Introduction
Safety protocols serve as the inviolable foundational framework for any scientific research project—especially in synthetic biology, where engineered microorganisms, recombinant materials, and experimental reagents are core to operations. While such biological tools drive innovation in the field (and are central to our project), their potential risks demand rigorous safety oversight and proactive risk mitigation. Against this backdrop, our project strictly complied with international guidelines (e.g., iGEM Safety Policies) and institutional laboratory regulations, with every step of chassis organism manipulation (e.g., E. coli BL21(DE3) culture), recombinant plasmid handling (e.g., piGEM25 series plasmid construction), and chemical reagent use (e.g., NaOH for biomineralization) aligned with highest safety and reliability standards. Throughout the entire experimental cycle, we implemented these protocols systematically: from sterile inoculation of bacterial cultures and biosafety cabinet-based plasmid transformations, to proper disposal of biohazardous waste and chemical residues. This meticulous adherence ensured not only the safety of researchers and the laboratory environment but also the standardization and reproducibility of every experimental procedure—critical for validating our project’s results.
Safe Project Design
1. Project Design
Our project addresses the critical challenge of plastic pollution by developing a closed-loop solution for PET recycling, encompassing two integrated components: efficient enzymatic degradation of PET and high-value upgrading of its degradation products.
In the first component, we tackled the limitations of free PET-degrading enzymes—poor recoverability and low stability—by engineering an immobilized dual-enzyme system (TfCa and Tfh) anchored to functionalized magnetic nanoparticles. Using K5C (a fusion protein of ELP and SpyCatcher) as a template, we synthesized the magnetic carrier K5C@Fe₃O₄ via biomineralization. To achieve ordered enzyme immobilization, we leveraged two orthogonal covalent self-assembly systems: SnoopTag / SnoopCatcher and SpyTag / SpyCatcher. We successfully constructed three plasmids (piGEM25_01, piGEM25_02, piGEM25_03) for expressing the required proteins, verifying their soluble expression and purification. Through optimization, we identified 3.0 M NaOH as the optimal mineralization concentration, and a sequential immobilization strategy (TfCa first, then Tfh) yielded the highest PET degradation efficiency, with the product TPA reaching 1.2 mM. Notably, the system retained 61% of its initial activity after 10 recycling cycles, demonstrating robust operational stability and reusability—key for practical scalability.
The second component focused on upgrading and recycling PET’s degradation products, ethylene glycol (EG) and terephthalic acid (TPA), to close the recycling loop. For EG, we employed a "bioinformatics-guided design + wet-lab validation" approach. Comparative genomics analysis of E. coli BL21 (DE3) against MG1655 (a known EG-metabolizing strain) revealed >99% shared core orthologous groups and structural collinearity in EG-metabolic genes (e.g., gcl, fucO), confirming BL21 (DE3)’s latent EG-metabolizing potential. We then reconstructed its genome-scale metabolic model (supplementing the full "EG→PEAs" pathway) and used flux analyses (FBA, FVA) to identify key regulators (fucO, aldA, gcl) for strain engineering. In wet experiments, we constructed plasmid piGEM25_04 (carrying these key genes) and transformed it into BL21 (DE3) to generate the EG-BL21 strain, which grew on EG as the sole carbon source (OD₆₀₀ ≈ 0.8 after 72 h, consuming ~22 mM EG). Co-transforming piGEM25_07 (for PEA monomer synthesis) and piGEM25_08 (for monomer activation/polymerization) yielded the PEA02 strain; Nile red staining and fluorescence detection (strong emission at 610 nm) confirmed EG was upgraded to PEAs, a biodegradable plastic.
For TPA, we targeted high-value conversion by constructing plasmid piGEM25_09 (carrying TphA1, TphA2, TphA3, and TphB), achieving soluble enzyme expression in BL21 (DE3). At an optimized OD₆₀₀ of 60, HPLC detected 1250 μM protocatechuic acid (PCA)—a pharmaceutical intermediate—from TPA. This not only mitigated microbial metabolic inhibition caused by TPA accumulation but also enhanced its resource value.
Together, our work demonstrates a holistic PET recycling system, with future efforts focusing on optimizing enzyme ratios, linker structures, and strain performance to advance real-world applicability.
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 China.[2] 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 graduaOur team is composed of undergraduate and graduate students. At the outset of the team's establishment, two graduate students, Qifa Jiang and Ruming Liu, 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.