Biological Safety Concerns Regarding E. curi

Identifying Safety Issues in Our CRISPR Project

Working in a lab can be fun but also dangerous if safety for the student, society, and environment are not addressed. Therefore, the safety issues of these three important entities will be examined under the scope of risk assessment. To assess risk for a biological material many factors have to be considered, which include but are not limited to: the microbe's pathogenicity and ability to survive outside of the lab, public awareness of the biological agent, availability of a prophylaxis, environmental impact, and ability to monitor biological material that are potentially dangerous.

We assess risk for the biological material involved in our project and its potential impact to the researchers, public society, and environment by taking into account two components of risk: probability and hazard. With probability, we ask ourselves:

• What are the likely and unlikely (including worst-case) scenarios of conducting research in our project?
• Is there a potential risk that may compromise in any way researchers, society, or the environment? How likely that risk comes to fruition?

Questions to consider about hazards associated with biological material risk are:

• If the project works, what may be the potentials hazards to researchers, society, and the environment?
• By what mechanism may the engineered organism of the project affect researchers, society, and the environment?
• If the engineered organism is used outside the lab, how may natural mutations to the organism's DNA affect and alter the functionality and concomitant risks to researchers, society, and the environment?

We assess biological safety risks with these questions in mind in an effort to make our project acceptably safe according to the standards set by organizations, such as the World Health Organization (WHO), the Convention on Biological Diversity, the National Institute of Health (NIH), American Biological Safety Association, Center for Disease Control, among other risk management groups, and conferences with an emphasis in synthetic biology and safety such as the SB5.0 conference. In addition, complying with these safety standards will make the project and its future directions more approachable for acceptance by the public society.

Researcher Safety

Ensuring the safety of the USC iGEM lab and its researchers is critical to the success of the program. We have therefore taken initiative that each member understands and is aware of the following:

• Physiological and environmental risks associated in experimenting with a certain biological material.
• Course of action to take, as standardized and/or recommended by risk management groups, if the biological material is inappropriately exposed to the researcher and/or the environment.
• Knowledge of lab equipment to utilize to reduce the possibility of inappropriate biological material exposure and protect the researcher if such exposure occurs.
• Knowledge of safety protocols and location of safety equipment in case of inappropriate exposure, including emergency situations.

The laboratory, where the USC iGEM team works, is certified for Biosafety Level 1. Researchers are required to wear personal protective equipment (PPE) which include goggles, gloves, and lab coats so that the eyes, hands, and body, respectively, are always protected from possible exposure to a biological material. Potentially harmful biological materials and chemicals are used daily in the lab. Below is a description of each one we encounter on a daily or limited basis as well as the measures taken to comply with USC's stringent safety standards so that we maintain the highest quality of our safety:

• Ethidium Bromide (EtBr) is an aromatic chemical agent often used for staining of DNA during agarose gel electrophoresis. It binds with DNA and fluoresces with an orange color under UV light. EtBr may be a mutagen and carcinogen since it is believed to intercalate with double-stranded DNA, therefore inhibiting the DNA from undergoing important biological processes such as replication and transcription. Special care is definitely taken when handling EtBr. Proper PPE is used, and disposal of the chemical is handled by the researchers in accordance with EtBr's Material and Safety Data Sheet (MSDS). Pipette tips exposed to EtBr are ejected to a chemical waste basket, which also include used agarose gels that contain a residual amount of EtBr. In addition, nitrile gloves are disposed of after pipetting EtBr to the agarose gel solution to prevent secondary exposure of EtBr.
• Ultraviolet (UV) light is used for imaging of stained DNA in agarose gel electrophoresis. However, unprotected and direct exposure of UV light is known to be harmful to the skin and eyes and may lead to skin cancer and eye-related problems. UV light damages DNA by multiple mechanisms. One involves the binding of two adjacent thymine nucleotides in the DNA sequence to make a thymine dimer, which results in distortion and reduced functionality of the DNA. When imaging DNA from a gel, researchers ascertain that the UV light is turned off and only turned on when the gel has been properly placed inside the imaging machine and its door closed so UV light exposure is shielded from the researcher. Similar steps are taken when taking the gel out. In gel extraction, a protective shield is worn protecting the face from UV light exposure.
• Descendants of two E. coli isolates, B and K12, have been used as tools and model organisms in our project, in particular, strains BL21(DE3) from the B isolate and DH5a and HT115 from the K12 isolate. These strains are known to be non-pathogenic and are extremely unlikely to survive outside of the lab or in human hosts, despite some conferring a variety of basic antibiotic resistances (e.g. ampicillin, kanomycin, chloramphenicol, and streptomycin). Nevertheless, prevention of contact from the bacteria is done by requiring the researchers to wear proper PPE and decontaminating all laboratory materials exposed to the bacteria with 10% bleach before disposal.

To ensure the researchers of USC iGEM are acceptably safe from physical harm by biological material or chemical exposure, each team member attended a Safety Orientation Course conducted by USC's Career and Protective Services (CAPS). In addition, the protocols used in our project have been approved by the Institutional Biosafety Committee.

Public Safety

Although the E. coli strains we are using are non-pathogenic, we remain aware that our project can potentially be a concern for public safety. By addressing this issue early, we lower the risk of fear and mistrust that can be met by the scrutiny of the general public when introducing a new biotechnological tool to the marketplace and diminish the uncalculated possibility of causing sever physical and non-physical harm. Our approach to public safety can be categorized as follows:

• How may our immediate surroundings, which include the university campus and its neighboring communities, be affected by our project?
• How may Greater Los Angeles as well as the general public be affected by our project?

The USC iGEM lab is located within the USC campus, one that invites more than 30,000 students daily. In addition, the campus is located merely miles from Downtown Los Angeles and other neighboring cities where millions of people reside. The risk for infection from the bacteria we use is extremely low since we use strains that are engineered for laboratory experiments. These strains would be unable to survive in the outside environment. However, we still do the following to minimize this risk as much as we can:

• Before entering and after exiting from the lab, hands are washed with hot water and soap for at least 30 seconds.
• Gloves are always removed upon leaving the lab.
• No food or drinks are brought inside the lab.

These simple procedures are implemented to ensure that the public in our immediate surroundings is respected with our safety concerns for them.

At first, it may seem difficult to judge the effect of our project on public safety on a national level. However, the CRISPR system in our model is designed to counteract potential spreading and resistance of harmful microbes. As such, our project may in fact increase public safety and health.

Environmental Safety

The safety of the surrounding environment should not be compromised. The E. coli strain is non-pathogenic and equipment and reagents are disinfected with the proper chemicals when cleaning materials to avoid spreading and contamination.

BioBrick Safety Issues

No, none of the new BioBrick parts that we made raise any safety issues. We designed the Cas3 and CasO BioBricks using K12 and B isolates, both of which are non-pathogenic.

Biological Safety Provisions

At USC, the Department of Career and Protective Services (http://capsnet.usc.edu/LabSafety/biosafety/) oversees that all research being done here, especially in biology, is safe. Initially, our team completed a laboratory safety class with the department before beginning work in the laboratory.

Our training included the following:

• Shared testimonials of accidents that have occurred in a laboratory and how we reacted to those accidents.
• Participated in an activity involving how to use a MSDS.
• Reviewed the meaning of common signs in the laboratories.
• Reviewed what personal protective equipment we should be using.
• Reviewed procedures during emergencies, including how to use an eye-wash station, shower, and how to dispose of a fire.
• Learned where to dispose of all hazardous laboratory materials, biological waste, and chemical waste.
• Received a certificate of our training.

In addition to the training, the iGEM researchers also made sure to:

• Wear gloves, lab coats, and goggles in the laboratory at all times.
• Take off all personal protective equipment when exiting the laboratory to work in the office.
• Not eat in any part of the laboratory except in the office.
• Clean up and organize the laboratory benches after each day of research.

We also spoke with the department about our specific project on CRISPR, and they saw no safety issues within our laboratory.

Thoughts on How to Improve Safety in iGEM

iGEM teams could test antibiotic sensitivity in all bacteria strains periodically, making sure that these strains will not survive an antibiotic that we expect the strains to be sensitive to. Parts, devices, and systems could be made even safer by having iGEM require mandatory laboratory safety check-ups during the summer as well as submit documentation of the safe practices employed.