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Safe Practices in the Lab

Our team conducted our work on the Cornell University campus, in the Biomedical Engineering Instructional Lab in Weill Hall, as well as in the [http://angenent.bee.cornell.edu/molecularlab.html Angenent Lab] in the Department of Biological & Environmental Engineering in Riley-Robb Hall. All work was conducted in a BSL 1 laboratory: this means that the strains we are using (Escherichia coli DH5a and WM3064, Shewanella oneidensis MR-1 and JG700, and Pseudomonas putida) are non-pathogenic and well-characterized. Before gaining access to the lab space, team members were required to: 1) complete chemical waste disposal training, 2) attend an orientation with Todd Pfeiffer, the Weill Hall Facilities Director, regarding the Cornell Environmental Health & Safety guidelines, and 3) receive specific training from [http://www.bme.cornell.edu/people/adj-profile.cfm?netid=sda4 Dr. Shivaun Archer], the manager of the BME Instructional Lab, for the equipment in the lab.

Our standard lab practices were in compliance with the World Health Organization’s Biosafety Level 1 [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf guidelines]. Many of our basic lab practices are described below.

• Researchers were required to wear gloves while in the lab space, and to remove one glove when going into lab common areas.
• No gloves were allowed to leave the lab space, and no food or drink was allowed into the lab space.
• Within Weill Hall, microbiological samples were transported only via the internal service elevator, to avoid contamination of public areas.
• Any materials transported between the two on-campus lab spaces were held in secondary containment during transport.
• All flammable liquids were kept in a flammable storage cabinet.
• The lab contained distinct waste containers for general waste, biohazard waste, biohazard sharps, and non-biohazard broken glassware.
• Liquid bacterial waste was treated with bleach before being discharged into sanitary sewage.
• Benchtops were decontaminated with ethanol before and after lab work was conducted. Tools that came into contact with bacteria were soaked in ethanol and flame-treated before and after use.
• An autoclave was used to decontaminate growth media, glassware, tubes, pipette tips, etc. All lab members were trained in proper autoclave use by the Weill Hall Facilities Director in order to avoid dangers to researcher safety.
• An emergency shower, eyewash, and first aid kit were available withing the lab space in case of emergency.
• Lab notebooks were maintained in a taped-off no-glove area.

Safety procedures specific to our project, i.e. separate from best microbial practices, were followed during testing with arsenic and naphthalene. We modified EH&S standard operating procedures for arsenites, arsenates, and naphthalene for our specific experimental needs. Most importantly, all work with arsenic and naphthalene was conducted by team members wearing appropriate PPE in a taped-off, designated area, and with equipment labeled as arsenic- or naphthalene-contaminated. Waste was collected separately, labeled appropriately as a cancer-hazard and disposed of through the Cornell EH&S.

Similar precautions were used in working with Ethidium Bromide - all EtBr-staining was done in designated boxes, and in taped off areas used only for running and staining gels. Gels were visualized on a UV light box protected from EB contamination using saran wrap, which could be easily disposed of in the biohazard bin rather than risking ineffective decontamination of the UV light box. Team members were protected from exposure to UV rays with a UV shield. Facial shields were also available for use when needed.

Material Safety Data Sheets for these three hazardous compounds, as well as all other hazardous compounds used in the lab, were readily available in a binder in the lab space, as well as in the team’s shared online file folder.

Is there a local biosafety group, committee, or review board at your institution? (Safety Question #3)

Yes, [http://sp.ehs.cornell.edu/Pages/Home.aspx Cornell University Environmental Health & Safety] oversees safe procedures in on-campus laboratory research. The Cornell EH&S mandates training required of researchers before they can gain access to lab spaces. Through the EH&S, all team members took a required course on Chemical Waste Disposal.

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

We have been in contact with the EH&S at our university about best practices for testing with arsenic. We have consulted them about disposal of carcinogenic wastes, as well as about how to keep researchers safe by using secondary containment and designated equipment. The EH&S has expressed satisfaction that our standard operating protocols and waste disposal plan will ensure researcher safety, as well as public safety.

Safety Concerns for Our Project

We are working with biosafety level 1 organisms - Escherichia coli DH5α and WM3064 (a conjugation-enabled DAP auxotroph), Shewanella oneidensis MR-1 and JG700 (an mtrB knockout strain), and Pseudomonas putida. None of these strains pose a threat to researcher or public health before modification. The pathways that we are modifying in S. oneidensis JG700 are natural pathways already found in Shewanella and Pseudomonas, so they do not raise any safety concerns. As our project aims to detect the unwanted presence of toxic compounds in the environment, our device could not easily be used to negatively impact the public or the environment, and thus does not pose any foreseeable biosecurity risk.

Would any of your project ideas raise safety issues in terms of: (Safety Question #1)

• researcher safety,

As we were working at BSL 1, the primary concern in terms of researcher safety was testing with our toxins of interest. Please see safe lab practices and standard operating procedures for arsenic and naphthalene, above.

• public safety,

Again, our project itself could not be easily used to negatively impact the public. However, even when a research project does not pose a security threat, the conducting of research too must be done in a way that protects public interests and safety. All bacteria and waste were disposed of properly, in biohazard bags and through the Cornell EH&S, rather than in common garbage cans, or decontaminated with bleach before being poured down the drain.

Additionally, in order to make our biosensor field-deployable, i.e. suitable for placement in water that will be drinking water, our device includes a thorough effluent filtration system to prevent the escape of our modified strains into the environment. We’re using a safe species and safe modifications that would not pose any foreseeable threat to native species or biodiversity. Finally, though our current selective pressure on our engineered strains is antibiotic resistance, the final device would use strains that were auxotrophs for some essential nutrient or that have our genetic modifications integrated into the bacterial chromosome; this would avoid the possibility of conferring antibiotic resistance to harmful bacterial strains in water. In the case of auxotrophic selection, our strain would be mutated such that it is an auxotroph for two essential nutrients: one of these nutrients would be provided in the media, so that the strain could only survive inside our bioreactors. Selective pressure for the plasmid would then be maintained through the second essential nutrient, which the plasmid would enable the bacteria to synthesize.

• or environmental safety?

We do not foresee any adverse effects of deploying our device in natural streams and lakes. As detailed above, we have thought carefully about how to prevent our modified strains from getting into the environment. However, because we are knocking out mtrB - an essential component of S. oneidensis’s respiration pathway - and putting its production under the control of an inducible promotor, it is likely that our modified strains would be less able to compete than the wild type organisms already in the lake. If our parts were to lose their function, through mutation or other means, the most likely outcome would be that our strains would lose their capacity for facultative anaerobic respiration entirely, again leaving them less able to compete with wild-type Shewanella. There is the concern that antibiotic resistance could be horizontally transferred to other organisms in the wild, if our strain were to be released into the wild. This could again be addressed by selecting for strains auxotrophically.

Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? (Safety Question #2)

The only safety issue associated with our new BioBrick parts is researcher safety in testing the parts. We have documented our standard operating procedures above, which other teams can reference if they want to use our parts, or test other parts using arsenic or naphthalene.

We are also conducting extensive tests to assess the functionality of our BioBrick parts, to ensure that they behave as expected under different conditions.

Safety Concerns for iGEM Competition

Do you have any other ideas 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? (Safety Question #4)

While our team is fortunate enough to have a biosafety group readily available for on-campus consultation, we recognize that many other teams may not have comparable biosafety resources available to them. In addition to the documentation by teams of their own safe practices, which can be used by other teams as references, more explicit safety guidelines, a “safety checklist” for instance, could be provided by the iGEM competition itself. One possibility is a checklist that rates a given lab from “unsafe” to “very safe” based on the number of safety provisions satisfied by the user filling out the checklist - rather like the Internet sites which assess the strength of inputted passwords.

Many of the safety concerns we expressed above are the same concerns that many other iGEM teams also face. Mainly, the prominent use of antibiotic resistance in the construction and retention, via selective pressure, of novel genetic circuits poses a continued risk to public health due to the possibility of conferred antibiotic resistance. While the genes of antibiotic resistance can be carefully controlled in the lab setting, for any device to be used in a real-world setting, this possibility of horizontally transferring antibiotic resistance must be addressed. With this in mind, future iGEM teams should make an effort, where possible, to transition to other forms of selective pressures, such as limited growth media.