha does not contain the necessary F+ factor, we are not concern with our project spreading antibiotic resistance to other strains.

Although our biobrick is designed for to surface display zinc-finger binding domain on the surface to DNA, there is very little risk of double-stranded DNA found in the environment. Double-stranded DNA cannot remain stable for a long period of time to pose any considerable target for our zinc finger binding proteins. Thus, threats to environmental quality are low.

Potentially Health-Risk Reagents/Equipment Used in the Lab

In our project, we constantly come in contact with the following reagents/equpiment that could pose potential health and safety concerns for our team members, environment, and security. All safety procedures and guidelines are assigned by OSEH.

• Ethidium bromide is used as an intercalating agent and a dye when testing double stranded DNA. While used in highly dilute solutions, this compound possesses some acute toxicity. Everyone handling it in its stock form must wear nitrile gloves and kept stored under a fume hood. Ethidium bromide is utilized in minuscule quantities and poses little environmental danger at its functional concentration. All liquid waste that comes in contact with this compound is disposed in dedicated jugs, and solid waste collected in marked containers as required by OSEH.
• Hydrochloric acid in the concentrations used is potentially dangerous to the public if mishandled. HCl possesses both caustic and reactive properties and is capable of evolving gaseous chlorine on contact with certain compounds, as well as producing certain harmful inorganic salts such as copper (I) chloride. In our lab, we sparingly use hydrochloric acid in 1M and 6M concentrations to clean DNA from cuvettes and purification columns. Concentrated solutions of HCl are handled with long sleeves, gloves and stored in either a fume hood with diaper pads or a lockable acid storage cabinet. If released into the environment, it would poses little long-term hazard due to its reactivity and subsequent short half-life. However, it could potentially alter the ecosystems by changing the chemical makeup of soils and groundwater if released in sufficient quantities. Also, large amounts of concentrated hydrochloric acid can potentially be used to produce more toxic compounds such as gaseous chlorine and mustard gas and be used as weapons, if the individuals or states in question possess sufficient knowledge and equipment. However, under OSEH guidelines, we are to dispose all acid waste in separately labeled glass jug from other liquid waste. Once jug is full, it would be picked up by OSEH. It is highly unlikely the bacteria strain we use can be made into bio-weapons without extensive modification.
• Sodium hypochlorite is used in household concentrations as a means of destroying organisms and organic material before disposal. This compound is handled with gloves on a dedicated bench in small amounts.
• Solutions and plates containing rich media such as LB, SOB, and SOC are frequently used for the growth of E.Coli for purification and DNA extraction. Rich media such as LB, if released into the environment in large quantities, has the ability to disrupt ecosystems through the facilitation of abnormal bacterial and fungal growth. However we adhere to the OSEH regulations of proper waste disposal (as mentioned above). Leftover stocks and plates are autoclaved and disposed of separately from other chemical waste.
• Bunsen burners are used in conjunction with flammable 70% ethanol solutions for the purpose of sterilizing certain laboratory implements such as plate spreaders and inoculation loops; such methods are capable of causing burns if misused.