May: Project Launch and Basic Preparation
Week 1
After the recruitment of the campus iGEM team, 13 cross-college members (from the School of Life Sciences, School of Medicine, School of Public Administration, and School of Mechanical and Electrical Engineering) formed the UESTC-China 2025 Team. The first offline meeting clarified the division of responsibilities: the student team leader coordinates overall work, the experimental team is divided into dry and wet lab groups, the hardware group is responsible for the development of detection equipment, and the HP (Human Practices) group focuses on social connection. Meanwhile, the core research topic—"PET Biodegradation and High-Value Conversion of EG to PEAs"—was confirmed with PIs (Principal Investigators), responding to the "resource utilization" requirement in the 14th Five-Year Plan for Plastic Pollution Control.
The Dry Lab Group downloaded genomic data of BL21 (DE3) and MG1655 from NCBI and BiGG databases; the Wet Lab Group inventoried laboratory reagents and confirmed the list of TfCa/Tfh enzyme genes and pET-30a vectors required for PET degradation; the HP Group collected policy documents on plastic pollution control and planned to conduct a background sharing session next week.
Week 2
The entire team launched a literature research campaign: the Dry Lab Group intensively read literature on comparative genomics and metabolic model reconstruction, and conducted a trial run of QUAST quality control for the MG1655 genome; the Wet Lab Group focused on the "magnetically immobilized dual-enzyme system for PET degradation" scheme, referring to the technical route of "K5C@Fe₃O₄ carrier + covalent immobilization of dual enzymes"; the Hardware Group investigated the cost of traditional spectrophotometers (at the 10,000-yuan level) and initially planned a low-cost alternative scheme of "Arduino + LED array + spectral sensor."
The HP Group completed a sharing session on "Current Status of Plastic Pollution and Technology Comparison," focusing on the non-degradable characteristics of PET and secondary pollution from traditional landfilling/incineration, and emphasized the project's dual goals of "degradation + upcycling."
Week 3
After three special discussions, a monthly implementation framework was determined:
- Dry Lab: Complete comparative genomics evaluation and metabolic reconstruction from May to June, and conduct flux analysis in July;
- Wet Lab: Conduct PET degradation in June, EG detection and first-stage metabolism in July, and second-stage EG metabolism and PEA synthesis in August;
- Hardware: Design the scheme in June, purchase and build equipment from July to August, and optimize the program in September;
- HP: Launch popular science activities in July, conduct field research in August, and summarize results in September.
During the same period, the team communicated with Chengdu Maike Macromolecular Materials Co., Ltd. and learned about the industry's core concerns—"cost control" and "enzyme/strain stability"—which provided a practical basis for subsequent experimental design.
Week 4
The Dry Lab Group completed preprocessing for Topic 1: unified gene annotation of BL21 (DE3) and MG1655 using the Prokka tool, solving the problem of inconsistent naming formats in raw data; the Wet Lab Group ordered TfCa/Tfh gene synthesis fragments, prepared and sterilized LB medium, and prepared for plasmid construction in June; the Hardware Group completed 3D modeling of the motor base and cuvette base using SOLIDWORKS, and selected black nylon material to prevent light reflection interference in spectral detection.
The HP Group compiled a "PET Treatment Technology Comparison Table," clarifying the project's core advantages—the mild conditions and high-value product characteristics of "bioenzymatic hydrolysis + engineered bacteria conversion"; the Wiki Group started learning basic frameworks and familiarized themselves with MediaWiki typesetting rules.
June: Theoretical Foundation and Preliminary Experiments
Week 1
The Dry Lab Group launched genome analysis: evaluated the assembly quality of BL21 (DE3) and MG1655 genomes using QUAST, confirming no abnormal fragments or contaminated contigs, and consistent continuity with public references; completed whole-genome alignment via progressiveMauve and initially observed that EG metabolism-related genes (gcl, fucO) exist in both strains.
The Wet Lab Group constructed PET degradation plasmids: amplified piGEM25_01 (expressing K5C protein, containing ELP and SpyCatcher) via PCR, and gel electrophoresis showed the expected band at approximately 750bp, which was sent for sequencing verification; prepared 0.1M FeCl₃ solution for magnetic carrier preparation.
The Hardware Group designed a PCB circuit (including 6 narrow-band LEDs and an HEF4017BT counter), generated a 2D rendering using Altium Designer, and determined the pin distribution; the HP Group contacted Wuhou District Urban Resource Treatment Center and Sangzhi Landfill, and initially confirmed the research schedule in August.
Week 2
The Dry Lab Group completed homologous clustering: identified 3,920 orthologous groups using OrthoFinder, of which 3,901 were core groups shared by the two strains, 3,755 were single-copy orthologs, and only 19 were strain-specific groups (57 genes, accounting for ~0.7%). This confirmed that the coding difference between BL21 (DE3) and MG1655 is limited, and both have EG metabolism potential.
The Wet Lab Group obtained correct sequencing results of piGEM25_01, transformed it into BL21 (DE3) competent cells, and spread them on Kan-resistant plates; induced K5C protein expression with 0.5mM IPTG and planned purification next week.
The Hardware Group sent the PCB board for prototyping and purchased Arduino UNO R3 and Mega2560 development boards simultaneously; the HP Group improved the "Educational Popular Science Plan" and clarified that campus classes and hospital popular science activities would be launched first in July.
Week 3
The Dry Lab Group screened baseline metabolic models: compared 5 models including iECD_1391 and iEC1356_Bl21DE3, and finally selected iB21_1397 (containing 1,943 metabolites and 2,741 reactions, supporting the COBRApy tool) to lay the foundation for supplementing the "EG→PEAs" pathway.
The Wet Lab Group purified K5C protein: adopted a two-step purification method using ITC thermosensitive purification, and SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) showed a clear band at 55kDa; prepared K5C@Fe₃O₄ magnetic carriers (mineralized with 3.0M NaOH, pH=9.0) with a magnet recovery rate ≥90%.
The Hardware Group received the PCB prototype and checked for no pin deviations; the HP Group conducted the first popular science activity—"The Metamorphosis of Plastic" class—at Pidu District People's Hospital, explaining medical plastic classification and PET characteristics to the children of hospital staff.
Week 4
The Dry Lab Group supplemented the "EG→PEAs" pathway: extracted 6 key reactions from KEGG, BiGG databases and literature, added 5 metabolites (including ethylene glycol, (R)-3-hydroxybutyryl-CoA, PEAs, etc.), and updated GPR associations (e.g., fucO gene corresponding to glycolaldehyde dehydrogenase). The final model contained 1,948 metabolites and 2,747 reactions.
The Wet Lab Group constructed a "magnetically immobilized dual-enzyme system": adopted the sequential immobilization method of "first immobilizing TfCa, then Tfh". After 12 hours of reaction at 37℃, the TPA yield reached 1.2mM (30% higher than the co-immobilization method); the enzyme activity retention rate after the first recovery was ≥95%.
The Hardware Group tested Arduino dual-board serial communication (UNO-TX→Mega-RX1, UNO-RX←Mega-TX1, common GND), and the Serial Monitor could stably transmit data; the HP Group compiled a "PET Biodegradation Technology Roadmap" to prepare materials for subsequent popular science activities.
July: Theoretical Deepening and Experimental Connection
Week 1
The Dry Lab Group conducted Flux Balance Analysis (FBA): set the goals of "EG as the sole carbon source" and "maximizing biomass". The results showed that the reaction fluxes of EG uptake (EX_eg_e), glyoxylate cycle (GLXCL), and glycolysis (ENO, PGM) were relatively high, confirming that EG could normally flow to acetyl-CoA and provide precursors for PEA synthesis.
The Wet Lab Group launched EG detection: prepared gold nanoparticles (boiled 1mL chloroauric acid + 49mL water, added 2mL 1% sodium citrate, heated for 30min, and the solution turned wine red); prepared 0, 10, 20, 30, 40, 50, 100, 150mM EG-LB solutions and planned to construct a standard curve.
The Hardware Group purchased AS7341 spectral sensors and 42-type stepper motors, assembled the frame, fixed the nylon base and acrylic frame with M5 screws, and the cuvette slot was adapted to 1cm quartz cuvettes; the HP Group conducted "Plastic ID Card" popular science activities at the primary school affiliated to the university, teaching children to identify the "No. 1" mark at the bottom of PET bottles.
Week 2
The Dry Lab Group completed Dynamic Flux Balance Analysis (dFBA): set the initial EG concentration at 10mmol/L and simulated the 25-hour growth curve—the bacterial growth rate reached 0.06~0.065 1/h in the early stage, and OD600 increased to above 0.4 over time, consistent with the subsequent wet experiment strain growth trend.
The Wet Lab Group constructed an EG detection standard curve: mixed 0.8mL gold nanoparticle solution with EG solutions of various concentrations, measured the absorbance at 525nm using a UV-Vis spectrophotometer, and obtained the linear equation y=0.0025x+0.7085 with a correlation coefficient R=0.988; constructed the EG-metabolizing strain EG-BL21 (DE3) and introduced the piGEM25_04 plasmid containing gcl/garR/glxK genes.
The Hardware Group debugged the light source system: Arduino drove LEDs to light up in turn (390~660nm), and the AS7341 sensor could collect light intensity at each wavelength; solved the stepper motor jamming problem (adjusted the A4988 driver current to 0.8A); the HP Group formulated the detailed schedule for August field research: Wuhou District Treatment Center (7th), Sangzhi Landfill (15th), Everbright Incineration Plant (22nd), Badagongshan Nature Reserve (28th).
Week 3
The Dry Lab Group conducted Flux Variability Analysis (FVA): fixed the product yield to 90% of the optimal value and calculated the upper and lower limits of reaction fluxes. It was found that the reaction fluxes corresponding to gcl, fucO, and aldA genes had small fluctuations and large mean values, clarifying the subsequent strain modification targets.
The Wet Lab Group verified the growth of EG-BL21 (DE3): after 72 hours of cultivation at 37℃ in M9 medium with EG as the sole carbon source, the OD600 reached 0.8 (wild-type BL21 barely grew); the gold nanoparticle method was used to initially detect EG consumption, which decreased by approximately 20mM compared with the initial concentration.
The Hardware Group tested the light intensity-absorbance conversion (Beer-Lambert Law), and the calculation of data at 6 discrete wavelengths was correct; the HP Group reached a cooperation with the Jilin University iGEM team and planned to jointly develop the popular science picture book E. coli Magic Factory.
Week 4
The Dry Lab Group conducted Flux Distribution Comparative Analysis (FDCA): compared the flux differences between "maximizing biomass" and "maximizing PEAs", and determined fucO, aldA, gcl, glxR, garR, and glxK as key regulatory genes.
The Wet Lab Group verified EG consumption using HPLC: after 72 hours of cultivation of EG-BL21 (DE3), the EG concentration decreased from 50mM to 28mM, with an actual consumption of 22mM; launched the construction of PEA synthesis plasmids (piGEM25_07 for monomer synthesis, piGEM25_08 for monomer polymerization).
The Hardware Group purchased an OLED display (replacing the original large-size screen) and rewrote the display code; the HP Group organized feedback from the first popular science activity and adjusted the subsequent content to "plastic recycling value closer to daily life" (HP wiki.docx 2.2); the Experimental Group prepared for the initial stage of TPA conversion: designed the piGEM25_09 plasmid (expressing TphA1/A2/A3, TphB).
August: Experimental Breakthrough and Practical Implementation
Week 1
The Wiki Group launched the construction of the first half: completed the writing of the "Experimental Section" (PET degradation, EG detection) and "Dry Lab Section", inserted the dual-enzyme system schematic diagram of the PET degradation summary from the Experimental Group and the standard curve diagram from EG detection-Wiki, and unified the terminology format.
The Wet Lab Group completed the construction of piGEM25_07/08 plasmids: piGEM25_07 (containing phaA, phaB, panD) and piGEM25_08 (containing phaC, Pct540, Act) were verified by PCR (correct band position) and sequencing; induced phaA protein expression, and SDS-PAGE showed the target band at 40kDa.
The Hardware Group tested the sensor stability: detected the same sample 10 consecutive times, with an absorbance RSD < 2%; the HP Group conducted research at Wuhou District Urban Resource Treatment Center and identified pain points such as mixed compression of PET and takeaway boxes, and no recycling channel for "low-value waste plastics".
Week 2
The Wiki Group improved the "Dry Lab Section": detailed the principles and results of four methods (FBA, dFBA, FVA, FDCA), inserted the flux analysis diagram from the bioinformatics Topic 3 Wiki summary; wrote the framework design and circuit connection for the "Hardware Section", and attached the PCB pin distribution diagram.
The Wet Lab Group co-transformed piGEM25_07/08 into BL21 (DE3) to construct the PEA02-BL21 (DE3) strain (dual plasmids); separately transformed piGEM25_08 to construct PEA01-BL21 (DE3) (requiring additional monomers), and spread them on double-antibody (Cm+Amp) plates.
The Hardware Group solved the display data delay problem by adjusting the serial baud rate from 9600 to 115200; the HP Group conducted research at Sangzhi County Landfill, observed the leachate nitrification tank (treating highly toxic ammonia nitrogen by biological method), and confirmed that the biological method is suitable for grassroots scenarios.
Week 3
The Wiki Group typeset the first half: unified the title format and image labels, added anchor links; corrected the Dry Lab terminology "flux variation" to "flux variability".
The Wet Lab Group verified PEA synthesis: after 72 hours of cultivation, PEA01, PEA02, and the control group (without inducer) were stained with Nile Red—PEA01/02 showed red, while the control group was colorless; PEA02 showed a strong fluorescence emission peak at 610nm under excitation at 530nm; Experimental Group TPA conversion: transformed piGEM25_09 into BL21 (DE3), and verified the soluble expression of TphA1/A2/A3 and TphB via SDS-PAGE.
The Hardware Group tested EG concentration detection: calculated by substituting into the standard curve, with an error < 5%; the HP Group confirmed the picture book framework with the Jilin University team—"Koala + little girl visiting the E. coli factory" to demonstrate the PET→PEAs process.
Week 4
The Wiki Group completed the preliminary review of the first half draft: supplemented the number of experimental repetitions; the Wet Lab Group optimized PEA synthesis conditions: 0.3mM IPTG induction resulted in a 15% higher yield than 0.5mM; Experimental Group TPA conversion: tested the effect of different OD600 on PCA yield, and PCA reached 1250μM when OD600=60.
The Hardware Group assembled a portable detection device (added a protective case); the HP Group made "Scientific Term Challenge" guessing cards (including terms such as "PET degradation" and "magnetically immobilized enzyme") and tested them in the community; the Wiki Group collected HP research data to prepare for writing the second half.
September: Result Integration and Promotion
Week 1
The Wiki Group launched the writing of the second half: inserted Nile Red staining and fluorescence spectrum diagrams in the "PEA Synthesis" section; detailed the construction of piGEM25_09 and PCA yield in the "TPA Conversion" section; wrote the application of the cubic spline interpolation algorithm (resampling 6 discrete points into a smooth curve with 1nm intervals) in the "Hardware Results" section.
The Wet Lab Group organized core data: PET degradation (61% activity retained after 10 cycles), EG metabolism (22mM consumed in 72h), PEA synthesis (yield 0.8g/L), TPA conversion (PCA 1250μM), and formed a summary table; the Hardware Group recorded a demonstration video (completing "sample addition→detection→concentration output" in 3 minutes).
The HP Group conducted research at Everbright Environmental Energy (Zhangjiajie) Co., Ltd., learned about the incineration characteristics of PET (risk of increased HCl emissions from chlorine-containing mixed combustion), and confirmed the complementary value of biological pretreatment; the Game Development Group used HTML5 (Canvas), CSS (Tailwind CSS), and JavaScript to develop Polymerization Rebirth: Plastic Alchemist, with the core mechanism of "catching PET fragments→enzymatic hydrolysis→bacterial conversion→earning funds".
Week 2
The Wiki Group wrote the "HP Results" section: divided into "Field Research" (pain points of 4 locations) and "Educational Popular Science" (6 activities, 3 types of materials), and attached key data such as "200 yuan/ton treatment cost" from Sangzhi Sanitation Office; the Game Development Group improved the "loss penalty" mechanism.
The Hardware Group optimized the program: burned the cubic spline interpolation code, and the spectral curve coincidence with the traditional spectrophotometer was ≥95%; the HP Group held a laboratory open day, invited high school students to visit the PET degradation experiment, and focused on demonstrating the magnetic enzyme recovery process.
Week 3
The Wiki Group completed the draft of the second half: detailed the design logic of Polymerization Rebirth (corresponding to the project's technical route) in the "Game" section; integrated Dry/Wet Lab, Hardware, and HP results in the "Conclusion" section, emphasizing the "PET→EG/TPA→PEAs/PCA" closed loop; unified the terminology expressions such as "acetyl-CoA" and "protocatechuic acid" during proofreading.
The Hardware Group organized technical documents (3D modeling drawings, Arduino code, PCB design drawings) and attached them to the Wiki download area; the HP Group completed the "PET Biological Treatment Technology Implementation Proposal" and proposed a promotion idea of "starting from PET-enriched scenarios in scenic spots/commercial areas"; the Experimental Group supplemented the stability test for TPA conversion: the RSD of PCA yield in 3 consecutive batches was <8%.
Week 4
The Wiki final version was finalized: all sections (Experimental, Dry Lab, Hardware, HP, Game) had a unified format, high-definition images, and cited document contents with source labels; the supervisor reviewed and confirmed "accurate data and coherent logic".
The Hardware Group completed the final test: wavelength accuracy ≤±3nm, absorbance resolution 0.01AU, and the cost was 2 orders of magnitude lower than traditional spectrophotometers; the HP Group summarized the annual results: field research covered the entire chain of "compression-landfilling-incineration-ecology", popular science reached more than 500 people, and materials were recognized by communities/schools; the entire team recorded a project summary video, reviewing the journey from May to September, and emphasizing the concept of "technology for good, recycling utilization".
