Introduction
Safety regulations are the foundational framework for any research project. In synthetic biology, although the application of engineered microorganisms is increasingly widespread, careful consideration of safety measures remains critical. Therefore, in this project, we strictly adhered to established safety standards to ensure that the handling of chassis organisms and reagents was safe and reliable. Throughout the experimental cycle, we consistently followed laboratory safety protocols to ensure the safety and standardization of each procedure.
Safe Project Design
1. Project Design
Our work covers two parts.
In the first part, the specific detection of four types of microplastic PP, PS, PET and PLA by anchoring peptides. We constructed four plasmids that each express an anchoring peptide labeled with His-tag and GFP and transformed it into E. coli (BL21 DE3). We purified the anchored peptide linked to GFP and observed its affinity and specificity to the corresponding type of microplastic by fluorescence microscopy. Then, GFP was removed by TEV enzyme digestion, and the four anchoring peptides were further purified. Finally, we combined each of the four anchoring peptides with colloidal gold for the identification of the corresponding microplastics by colorimetric assay.
In the second part, we completed the upcycling of PET in two steps. Firstly, to promote the biodegradation of polyethylene terephthalate (PET), we adopted an innovative strategy to expose key enzymes to the cell surface of E. coli (BL21 DE3) through a surface display system. Specifically, we designed a fusion protein consisting of sequences of membrane proteins Lpp-OmpA, Z1-PETase, and MHETase, and sequentially attached these sequences to the expression vector pET30a. Through this design, we can degrade PET into an intermediate product TPA (terephthalic acid), which can easily enter into the cell and then be modified by a multi-enzyme system.
Secondely, we convert TPA into a high value product vanillin through a multiple enzyme system, we transformed three plasmids into BL21 (DE3): The first plasmid contains four genes responsible for the degradation of TPA to PCA, which are three genes encoding three subunits of terephthalate 1, 2-dioxygenase and one gene encoding 1, 4-dicarboxylate acid dehydrogenase. The second plasmid contains catechol O-methyltransferase genes and carboxylate acid reductase genes that mediate the conversion of PCA to vanillin. In addition, the third plasmid contains genes that encode the cofactor of phosphopantetheinyl transferase. Together, these seven genes complete the cascade reaction that converts TPA into vanillin.
2. Kill Switch
In the design of the kill switch, we adopted the scheme of the last UESTC-China team1.
At the core of the device is the utilization of the E. coli hokD gene, whose overexpression disrupts cell membrane integrity, leading to cell death. Research has demonstrated that the hokD gene can be engineered to design effective bacterial suicide systems. To prevent unintended cell death due to basal hokD expression, the project employs a two-tier regulation system. The hokD gene is placed under the control of the T3 promoter (phi4.3), while the T3 polymerase, necessary for activating the T3 promoter, is regulated by the pBad promoter. The pBad promoter, in turn, is repressed by the AraC protein in the presence of glucose. In glucose-rich conditions, AraC inhibits the expression of T3 polymerase, thereby preventing hokD expression and allowing normal bacterial growth. When released into a glucose-depleted environment, AraC repression is lifted, enabling T3 polymerase to drive hokD expression, which triggers the cell death pathway and initiates the bacterial self-destruction mechanism.
3. Hardware Protection
We incorporated UV lamps into the hardware. In the event of a leak, the switch can be turned on to perform UV disinfection both inside and outside the device, ensuring that no personnel in the vicinity are exposed to the radiation.
Laboratory Safety
1. Laboratory Risk Assessment
iGEM is the Heart of Synthetic Biology. In the process of experience, we must face considerations related to gene safety. Hence, in preparation for the experimental procedures, we conducted an exhaustive review of the 'Genetic Engineering Safety Management Measures' mandated by the Central People's Government of the People's Republic of China2. We pledge to execute the experiment with unwavering adherence to these regulations to guarantee the safety and reliability of our experimental procedures.
We use non-toxic E.coli as the chassis organism for this experiment project. E.coli is a strain listed on iGEM's official website, so it has high safety and recognition, low pathogenicity, and no public health risk. According to the "pathogenic microbiology laboratory Biosafety management Regulations", E.coli belongs to the third class of pathogenic microorganisms, that is, can cause human or animal diseases, but under normal circumstances do not pose serious harm to people, animals or the environment, the risk of transmission is limited, rarely cause serious diseases after laboratory infection, and have effective treatment and prevention measures of microorganisms.
In the gene editing module, we studied the risks of gene drives, and made sure we had eliminated it in our project.
Finally, we evaluate the safety level of the laboratory, and the evaluation result is: a biosafety level laboratory. Our laboratory is a basic laboratory, often for basic teaching and research laboratories, dealing with risk level 1 microorganisms.
To sum up, the experimental project and the laboratory have a high safety factor.
2. Experimental Operation Training
Our team is composed of undergraduate and graduate students. At the outset of the team's establishment, two graduate students, Zhong Linling and Liu Ruming, who are rich in experimental experience, conducted biological safety training for undergraduate students. The training content encompassed relevant safety concepts, basic emergency procedures, and the handling of microorganisms. In the subsequent experiments, the graduate students also provided practical guidance and supervision to guarantee the safe progress of the experiments.
3. Laboratory Requirements
(1) laboratory entry disinfection, not eat in the laboratory, allowed to drink beverages, if you want to eat, please eat outside the laboratory or the canteen;
(2) Make a plan before the experiment, conceive the process well, it is best to write it down to reduce the probability of error;
(3) The experiment should be carefully conducted, strictly in accordance with the operating procedures, and pay attention to changing the habits of life;
(4) Wear gloves to disinfect before operating in biosafety cabinet, to prevent contamination of other bacteria;
(5) The experimental operation must wear a lab coat, mask, and gloves;
(6) After the experiment is completed, the experimental platform must be cleaned, the instrument should be placed in the designated place, the pipette should be adjusted to the maximum range, the waste liquid cylinder should be dumped and washed, and the power supply of the instrument should be turned off.