Our laboratory work was conducted at the Synthetic Biology and Biofabrication Laboratory within National Taiwan University's Department of Biochemical Engineering. This lab is a Biosafety Level 1 facility managed by Professor Hsuan-Chen Wu, only permitting the use of agents that, at most, pose minimal health risks upon exposure. All safety protocols were also carefully adhered to under the supervision of graduate students and trained lab technicians. By working with common E. coli strains such as NEB Stable and DH5α, we minimized any biosafety or health concerns that might have arisen from using other unapproved E. coli strains. Disinfection with bleach and alcohol for glassware, the use of laboratory gloves, and constant cleaning with rubbing alcohol ensured the minimization of any other risks of biosafety.

To abide by basic biosafety regulations, we washed our hands coming in and out of the laboratory while protecting our eyes, mucous membranes, open cuts, or wounds from any contact with biohazard materials. We also refrained from eating or drinking within the lab vicinity. We tied back long hair and disinfected equipment with 70% ethanol before use. We disinfected all disposable pipette tips and tubes by soaking them in a 10% bleach solution for 20 minutes before disposing of them. We disposed of any containers used for growing bacteria into a biohazard waste bin, which was autoclaved afterward, and we cleaned our workspace with 70% ethanol after every use.

Before beginning lab work, we developed experiment plans that our instructors and advisors carefully reviewed and approved to mitigate unnecessary risks. Alongside safe and well-organized lab practices, we completed four hours of online training provided by the Taiwanese government's Taiwan Centers for Disease Control. This training covered laboratory safety, risk groups, risk assessment, biosafety program management, biosecurity, and other relevant topics. To organize all of our plans, they were uploaded to a shared drive and documented in a loose-leaf binder kept in the lab to ensure that instructors and team members were informed of experiment details and schedules. We followed all general lab safety guidelines of the Synthetic Biology and Biofabrication Laboratory, using equipment with caution. Liquids were all stored beneath eye level. Microorganism-related safety procedures were conducted in laminar flow hoods, and glassware was disinfected with diluted bleach and 95% alcohol after use. In the event of bacterial contact with our workspace, rubbing alcohol or UV light treatment was promptly applied to the affected areas and cleaned thoroughly.

The bacterial strains we used in experimentation are commercial DH5ɑ, and the minCDE knocked-out E. coli BLR(DE3). The former is for plasmid amplification and storage while the latter is for protein expression and implementation. E. coli DH5ɑ and BLR(DE3) strains pose little harm to adult humans even in the case of accidental environmental contamination and skin contact. Nevertheless, we still closely followed general lab safety procedures to minimize danger to ourselves. Since our product is to be applied to the environment, we want to mitigate its infecting or invasive effects on the environment—our choice of the specially engineered E. coli BLR (DE3) strain can be filtered to retain only non-replicating minicells (Baker et al., 1979). Upon completion of any experiments directly involving the use of bacterial strains, all waste is disposed of in biohazard waste bins.

In the future, through the use of ClearColi™ in the future, we will also be able to get rid of the extracellular toxins that E. coli BLR(DE3) still contains (Mamt et al., 2013). This lack of surface toxins will allow for more ease of mind when using the product, especially for consumers who are not well-informed on microorganism biosafety. This further reinforces our commitment to biosafety for consumers.

The active ingredient we are synthesizing, borneol, is admittedly irritating in high concentrations and at low purity and has a low risk in the context of our project. Our biosynthesis uses enzymes that produce L-Borneol, which is the safest form of the many borneol isomers; furthermore, the concentration of borneol produced enzymatically is low, causing less irritation to the human skin (Vainer et al. 2023). Other than L-Borneol, ingredients in the fragrance pose no threat to the safety of its surroundings as we plan to use commercial fragrances that the Taiwan Food and Drug Administration has approved. Due to the nature of minicells which lack a method of replication, L-borneol does not accumulate to high concentrations in the product, which offers a balance between the longevity of the fragrance’s effects. Finally, the product never left containers that were approved to handle laboratory-grade chemical compounds.

We plan for biosynthesized L-borneol to attach to odor receptors 9 and 49 of common mosquito species, which affects the mosquito through neural signals that cause disgust to the smell (Vainer et al. 2023). Based on the review of scientific literature on the subject, borneol’s effects on the mosquito are temporary and do not cause physical or mental damage. Therefore, our product will not cause ecosystem-wide detriments as it does not directly cause the widespread death of mosquitoes.