Safety

Because we are working to create a project that will be consumed by other people, we take safety very seriously. We extensively researched the safety and possible risks of both the organism we are modifying, Lactococcus lactis cremoris, and the four compounds we are synthesizing.

Also, through our talks with US government officials about the approval process for this project, we were able to compile a guidebook to help other American teams. With the knowledge and insights we gained from interviews with FDA regulators and industry professionals, we hope to increase accessibility for this type of project for future iGEM teams.

Lactococcus

Lactococcus is a BSL-1 bacteria, which is important because not only does that allow us to work on it in our lab, but it also means that it poses no significant threat to humans. Furthermore, Lactococcus lactis cremoris is frequently used in the cheese industry for the acidification of milk. Thus, we can be certain that this organism creates no potentially harmful compounds and is safe to eat. In addition to the fact that some of the pathways we tried to express were only documented in Lactococcus, we chose this bacteria because of its rigorously tested safety.

Synthesized compounds

When handling any of the chemicals we produced in the lab, all of the participants were briefed on the possible side effects of inhaling, swallowing, or coming in contact with the chemical. They were required to wear gloves while measuring and dissolving the chemical in water for the smell test.

Theobromine

3,7-dimethylxanthine, commonly referred to as theobromine, is one of the most highly concentrated chemicals in chocolate, with about ½ gram per 100g of dark chocolate. Clinical studies on the effects of theobromine consumption showed it having an effect similar to caffeine, but only at a dose of 700 mg or greater. This means that the average person will only be affected by theobromine when consuming more than 140 grams of chocolate, and these effects would only mimic that of consuming ~70 mg of caffeine (Baggott et al. 2013).

2-Phenylethylamine

2-phenylethylamine is a chemical in chocolate that doesn’t affect the smell or taste but is instead attributed to the feeling of elation that some experience when consuming chocolate. When consumed in large quantities, the 2-phenylethylamine that binds to the dopamine receptors in the brain can cause a “high” which can be attributed to symptoms like muscle spasms and confusion (Horne et al. 2021). Due to its adverse effects on aquatic life, 2-phenylethylamine cannot be flushed down the drain or thrown away, so any equipment or gloves that came in contact with the chemical or solution it was dissolved in were properly labeled and stored in our biohazard storage to be properly disposed of by CLYM Environmental, an LLC that specializes in disposing biohazards.

HDMF (Furaneol)

There is about 100mg of 4-methyl-2,5-dimethylfuran-3-one (HDMF) per 100g of dark chocolate. The only clinical studies on the effect of HDMF showed that inhaling it could increase the heart rate and blood pressure slightly, alongside a slight decrease in oxidative stress levels in mice (Yokoyama et al. 2020). HDMF has long been GRAS (Generally Recognized as Safe) by the FDA due to its presence in many different types of fruit, so its presence in chocolate will not be harmful to consumers.

Pyrazine

Pyrazine only exists in trace amounts in chocolate; roughly 500 micrograms per 100 grams of dark chocolate. Some research done in the mid-late 2000s showed pyrazine to have some pharmaceutical effects as an anti-thrombogenic and antimicrobial, but due to its very low concentration, it’s unlikely that consumers of chocolate with experience any effects related to pyrazine (Janssens et al. 2019).