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Safety is always a top priority in our projects. We fully studied the relevant laws and regulations of microbial safety, and also discussed industrial safety issues with experts and professors in the field of wastewater treatment, and finally set reasonable safety indicators for our experimental projects.

We strictly abide by the regulations of the government and the school concerning laboratory safety, and carry out experimental activities under the guidance of the official. Every wet Lab team member has completed professional safety training prior to entering the lab, including:

Primary PI Jiazhang Lian is our main experiment instructor, with rich experience in microbial experiment operation, and can give us suggestions at any time. Tanglei Zhang, a doctoral student, as our instructor, will guide us to carry out experimental operation and ensure the safety of the experiment.

During the experiment, we complied with the Biosafety Law of the People's Republic of China and considered biosafety issues from the aspects of public health, biological resources, ecosystems, and biodiversity. We used Escherichia coli and Saccharomyces cerevisiae in the experiment, both of which are common laboratory model strains. Saccharomyces cerevisiae is Generally Recognized As Safe (GRAS), and its biosafety is fully guaranteed. At the same time, we will also carry out genetic modification in accordance with conventional operations to avoid biosafety risks. Finally, we also designed measures such as biological suicide switches to further enhance safety precautions.

In order to apply engineered yeast to actual industrial production, we need to pay special attention to the safety of GMOs. Since the application object of our engineered yeast with lanthanide rare earth adsorption capacity is the wastewater containing a large number of metal ions such as rare earth ore leaching solution, rare earth mine wastewater or industrial wastewater, we hope to select a metal ion as a "signal" to control the suicide switch of engineered yeast and prevent cell escape.

In the previous investigation, we found that most sources of mine or industrial wastewater often contain a certain concentration of copper ions, which is related to the wide abundance of copper ions in the Earth's crust. Taking conventional industrial wastewater as an example, the Ministry of Ecology and Environment, PRC limits the discharge standard of total copper in conventional industrial wastewater pollutants to 1.0 mg/L[1], while the average concentration of copper ions in ordinary fresh water in the natural environment is about 3 μg/L, and only about 0.25 μg/L in seawater [2]. The large difference between the two allowed us to design a yeast suicide mechanism based on the copper ion concentration response, so that engineered yeast can spontaneously die after leaking out of the membrane bioreactor for wastewater treatment into the natural environment.

We introduced the RelE/RelB ( BBa_K185047 / BBa_K185048) system into engineered yeast, a toxin-antitoxin system that functions in Saccharomyces cerevisiae: RelE toxin is an endonuclease whose expression leads to strong growth defects in yeast; However, the expression of antitoxin RelB can be directly combined with RelE to neutralize its activity, reduce toxicity and prevent cell death [3].

In order to achieve the function of cell suicide, we introduced the copper ion inhibited promoter CTR3[4], which is an endogenous regulatory element of Saccharomyces cerevisiae. When the concentration of copper ions in the environment is high, copper ions bind to transcriptional regulatory factors and down-regulate the expression of its downstream RelE toxin gene by inhibiting the CTR3 promoter. In addition, we also used the weak constitutive promoter CYC1 to activate the expression of antitoxin RelE to avoid accidental cell death caused by the leaking expression of the CTR3 promoter. In this way, we can ensure that the expression of toxin RelE is inhibited and yeast can survive in the rare earth ore leaching solution rich in copper ions. Once released into the natural environment, the concentration of copper ions is too low to inhibit the expression of the toxin RelE, and the engineered yeast will die based on its toxicity to yeast.

In the end, we added a liquid waste treatment module for biosafety protection at the very downstream of the hardware design of the rare earth element biomining platform. Considering that the waste liquid treated by the membrane bioreactor flows downstream with a lower pH value and may contain accidental leakage of engineered yeast, direct discharge will bring environmental pollution and biosafety issues, so we set up a neutralization pool for pH regulation and a UV chamber for sterilization in the waste liquid treatment module. After the waste liquid treatment module treatment, indicators test qualified waste liquid, can be finally discharged.