During this year's iGEM project at UM-Macau, we used a biosafety level 1 lab with an open bench, biosafety cabinet and chemical fume hood. In addition, the lab and the surrounding area were equipped with safety equipment as shown in the figure below.

Before participating in laboratory activities, to make sure UM-Macau members know the basis of laboratory rules and how to discard waste correctly, all wet lab group members need to complete the safety training provided by the University and pass the examination. According to standard practice, laboratory personnel are always required to read Material Safety and Data Sheets (MSDS) prior to experimentation, and at our university we can use a digital platform for this function by using our student’s account. Moreover, our project will be monitored and assisted by two supervisors and a biosafety officer (technician) from Faculty of Health Sciences, when we have the wrong operation.

In our faculty, after formally entering the laboratory we need to be fully aware of laboratory safety protection, personal safety protection equipment and emergency response as well as to understand the Global Harmonized System (GHS) and National Fire Protection Association (NFPA). Laboratory personnel must use proper Personal Protective Equipment (PPE), such as lab coats and gloves, to reduce accidents and injuries in the workplace. When handling waste generated during experiments, our wet lab group members should strictly sort the waste into appropriate waste bins (including sharp waste bins, needles and syringes should be disposed of in sharp waste bins, and broken glass should be disposed of in broken glass bins), and fill out the waste disposal form posted on the wall of the room after the waste has been disposed of. After the experiment, all the chemical waste, bacteria or cell will be inactivated to avoid their pollution or spreading in the environment.

In our project, we have used the DH5-alpha and BL21 Escherichia coli strain as our engineered organism. E. coli bacteria are common in the natural environment, especially living in the intestines of creatures. Also, it has been the most common bioengineered organism in laboratory studies. However, nonstandard operations might cause diseases to the lab user. Therefore, strictly following the experiment guidance and disinfecting in time is important to the our team members’ health. All our experiments will be progressed inside the laboratory and we will kill the bacteria with disinfectant or autoclave after we finish the experiment. Morever, we also have the safety module to make sure we will not release them into the environment, the details will show in the description of “Safety of our functional module”.

The safety of the engineered bacteria in the environment and in patients themselves are also important considerations in the realisation of the most important functional modules of our project. Our safety module is divided into two main parts: in vitro and in vivo.

The toxin/antitoxin system, widely used both inside and outside of the iGEM competition, is a proven and robust system for building flora suicide switches. When our implanted intestinal flora is expelled from the body, we ensure that it will not be potentially contaminated with other natural environmental microorganisms due to the uncontrolled horizontal gene transfer of our additional adhesion and releasing modules to the intestinal flora. Based on the human body temperature of approximately 37° Celsius, we chose a temperature controlled suicide switch based on Part:BBa_K3247005 RNA thermometer NoChill-06. When our engineered bacteria are excreted from a patient's body, the module that manipulates the expression of the antitoxin stops due to the temperature deviation from 37° Celsius, terminating the inhibition of the toxin, and realising its suicide in the natural environment after it is expelled.

In vivo, a safety module is also be constructed independently from the in vitro safety module, to ensure that the engineered bacteria that we put into the module do not become the dominant species with potential negative effects in the gut and destabilise the patient's intestinal ecosystem due to the additional modules added. Unlike the in vitro safety module, the in vivo safety module is based on the quorum sensing of bacteria and the phenomenon of quorum quenching. When the population density of the bacteria exceeds the threshold, the bacteria needs a signalling molecule called acylated homoserine lactone (AHL) to regulate the proliferation signalling pathway at a certain concentration. Based on the species of probiotics we choose to put into practice and the specificity of AHL signalling molecules of different strains, we will select AHL inhibitor molecules based on the strains we choose to put into practice to achieve the control of the population density of the bacterial colony under the mediation of the concentration of AHL itself. The AHL inhibitor controls the population density of AHL without killing it all at once, but at the same time stabilises it so that it can continue to perform its therapeutic function against celiac disease in patient's small intestine.

In summary, by building the safety modules in vitro and in vivo, we will be able to minimise the potential hazards present in our engineered bacteria.