The goal of Nuscent’s project is to engineer E. coli to produce pleasant-smelling compounds: menthol and linalool. The goal is for these genetically engineered microbes to be added to compost, where they will help to improve the odor associated with compost and deter vermin, which will improve the adoption of composting in urban settings. This project focuses specifically on engineering and testing the microbes in a lab setting, to determine I) if these compounds can be made, II) how long these microbes can continue producing in culture, and III) how long the engineered microbes survive in culture.

We used Escherichia coli strains DH5a and BL21(DE3), which are both commonly used in the laboratory and have a high transformation efficiency. These strains are considered to be non-pathogenic, which means they are unlikely to infect and survive in human hosts. In addition, these strains’ hazards are limited and can be handled in laboratories rated as biosafety level 1. Before the disposal of liquid bacteria cultures, the cultures were inactivated with an equal volume of 30% bleach and allowed to sit for 30 minutes before disposal.

Linalool and menthol are highly flammable chemicals, but when used properly in a chemical hood and stored in the flammables cabinet, they pose little risk to individuals. other hazardous chemicals include ethanol, Sybr Safe DNA Stain, and acetonitrile (for HPLC) all follow the same safety logic. These were used in the preparation of antibiotics, the extraction and preparation of DNA for cloning, and as HPLC solvents, which are all common laboratory procedures that have well established safety protocols.

All survey participant answers were kept anonymous.

Though outside the scope of the current project, we eventually aim to develop strains that could be added to urban compost containers. Nuscent would need to make sure to design and test containment procedures (e.g. kill switches) to make sure that strains could not escape beyond their intended compost containers. Institutional Biosafety Committee (IBC) approval would be required for testing of these aspects outside the laboratory. Since a fully developed project would involve the sale of engineered bacteria to the general public in a device that would sit in one's house, it is very easy to imagine a scenario where an untrained or unaware customer would accidentally spread the bacteria from its containment and spread it around a house or environment. However, these risks could be mitigated by encouraging good sanitation and hand washing techniques before and after using the product as well as the killing of the bacteria before the disposing of the compost, and development of biocontainment strategies (e.g. kill switches).

Saige Mcginnis – the Department Safety Officer for the Bioengineering Department – is one of the safety experts that has guided Nuscent through the project. All laboratory projects are routed through her to be vetted for safety. Our faculty advisor, Dr. Benjamin Woolston, supervises the lab sessions, acts as consultants on all major lab decisions, and steps in to mitigate any potential risks. The team also has a PhD advisor, William Gasparrini, who also acts as a lab supervisor. All of the lab supervisors are trained in all Northeastern safety procedures. Since undergraduate students are not permitted to be in the lab alone, the team was always accompanied by Gasparrini and Woolston, who were always able to troubleshoot any problems that arose. All students in the lab were required to go through an intensive lab safety training and lab tour before entering the lab space.

In the event of a spill or accidental exposure, the faculty advisor, the Northeastern Office of Academic and Research Safety, and the Northeastern University Police Department would be involved. Finally, our group consists of a diverse group of students with a wide range of skill levels, so those with more experience were able to assist those with less experience.

Several National and local standards govern laboratory safety practices at Northeastern. Nationally laboratory safety practices are governed by OSHA under General Industry standards 29 CFR 1910 Subparts I, J and Z. They are also linked here:

• https://www.osha.gov/laboratories/standards

Although Massachusetts has its own sector of safety regulations, these only apply to state and local workers. Private sector employees (IE Northeastern) are governed under Federal Regulations.

Finally the Northeastern Bioengineering Department has their own safety regulations which are linked here:

• https://bioe.northeastern.edu/wp-content/uploads/sites/2/pdfs/bioe-safety-plan.pdf

All safety steps Nuscent undertook were in line with the Northeastern laboratory safety policy, which is derived from the OSHA workplace standards for best lab practices and NIH recombinant DNA guidelines. Included in this policy:

• All lab members must take the necessary laboratory training taught by a licensed professional. These trainings provide an overview of the best practices to ensure safety in a lab environment as well as real life examples on what can happen if lab safety protocols are not followed properly.
• Conducting several lab training sessions within the first week of being in the lab in order to ensure every member of the team has hands-on experience with laboratory safety processes before experiments begin.
• A graduate student will be in the lab at all times in order to ensure all proper safety guidelines are being followed.

Figure 1: Airflow in the biosafety cabinet reduces exposure to harmful particles, ensuring a safe workspace for laboratory personnel.