Before we jumped into the actual experiments, all of us had to complete a mandatory online lab safety course run by our supervisor. To really hammer the points home, they also gave us a quick in-person refresher briefing right before we started any work. And to make sure everyone was actually paying attention, we even took a little quiz on the safety rules. We took lab safety super seriously throughout the whole project.

On the biosafety front, we were extra cautious, strictly following iGEM policies. Complied with the iGEM Safety Policies, we chose only E.coli as our experimental strain, which is a relative safe bacteria compared with other strains like Bacillus subtilis. Crucially, we never let any bacteria escape into the environment. Thanks to playing by all these rules, we managed to get through the entire experimental period without a single safety hiccup.

The safety training we received from our supervisor are the followings.

• Wear gloves, lab coat and googles(gloves not touching face or hair, also take off lab coat when leaving the lab)
• Keep long hair bundled up to avoid touching reagent.
• Wear closed-toe shoes to avoid reagent hurting feet
• Transfer solutions quickly when using explosive reagents（e.g. Na2S）, avoiding it reacting too much with oxygen in the air.
• No running or horseplay in the laboratory since they may bump into others conducting the experiment and this may cause reagent splashing.
• Wash hands both before and after the experiment to keep personal hygiene.

• There is card access control at the entrance of the laboratory so that unrelated personnel can not enter.
• We should use fuming cupboard when doing experiment with harmful substances. Keep viewing window not raising too high, keep wind speed inside around 0.5m/s to make sure toxic gas does not escape.
• The laboratory is equipped with safety shower and emergency eyewash in the lab, so that when someone’s body or eyes are contaminated with chemicals, he can wash himself quickly.
• Throw waste generated in the laboratory into the yellow biohazard waste bin so that they can be specially treated and disposed. As a result, it wont cause pollution to the environment.
• Throw the sharps, for instance, syringe needle and broken glass into Puncture Resistant Rigid Box.
• The laboratory is equipped with various kinds of fire extinguishers in order to respond to fires with different sources of ignition

• The safety of Escherichia coli BL21(DE3): This strain is a commonly used non-pathogenic host in laboratories. It lacks the genes for endotoxin synthesis and requires a plasmid antibiotic marker to maintain the expression system, thus it is safe.
• Protein toxicity assessment: SQR and FCSD are endogenous metabolic enzymes in the human body. We will detect whether their expression in Escherichia coli forms inclusion bodies or triggers host stress responses.
• Plasmid stability: It will be verified that the plasmid retention rate is > 95% after 50 consecutive passages to avoid horizontal transfer of antibiotic resistance genes.
• Drug application safety

Oral delivery system: Use enteric-coated capsules to protect proteins from degradation by gastric acid and verify the absorption efficiency of recombinant proteins by intestinal epithelial cells.

Target population definition: Patients with a BMI of 28 or above who have no response to traditional medications. Also, it is prohibited for minors to take it to avoid excessive medical treatment for those with mild obesity.

Price policy: The cost of E. coli fermentation is relatively low, so the pricing will be lower than that of similar biological drugs, thereby enhancing accessibility in middle- and low-income countries.

No animals were involved in this experiment.

1. Rosano, G. L., & Ceccarelli, E. A. (2014). Frontiers in Microbiology, 5, 172.
2. Szabo, C. (2018). Nature Reviews Drug Discovery, 17(3), 185-203.