Lab safety

Our team attaches great importance to laboratory safety. Students are not permitted to enter the labs without passing the safety test, and no one is allowed to conduct experiments alone in the lab. White lab coats, lab gloves, and masks are required to wear when our team members work in the lab. After the experiments, used instruments and equipment are thoroughly sterilized before being discarded or put back into use, ensuring no leaks occur. Garbage classification is strictly enforced. For example, dry and wet garbage is separated before being abandoned. The laboratories are equipped with emergency handling devices such as fire extinguishers in case of accidents.

The safety of the product to the human body

Since our product is a food product, the first thing we had to consider was that the substances in the meal replacement shakes would not pose a health risk to humans. The first problem we faced was the selection of engineered bacteria. Although E. coli is the most commonly used engineered bacteria in the lab, it was obvious that E. coli could not be in our product. This is because pathogenic E. coli may cause food poisoning, acute diarrhea, extraintestinal infections and other gastrointestinal diseases if they enter the human body. In contrast, lactobacillus, as an essential probiotic, not only does not harm human health, but also possesses the function of promoting digestion and enhancing human immunity. Therefore, we chose Lactobacillus as the chassis bacteria. After literature research and discussion, we chose L. delbroeckii subsp. bulgaricus as the chassis bacteria. Because L. delbroeckii subsp. bulgaricus is the model strain in lactic acid bacteria and well studied. And this strain is widely used in food products with high fermentation capacity without producing metabolites that are harmful to health.

In addition, we had to make sure that there were no other substances in the product that could be harmful to human health. First of all, we selected the appropriate functional and flavor substances and ensured that no potentially harmful intermediates were produced in the synthesis pathway of these substances. For the 2x2 transfer switch part, we initially found two classical bistable switches as a backbone, the cl and cro type bistable gene regulation circuits and the tetR and lacl type bistable gene regulation circuits. However, the design of CI/Cro circuit is too cumbersome, and the tetR/lacl circuit requires the use of IPTG as a regulator, which is harmful to human body. Therefore, we abandoned the original design and turned to find other regulators that are not harmful to humans. We finally chose light and temperature during strain culture as the regulating factors for the bistable switch.

Prevention of leakage of engineered bacteria

As a qualified synthetic biology project, we need to ensure that the gene-edited engineered bacteria do not leak into the environment causing genetic contamination. Therefore, we designed the suicide switch system. After discussion and literature research, we chose the oxygen concentration change as the factor to activate the suicide switch. First, our chassis bacterium Lactobacillus is anaerobic, and the increase of oxygen concentration itself has an inhibitory effect on the growth of Lactobacillus. Secondly, compared with some other factors such as light variation, temperature variation, etc., oxygen concentration variation can encompass a wider range of applications. The optimum working temperature of lactic acid bacteria is 37°C. If we use temperature change as a factor to start the switch, the suicide switch will fail in an environment where the temperature is comparable to the working temperature of lactic acid bacteria (e.g. summer). If we use exposure to sunlight as a factor to initiate the switch, then the suicide switch will fail at night or in other shady environments.

Our suicide switch system consists of the FNR-based regulation of the HIP-1 promoter with the ccdA-ccdB toxin-antitoxin system. The antitoxin ccdA is downstream of the HIP-1 promoter, which can be repressed by binding to the FNR. Under hypoxic conditions, the FNR protein forms a homodimer that induces the initiation of downstream ccdA expression; under normoxic conditions, the FNR dimer is depolymerized, the initiation of ccdA transcription is repressed, and the toxin ccdB acts to kill the engineered bacteria.

In addition, the meal replacement machine we designed also contains devices to prevent leakage of the engineered bacteria. First, the meal replacement machine has a UV sterilization device in it. When the engineered bacteria have produced enough flavor and functional substances, the UV sterilization system can kill most of the engineered bacteria before the product is produced. The meal replacement machine also contains an air pump device, which can release a lot of gas in the system, which can make the original anaerobic fermentation system into an oxygen-rich environment, thus activating the suicide switch inside the engineered bacteria.

Go to our design page to learn more about our genetic circuit design!