Parts Contribution

A series of functional plasmids and core components have been successfully constructed, providing key support for PET degradation and product conversion:

In the PET degradation process, plasmids piGEM25_01/02/03 were constructed to enable the expression of magnetic carrier protein (K5C) and dual enzymes (TfCa, Tfh), achieving ordered immobilization and efficient recovery of the enzymes;
In the EG conversion and PEA synthesis process, plasmids piGEM25_04 (containing gcl/garR/glxK) and piGEM25_07/08 (PEA synthesis pathway) were constructed, verifying the soluble expression of key genes and the EG utilization capacity of the strain;
In the TPA conversion process, plasmid piGEM25_09 was constructed to express TphA1/A2/A3/B enzymes, realizing the conversion of TPA to PCA.

All plasmids were verified to be correct by PCR and sequencing.

Parts

Model Contribution

By forming a "model prediction - wet experiment verification" closed loop, this study bridges computational simulation and practical experiments:

First, Bioinformatics Project 1 took E. coli MG1655 as the reference, confirmed E. coli BL21(DE3) has EG metabolic potential via whole-genome alignment, homologous clustering and collinearity analysis (it retains related genes with good conservation), directly supporting BL21(DE3) as the chassis strain for subsequent wet experiments and laying a foundation for metabolic model construction.
On this basis, the second project integrated model reconstruction and flux analysis: it selected iB21_1397 as the baseline, supplemented 6 key reactions and 5 metabolites in the EG→acetyl-CoA→PEA pathway, corrected 1 erroneous reaction, constructed the complete-pathway GSMM (iBL21_EG-PEA) (verified to operate normally), and identified 6 core regulatory genes (gcl, glxR/garR, glxK, fucO, aldA) via FBA/dFBA/FVA/FDCA.

These results guided 3 groups of wet experiments (constructed baseline plasmid piGEM25_05, optimized plasmid piGEM25_04, expanded plasmid piGEM25_06); experiments confirmed the model-proposed minimal combination (gcl-garR-glxK) had comparable performance to the traditional four-gene combination, and fucO-aldA was non-rate-limiting—verifying the model and reducing trial-and-error costs.

Model

Education Contribution

A diversified science popularization system has been established to break down the cognitive barrier between scientific research and the public:

Online promotion: We popularize plastic classification and project progress through Xiaohongshu operations, co-develop the picture book E. coli Magic Factory with Jilin University, and design the game Polymer Rebirth, transforming EG metabolism and PEA synthesis into engaging content;
Offline activities: We carry out primary school "Plastic Metamorphosis" classes, waste PET handicraft activities in hospitals, and laboratory open days (where middle school students visit enzyme reaction devices), and conduct interdisciplinary exchanges with medical schools and software schools;
Practical case integration: Combining research cases from Sangzhi Nature Reserve and waste treatment plants, we convey the concept that "plastic is a resource rather than garbage."

This system covers audiences of different age groups and enhances public awareness of the environmental applications of synthetic biology.

Education

Hardware Contribution

Low-cost detection hardware and methods have been developed to support experimental verification and technology implementation:

2. A gold nanoparticle-based EG detection method was established, leveraging the linear relationship (R²=0.988) between the SPR peak intensity at 525nm and EG concentration. Combined with the self-developed system, it enables quantitative detection of EG, providing a convenient tool for evaluating the EG utilization efficiency of engineered bacteria.

Hardware