Safety in Design and Responsible Use of Synthetic Biology in Bioremediation

For an experiment working with PFAS chemical handling is extremely important as our team is very familiar with the hazards of such chemicals.

There are two main pathways to safe and responsible use of synthetic biology- safety in design, and safety in implementation.

• Safety in Design
1. To minimize the need to use PFAS chemicals, we opted to use 2,2-difluorohexanoic acid for testing our deflourinating constructs. By doing so, we reduce potential exposure and accidental release of these incredibly harmful chemicals.
2. Our construct is not designed for environmental release, it is meant to be used in a contained system where water/waste leaving the bacterial membrane or chamber would be filtered and sterilized of bacteria.
3. Our project was designed with modularity in mind. We planned to test metabolic and defluorination seperately. Our metabolic construct was tested on hexanoic acid, the base molecule for the 2,2-difluorohexanoic acid.
• Safety in Implementation
1. There are many aspects of the environment that need a solution to PFAS pollution, but the process of implementing a bioremediation solution requires a lot of thought into the safety of potential release of an engineered organism.
2. For water treatment, the bacteria would be used in conjunction with more traditional water filtration methods such as reverse osmosis. The bacteria would be contained within a biomembrane or bioreactor that would then go through separate filtration steps before returning to the water supply or for release.
3. Treatment for contaminated soil could be done by collecting contaminated soil (such as soil near airfield runoff) and using the bacteria to compost the PFAS chemicals. The barrels could then be heated in an industrial kiln similar to those in use at cement processing factories.
4. Our project is not intended for use in live organisms, and while there could be potential for engineering microbiome compatible bacteria, that is beyond the scope of this project.

Institutional Approval

At ASU, institutional approval begins with a proposal to explain what our goal is, the specific experiments we plan to conduct, and the materials necessary to the project.

ASU's institutional review board looked at the chemicals we would be using, the chassis, all of the potentially used parts, and how we plan to use them. From this, they make a justification of the safety precautions necessary for the experiment to proceed.

Before stepping into lab, extensive safety training was required by all team members.

1. After Institutional Biosafety Committee review, our project was deemed as BSL 1, however due to the lab we work in being BSL 2, the more stringent safety protocol applied to our daily work.
2. The ASU Environmental Health and Safety Office advised on disposal of fluorinated products as well as ensured hazardous material handling compliance.

Lab Safety

1. The first line of defense for lab safety is proper awareness, which begins with understanding location of emergency exits, eye wash stations, showers, fire extinguishers, and lists of relevant emergency response contact information.
2. All lab members were equipped with proper BSL 2 PPE, to include lab coats, gloves, goggles, long pants and closed toed shoes. A face mask is worn as necessary.
3. Sterile safety procedures and biosafety cabinet use was done in compliance with BSL 1/2 facilities.
4. Many chemicals used in lab present some hazard in one way or another, understanding chemicals being used and the hazards associated reduces the likelihood of a mishap.
1. Before working with any chemical, understanding the hazard symbols and location of Safety Data Sheets is imperative. Knowledge of the chemical leads to responsible use.
2. All chemicals are stored in appropriate locations, flammable chemicals are stored in flame lockers.
3. Hazardous PFAS chemicals (as well as our 2,2-difluorohexanoic acid) was stored in secondary containment, away from commonly used items.
4. Hazardous waste disposal of chemicals was taken into consideration, fluorinated chemicals received their own labeled capped waste jars and were not thrown away with the rest of the biohazardous waste.
5. Our biggest waste stream was solid and liquid media from bacterial cultures. Liquid waste was disposed of in jars, and equipment was rinsed with 10% Bleach and set aside for dishwashing.
6. Solid biological waste was disposed of in biohazardous containers and the bags were autoclaved before collection by EHS.