iGEM 2025 — Team uestc-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

Notebook

* [May](#May)
* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)

# **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.

![First team meeting group photo](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p1.webp)

Figure 1: Group photo of the first team meeting

## **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.

![Visit to Maike Macromolecular Materials](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p2.webp)

Figure 2: Visit to Chengdu Maike Macromolecular Materials Co., Ltd.

## **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.

![Hardware Group PCB 2D rendering](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p3.webp)

Figure 3: 2D rendering of the PCB designed by the Hardware Group

## **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.

![HP Group hospital popular science activity](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p4.webp)

Figure 4: Photo of the HP Group's popular science activity at the hospital

## **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.

![HP Group campus popular science activity](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p5.webp)

Figure 5: Photo of the HP Group's popular science activity on campus

## **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".

![HP Group research at Wuhou District Treatment Center](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p6.webp)

Figure 6: Photo of the HP Group's research at Wuhou District Urban Resource Treatment Center

## **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.

![Leachate treatment at Sangzhi Landfill](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p7.webp)

Figure 7: Photo of leachate treatment at Sangzhi Landfill

## **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.

![Draft of popular science picture book 'Disassembly Workshop'](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p8.webp)

Figure 8: Draft of the "Disassembly Workshop" in the popular science picture book

## **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".

![HP Group research at Everbright Incineration Plant](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p9.webp)

Figure 9: Photo of the HP Group's research at Everbright Incineration Plant

## **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.

![Laboratory Open Day photo](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p10.webp)

Figure 10: Photo of the Laboratory Open Day

## **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%.

![Screenshot of 'Polymerization Rebirth' game interface](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p11.webp)

Figure 11: Screenshot of the "Polymerization Rebirth" game interface

## **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".

![Wiki homepage screenshot](https://static.igem.wiki/teams/5979/uestcpic/wetlab/notebook/p12.webp)

Figure 12: Screenshot of the Wiki homepage

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

Engineering

* [PET Biodegradation](#PETBiodegradation)
  + [Cycle 1:Design and Construction of Key Plasmids for the Immobilized Dual-Enzyme System](#PETCycle1)
  + [Cycle 2:Optimization of System Assembly and Immobilization Strategies](#PETCycle2)
* [Upcycling of EG to PEAs](#EGUpcycling)
  + [Engineering E.coli to utilize EG as a Carbon Source:circle1](#EGCycle1)
  + [Synthesis and detection of PEA.circle1 and circle1](#EGCycle2)
* [Upcycling of TPA to PCA](#TPAtoPCA)
  + [Cycle 1: Optimization of the Expression Process with Cofactors](#PCACycle1)

# **PET Biodegradation**

## **Cycle 1: Design and Construction of Key Plasmids for the Immobilized Dual-Enzyme System**

#### Design

To ensure the robustness and efficiency of the assembly, we adopted a stepwise strategy for synthesizing the functional components. The pET-30a(+) vector was selected as the construction backbone for this stage, owing to its advantageous features, including a strong promoter for high-level expression and a multiple cloning site that facilitates the insertion of multiple genes.

#### Construction

Leveraging the pET vector framework, renowned for its high-level protein expression capabilities, we constructed three distinct expression plasmids:

1. piGEM25\_01: Engineered to express the K5C (ELP-SpyCatcher) fusion protein, which constitutes the functional surface of the magnetic carrier.
2. piGEM25\_02: Designed for the expression of the SpyTag-TfCa-SnoopTag complex, the hydrolase responsible for degrading the PET intermediate, MHET.
3. piGEM25\_03: Constructed to express the SnoopCatcher-Tfh complex, the primary PET hydrolase.

We strategically embedded multiple rigid GC linkers within these constructs to enhance their structural stability and optimize interaction kinetics.

Plasmid piGEM25\_02 comprises three key components linked sequentially: SpyTag, TfCa, and SnoopTag. These plasmids were then introduced into the high-expression host strain, E. coli BL21(DE3), using the heat shock method—a technique widely employed for transforming E. coli with exogenous DNA. Plasmid piGEM25\_03, which contains the SnoopCatcher and hydrolase Tfh components, was assembled using seamless cloning via homologous recombination. This method efficiently inserts multiple genes into a vector with minimal effort and time. The process involves two main steps: first, amplifying the target genes with specific primers, followed by overlap extension PCR. The genes were then recombined and integrated into the plasmid in vivo through direct transformation into E. coli DH5α competent cells. Finally, the resulting recombinant plasmid was introduced into the E. coli BL21(DE3) strain for expression. This optimized protocol significantly increases the yield of transformed cells, thereby enhancing overall transformation efficiency.

#### Testing

The successful construction of all three plasmids was first confirmed by PCR amplification and Sanger sequencing. Following induction with IPTG, protein expression was analyzed by SDS-PAGE, which verified the soluble expression of all components at their expected molecular weights. The K5C protein was successfully purified using Inverse Transition Cycling (ITC), a method that leverages the phase transition properties of its ELP domain. Subsequently, the purified K5C served as a template for the biomimetic mineralization process, yielding the functionalized magnetic carrier (K5C@Fe₃O₄).

#### Learning and Experimentation

The successful construction of the plasmids and expression of the core protein components validated our initial modular design. This foundational work demonstrated the capability of the selected vectors and expression system to produce the necessary bioparts. However, the mere production of these parts is insufficient; their effective assembly into a functional system is of paramount importance. This realization underscored the need to optimize the assembly conditions to maximize the catalytic efficiency of the final system. Consequently, the next phase of our work focused on optimizing the carrier preparation process and identifying the most effective strategy for enzyme immobilization.

---

## **Cycle 2: Optimization of System Assembly and Immobilization Strategies**

#### Design

Building on the components successfully constructed in the first cycle, we aimed to maximize the PET degradation activity of the assembled system. We identified two key parameters for optimization: the NaOH concentration used during the biomimetic mineralization of the K5C@Fe₃O₄ carrier, as this can influence surface properties and enzyme-binding capacity, and the immobilization strategy for the two enzymes (TfCa and Tfh) onto the carrier. We hypothesized that the method of enzyme attachment—whether sequential (stepwise) or co-immobilization (simultaneous) —could significantly impact the spatial arrangement and synergistic action of the enzymes.

#### Construction

The plasmids and protein components from the first cycle remained unchanged. We utilized these established components to systematically test different assembly protocols, ensuring experimental consistency and allowing us to isolate the effects of varying mineralization and immobilization conditions on the degradation process.

#### Testing

A series of experiments were conducted to determine the optimal conditions. First, the NaOH concentration in the mineralization reaction was varied, and the subsequent activity of the assembled system was quantified by measuring the concentration of the final degradation product, TPA, using High-Performance Liquid Chromatography (HPLC). Second, we compared two immobilization strategies: Group A (Sequential Immobilization: TfCa was immobilized first, followed by Tfh) and Group B (Co-immobilization: both enzymes were mixed with the carrier simultaneously). The degradation efficiency of each strategy was again assessed by TPA quantification. Finally, to evaluate the system's robustness, the reusability of the optimized magnetic enzyme beads was tested over 10 consecutive degradation cycles.

#### Learning and Experimentation

The test results provided critical data for system optimization. HPLC analysis revealed that a 3.0 M NaOH concentration during mineralization yielded the highest catalytic activity. Most importantly, we observed that sequential immobilization significantly enhanced degradation efficiency compared to co-immobilization, achieving a final TPA concentration of 1.2 mM. This suggests that an ordered, stepwise assembly is crucial for the optimal functional synergy of the dual-enzyme complex. Furthermore, the reusability tests demonstrated the system's excellent operational stability, as it retained 61% of its initial activity after 10 cycles. Based on these findings, future work will focus on exploring the ideal molar ratio of the two enzymes and optimizing the linker structures connecting the functional domains to further enhance system performance.

---

# **Upcycling of EG to PEAs**

## **Engineering E.coli to utilize EG as a Carbon Source:circle1**

#### Design

To enable BL21(DE3) to efficiently utilize EG as a carbon source, we first adopted the previously reported gcl–hyi–glxR–glxK quadruple gene knockout as a reference baseline. Building upon this, we retained gcl and glxK while introducing the isoenzyme garR to replace glxR and removing hyi. This optimization aimed to enhance the aldehyde flux at the key node and reduce genetic burden. Additionally, we evaluated the potential value of expanding the upstream fucO and aldA genes.

We constructed three distinct gene combinations for parallel testing:

1. The literature-reported four-gene combination gcl-hyi-glxR-glxK (expressed in piGEM25\_05);
2. The streamlined three-gene combination gcl-garR-glxK (expressed in piGEM25\_04);
3. The expanded combination gcl-garR-glxK/fucO-aldA (co-expressing piGEM25\_04 and piGEM25\_06).

#### Build

We employed a multi-round PCR approach combined with homologous recombination to construct the recombinant plasmid. First, high-fidelity PCR amplified the coding sequences of each gene, introducing homologous arm sequences at both primer ends. Concurrently, the pCDFDuet-1 vector was linearized via PCR, with corresponding homologous sequences introduced at both ends. Purified gene fragments and the linearized vector were mixed at a specific molar ratio. Homologous recombination was performed at 50°C for 30 minutes using a homologous recombination enzyme to complete the ligation. The ligation product was chemically transformed into E. coli DH5α competent cells and screened for resistance on LB plates containing the corresponding antibiotics. Positive clones were picked and sent to the company for Sanger sequencing validation. Correctly sequenced plasmids were extracted and transformed into E. coli competent cells via heat shock to obtain the final engineered strains. To directly test each engineered strain's EG utilization capability, multiple single colonies were picked from the resistance plates of each transformation system and inoculated into M9 selective medium with EG as the sole carbon source for cultivation.

#### Test

The constructed plasmids were individually transformed into BL21(DE3). The resulting engineered strains EG04-BL21(DE3) (expressing piGEM25\_04) and EG05-BL21(DE3) (expressing piGEM25\_05) both grew efficiently using EG as the sole carbon source. Specifically, after 72 hours of cultivation, EG04-BL21(DE3) reached an OD600 of 0.218 with an EG degradation rate of 28%; EG05-BL21(DE3) achieved an OD600 of 0.24 with a 24% EG degradation rate. Both strains exhibited comparable metabolic efficiency, but the three-gene combination employed in EG04-BL21(DE3) achieved streamlined optimization of the metabolic pathway. Furthermore, the co-transformed strain EG04-06-BL21(DE3) (expressing piGEM25\_04 and piGEM25\_06) and the blank control EV-BL21(DE3) failed to grow in medium where EG served as the sole carbon source.

Experimental results indicate that the engineered strain EG04-BL21(DE3) can grow using EG as the sole carbon source. However, the co-transformed strain EG04-06-BL21(DE3), which incorporates fucO-aldA into the gcl-hyi-glxR-glxK combination, fails to utilize EG as the sole carbon source. This is likely due to the substantial metabolic resources consumed by the co-expression of multiple genes. Therefore, we successfully conferred the EG utilization capability to E. coli—which normally requires a four-gene combination—using a three-gene combination (gcl-garR-glxK) guided by metabolic modeling. By integrating growth phenotype data with quantitative substrate consumption analysis, we validated the functionality and efficiency of this metabolic pathway. This breakthrough provides a clear direction for subsequent efforts to further optimize EG utilization efficiency.

## **Synthesis and detection of PEA：Cycle 1**

#### Design

To achieve PEA biosynthesis, we designed a dual-plasmid system: piGEM25\_08 (phaA-phaB-PanD) synthesizes ester and amide bond monomer precursors, while piGEM25\_07 (phaC-PCT540-Act) catalyzes monomer activation and polymerization. Preliminary experiments were conducted in LB medium, with plans to validate PEA synthesis by extracting and purifying intracellular polymers.

#### Build

Recombinant plasmids were transformed into E. coli BL21(DE3) and induced for expression in LB medium. After cell harvesting, intracellular polymers were purified using chloroform extraction combined with methanol precipitation.

#### Test

Following induction in LB medium, extracted polymer yields were extremely low and invisible to the naked eye. Fluorescence microscopy revealed hydrophobic particles stained by Nile red, but yields were insufficient for subsequent detection.

#### Learn

Preliminary results indicate: 1) PEA synthesis may be successful but yields are low. This necessitates optimizing control group design and detection methods.

## **Synthesis and detection of PEA：Cycle 2**

#### Design

Based on Cycle 1 results, we redesigned the experiment: 1) Replaced LB medium with MR medium due to its superior buffering capacity and more favorable conditions for polymer accumulation; 2) Optimized the extraction process.

#### Build

We cultured the engineered strain in MR medium, establishing three experimental groups: ① PEA01-BL12 (DE3) (transformed with piGEM25\_08 plasmid alone; requires exogenous monomer for PEA synthesis); ② PEA02-BL12 (DE3) (co-transformed with piGEM25\_07 and piGEM25\_08 plasmids) ③ Negative control (transformed with piGEM25\_08 plasmid only, without inducer). Key improvements included: reducing chloroform and methanol usage to enhance extraction yield and efficiency.

#### Test

Significant improvements were achieved after optimizing the medium and extraction method:

1. Effective yield increase: extracted polymer quantity markedly increased, with visible white flocculent precipitation
2. Nile Red staining: Specific staining observed only in experimental group
3. Fluorescence quantification: Concentration-dependent fluorescence enhancement demonstrated

#### Key learnings

The switch from LB to MR medium and extraction method optimization were critical to success. These improvements substantially increased PEA yield and extraction efficiency.

---

# **Upcycling of TPA to PCA**

## **Cycle 1: Optimization of the Expression Process with Cofactors**

#### Design

Given the very low vanillin yield from the TPA-to-vanillin conversion in the 2024 UESTC-iGEM project, we identified the low concentration of the precursor, PCA, as a likely cause. In our project, we optimized the expression conditions for the enzymes in the TPA-to-PCA pathway. Unlike the previous year's project, we supplemented the expression medium with FeCl₃, L-cysteine, and ZnSO₄ to enhance TPA degradation efficiency.

#### Construction

After 24 hours of induction, E. coli BL21(DE3) cells were concentrated and resuspended in M9 medium to a final OD600 of 60 in a total volume of 3 mL. Subsequently, 12 µL of a 500 mM TPA stock solution in dimethyl sulfoxide (DMSO) was added. The degradation reaction was conducted at 20°C for 24 hours with shaking.

#### Test

High-Performance Liquid Chromatography (HPLC) analysis successfully detected the target product, PCA, at a concentration of 1.2 mM.

#### Learn

The results demonstrated that the addition of the cofactors FeCl₃, L-cysteine, and ZnSO₄ led to a substantial increase in PCA production.

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Experiments

* [Culture](#Culture)
  + [1.Culture medium](#Culturemedium)
  + [2.Antibiotic](#Antibiotic)
  + [3.IPTG\L-arabinose](#IPTGLarabinose)
  + [4.Thiamine\RS-3-Hydroxybutyric Acid Sodium Salt\3-Aminopropionic Acid Solution](#OtherSolutions)
* [Breed Preservation](#BreedPreservation)
  + [1.Glycerol Stock](#GlycerolStock)
  + [2.Recovery](#Recovery)
* [Plasmid construction and Transformation](#PlasmidconstructionandTransformation)
  + [1.PCR](#PCR)
  + [2.Nucleic Acid Gel Electrophoresis](#NucleicAcidGelElectrophoresis)
  + [3.Gel Recovery](#GelRecovery)
  + [4.Homologous Recombination](#HomologousRecombination)
  + [5.Transformation](#Transformation)
* [Expression](#Expression)
  + [1.Bacterial Culture and Target Protein Expression](#BacterialCultureandTargetProteinExpression)
  + [2.Bacterial Culture Harvesting and Pellet Resuspension](#BacterialCultureHarvestingandPelletResuspension)
* [Verification](#Verification)
  + [1.Bacterial Lysis](#BacterialLysis)
  + [2.Nickel Column Purification](#NickelColumnPurification)
  + [3.Coomassie Brilliant Blue Assay for Protein Concentration](#CoomassieBrilliantBlueAssay)
  + [4.SDS-PAGE](#SDS-PAGE)
  + [5.Preparation of Bacterial Lyophilized Powder](#BacterialLyophilizedPowder)
  + [6.Purification of PEA](#PEAPurification)
  + [7.Nile Red Staining](#NileRedStaining)
  + [8.FTIR](#FTIR)
  + [9.TEM](#TEM)
  + [10.Purification of K5C via ITC](#K5CPurification)
  + [11.Bionic Mineralization for K5C@Fe₃O₄ Carrier](#BionicMineralization)
  + [12.Covalent Immobilization of Enzymes and PET Degradation](#EnzymeImmobilization)
  + [13.HPLC](#HPLC)
  + [14.Ethylene glycol（EG）detection](#EGDetection)

# **Culture**

## **1. Culture medium**

### **1.1 TB**

Cultivation of E. coli using TB medium while expressing proteins in experiments.

The TB medium was configured as follows.

| Ingredient | Quantity |
| --- | --- |
| YEAST EXTRACT | 24 g |
| TRYPTONE | 12 g |
| K2HPO4·3H2O | 12 g |
| KH2PO4 | 2.2 g |
| GLYCERINUM | 4 mL |
| ddH2O | To 1 L |

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Save

Detailed steps:

1. Calculation of substance requirements based on the volume of medium required.
2. Weigh the substances according to the calculation.
3. Add ddH2O to fix the volume and stir well to dissolve it.
4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. Store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4℃ and use it as soon as possible.

### **1.2 LB**

Cultivation of E.coli in experiments using LB medium (with appropriate antibiotics if needed).

The LB medium was configured as follows.

| Ingredient | Quantity |
| --- | --- |
| NaCl | 10g |
| TRYPTONE | 10g |
| YEAST EXTRACT | 5g |
| ddH2O | To 1 L |

If formulated as a solid medium, add an additional 15 g of agar powder (1.5%) before sterilization, then autoclave the configured medium.

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Pour Plate (if needed) → Save

Detailed steps:

1. Calculation of substance requirements based on the volume of medium required.
2. Weigh the substances according to the calculation.
3. Add ddH2O to fix the volume and stir well to dissolve it.
4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. In case of LB agar the inverted plate operation is carried out when the medium is cooled to about 45 ℃. The petri dish lid can be tilted to minimize condensate contamination, and the inverted plate operation is carried out in an ultra-clean bench next to an alcohol lamp.
6. Store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4 ℃ and use it as soon as possible.

### **1.3 M9**

1. Configure of 1 mol/L MgSO4: weigh 2.46g of MgSO4-7H2O and dissolve it in 10ml of ddH2O, autoclaved it.
2. Configure 1 mol/L CaCl2: weigh 2.191g of CaCl2-6H2O and dissolve it in 10ml of ddH2O, autoclave it.
3. Configure 5×M9 salt solution:

   | Ingredient | Quantity |
   | --- | --- |
   | Na2HPO4・H2O | 12.8g |
   | KH2PO4 | 3g |
   | NACL | 0.5g |
   | NH4CL | 1 g |
   | ddH2O | To 200ml |

   Autoclave standby.
4. Configure 20% glucose solution: weigh 4g glucose and dissolve it in 20ml of ddH2O, then filter with 0.22μm filter to remove bacteria.
5. Configure M9 culture medium

   | Ingredient | Quantity |
   | --- | --- |
   | 5×M9 | 200ml |
   | 1M MgSO4 | 2ml |
   | 20% Glucose solution | 20ml |
   | 1M CaCl2 | 0.1ml |
   | ddH2O | To 1L |

### **1.4 MR**

In the experiment, E. coli was cultured using MR medium (with appropriate antibiotics or inducers added as necessary) for fermentation studies.

The preparation method for MR medium is as follows:

| Ingredient | Quantity |
| --- | --- |
| (NH4)2HPO4 | 4 g |
| K2HPO4 | 6.67 g |
| Citric acid | 0.8 g |
| MgSO₄·7H₂O | 0.8 g |
| Glucose | 20 g |
| MOPS | 20.926 g |
| Trace metal solution | 5 ml |

Note that glucose and certain heat-sensitive supplements (e.g., MOPS, MgSO₄) must be sterilized by filtration and added only after the base medium has cooled. They must not undergo autoclaving.

The preparation process is as follows:

Calculation → Weighing → Dissolution → Aliquoting and Sterilization → Addition of Sterile Components → Storage

Detailed steps:

1. Calculate reagent quantities based on required medium volume.
2. Weigh reagents according to calculations. Heat-sensitive components like 3-(N-morpholinyl)propane sulfonic acid (MOPS), glucose, magnesium sulfate, and trace elements require separate filter sterilization.
3. Add deionized water to final volume and stir thoroughly until dissolved.
4. Seal flasks with breathable plastic wrap and leather bands, then sterilize in an autoclave.
5. Prepare 0.5 g/ml MOPS solution: Weigh 20.926 g MOPS, dissolve in 42 ml deionized water, and filter through a 0.22 μm membrane to remove bacteria.
6. Prepare a 0.5 g/ml glucose solution: Weigh 20 g glucose, dissolve in 40 ml deionized water by heating until completely dissolved, then filter through a 0.22 μm membrane filter to remove bacteria.
7. Prepare a 0.5 g/ml MgSO₄·7H₂O solution: Weigh 0.8 g of magnesium sulfate heptahydrate, dissolve in 1.6 ml of deionized water, and filter through a 0.22-micron membrane filter to remove bacteria.
8. Prepare trace element solution: Weigh 0.5 g ferrous sulfate heptahydrate (FeSO₄·7H₂O), 0.1 g calcium chloride (CaCl₂), 0.11 g zinc sulfate heptahydrate (ZnSO₄·7H₂O), 0.025 g manganese sulfate tetrahydrate (MnSO₄·4H₂O), 0.025g copper sulfate pentahydrate (CuSO₄·5H₂O), 0.05g ammonium molybdate tetrahydrate ((NH₄)₆Mo₇O₂₄·4H₂O), 0.001g sodium tetraborate decahydrate (Na₂B₄O₇·10H₂O), dissolved in 0.5M HCl solution, stirred until completely dissolved, then sterilized by filtration through a 0.22μm membrane filter. (Note: This formula yields 20mL of trace element stock solution; scale proportionally as needed. Add 5mL of this stock solution per liter of MR medium.)
9. Sterile addition of heat-sensitive components: After autoclaving the base medium, cool it below 60°C and perform sterile operations in a laminar flow hood. Add sterile-filtered glucose stock solution to a final concentration of 20 g/L; add sterile-filtered MgSO₄·7H₂O solution to a final concentration of 0.8 g/L; add sterile-filtered MOPS solution to a final concentration of 100 mM; add 5 mL of sterile-filtered trace element solution.
10. After thorough mixing, the liquid medium can be stored at 4°C and used promptly.

## **2. Antibiotic**

The following table shows the concentration of antibiotics and working solution used, which should be diluted 1:1000 at the time of use. Note that solid media need to be added after sterilization when cooled to approximately 45 ℃ to avoid inactivation of antibiotics.

| Ingredient | Concentration |
| --- | --- |
| Kanamycin | 100 mg/mL |
| Ampicillin | 100 mg/mL |
| Chloramphenicol | 50 mg/mL |
| Streptomycin | 50 mg/mL |

The preparation process is as follows.

Calculation → Weighing → Dissolving → Dispense

Detailed steps:

1. Calculation: according to the concentration and the required amount to calculate the antibiotic mass and sterile water volume to be weighed.
2. Weighing: according to the calculation results.
3. Dissolution: use a pipette gun ( the gun’s head needs to be sterilized ) to suck sterile water (ddH2O is sterilized by high-pressure steam ) and dilute to the corresponding volume.
4. Dissolution: use a pipette gun ( the gun’s head needs to be sterilized ) to suck sterile water (ddH2O is sterilized by high-pressure steam ) and dilute to the corresponding volume.
5. Sub-packaging: by using disposable syringe and 0.22 μM sterile syringe filter (bioshrap, catalog number: bs-pes-22 ) to filter and sub-pack the antibiotic into several 1.5/2 ml EP tubes and then store them in a - 20℃ refrigerator, and use it as soon as possible.

## **3. IPTG\L-arabinose**

### **3.1 Preparation of 0.5M IPTG Stock Solution**

1. **Personal Protective Equipment (PPE):**Put on appropriate PPE including lab coat, gloves, and safety goggles.
2. **Weighing IPTG:** In a fume hood, accurately weigh the required amount of IPTG powder to prepare a 0.5M solution. The amount needed can be calculated using the molecular weight of IPTG and the desired volume of the stock solution.
3. **Dissolution:**Add the weighed IPTG to a suitable volume of sterile water in a clean container. Stir gently until the IPTG is completely dissolved. Ensure that the solution is at room temperature to facilitate dissolution.
4. **Sterilization:**Filter the solution through a 0.22 µm sterile filtration system to remove any potential contaminants. This step should be done in a laminar flow hood to maintain sterility.
5. **Aliquoting:** Using aseptic technique, aliquot the filtered IPTG solution into 1 mL centrifuge tubes. Label each tube with the solution name, concentration, date of preparation, and any other relevant information.
6. **Storage:** Store the aliquoted IPTG stock solution at -20°C. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution.

### **3.2 Preparation of L-arabinose**

1. **Personal Protective Equipment (PPE):**Put on appropriate PPE including lab coat, gloves, and safety goggles.
2. **Weighing L-arabinose:** In a fume hood, accurately weigh the required amount of L-arabinose powder to prepare a 200mg/ml solution. The amount needed can be calculated using the molecular weight of L-arabinose and the desired volume of the stock solution.
3. **Dissolution:**Add the weighed L-arabinose to a suitable volume of sterile water in a clean container. Stir gently until the L-arabinose is completely dissolved. Ensure that the solution is at room temperature to facilitate dissolution.
4. **Sterilization:** Filter the solution through a 0.22 µm sterile filtration system to remove any potential contaminants. This step should be done in a laminar flow hood to maintain sterility.
5. **Aliquoting:**Using aseptic technique, aliquot the filtered L-arabinose solution into 1.5 mL centrifuge tubes. Label each tube with the solution name, concentration, date of preparation, and any other relevant information.
6. **Storage:**Store the aliquoted L-arabinose stock solution at -20°C. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution.

All of the aforementioned operations must be conducted within a biological safety cabinet in order to prevent contamination.

## **4. Thiamine\RS-3-Hydroxybutyric Acid Sodium Salt\3-Aminopropionic Acid Solution**

### **4.1 Preparation of Thiamine**

1. **Personal Protective Equipment (PPE):** Wear appropriate PPE, including a lab coat, nitrile gloves, and safety goggles. All weighing and dispensing operations must be conducted within a fume hood to prevent powder inhalation and contamination.
2. **Weighing Thiamine:** Precisely weigh the required amount of thiamine powder in a fume hood to prepare a 200 mg/mL solution. For example, to prepare 10 mL of stock solution, weigh: 200 mg/mL × 10 mL = 2000 mg, i.e., 2.0 g.
3. **Dissolution:** Add the weighed thiamine powder to an appropriate volume of sterile deionized water in a clean container. Vortex or gently stir until completely dissolved. Thiamine is highly soluble in water and dissolves rapidly at room temperature.
4. **Sterilization:** Filter the solution through a 0.22-micron sterile syringe filter into a sterile receiving tube to remove potential contaminants. This step should be performed within a biosafety cabinet or laminar flow hood to maintain sterility.
5. **Aliquoting:** Using aseptic technique, immediately aliquot the filtered thiamine solution into sterile 1.5 mL centrifuge tubes in single-use volumes (e.g., 1.0 mL/tube). Clearly label each tube with the solution name, concentration (200 mg/mL), preparation date, and expiration date.
6. **Storage:** Store aliquoted thiamine stock solution protected from light at -20°C. Avoid repeated freeze-thaw cycles to maintain solution stability and biological activity.

### **4.2 Preparation of Sodium RS-3-Hydroxybutyrate and 3-Aminopropionic Acid Solutions**

1. **Personal Protective Equipment (PPE):** Wear appropriate PPE, including a lab coat, nitrile gloves, and safety goggles. All weighing and aliquoting operations must be performed in a fume hood to prevent powder inhalation and contamination.
2. **Weighing RS-3-Hydroxybutyric Acid Sodium Salt and 3-Aminopropionic Acid Powders:** Precisely weigh the required amounts of RS-3-hydroxybutyric acid sodium salt and 3-aminopropionic acid powders in a fume hood to prepare a 400 mg/mL solution. For example, to prepare a 10 mL stock solution, weigh 400 mg/mL × 10 mL = 4000 mg of each powder, equivalent to 4.0 g.
3. **Dissolution:** Add the weighed sodium RS-3-hydroxybutyrate and 3-aminopropionic acid powders to an appropriate volume of sterile deionized water in a clean container. Vortex or gently stir until completely dissolved, forming a clear solution.
4. **Sterilization:** Filter the solution through a 0.22-micron sterile filtration system into a sterile receiving tube to remove potential contaminants. This step should be performed within a biosafety cabinet or laminar flow hood to maintain sterility.
5. **Aliquoting:** Using aseptic technique, aliquot the filtered sodium RS-3-hydroxybutyrate and 3-aminopropionic acid solution into sterile 1.5 mL centrifuge tubes in single-use volumes (e.g., 1.0 mL/tube). Each tube must be clearly labeled with the solution name, concentration (400 mg/mL), preparation date, and expiration date.
6. **Storage:** Store the aliquoted sodium RS-3-hydroxybutyrate and 3-aminopropionic acid stock solutions at -20°C. Avoid repeated freeze-thaw cycles to maintain solution stability.

---

# **Breed Preservation**

## **1. Glycerol Stock**

1. **Prepare a 50% glycerol solution:** Mix equal volumes of 100% glycerol and ultrapure water, and store at room temperature.
2. **Inoculate Glycerol Stock:** Transfer 500 μL of bacterial culture to a 2 mL tube, add 500 μL of 50% glycerol, and mix gently.
3. **Storage:** Use screw top tubes, label with strain and date, and store at -80°C to prevent unexpected opening.
4. **Stability:**Maintain stocks at -80°C for long-term stability; avoid repeated freeze-thaw cycles.

## **2. Recovery**

1. **Recovery of Bacteria:**Under sterile conditions, open the glycerol stock tube and use a sterile inoculation loop or pipette tip to scrape an appropriate amount of frozen bacteria, then streak onto an LB agar plate with or without the appropriate antibiotic.
2. **Overnight Cultivation:**Incubate the streaked LB agar plate at the optimal temperature for the bacterial strain to allow for growth.
3. **Isolation of Single Colonies:** After overnight incubation, pick individual colonies for further processing or analysis.

---

# **Plasmid construction and Transformation**

## **1. PCR**

Prepare a 25 µl reaction system using PCR tubes (ignite an alcohol lamp, no need to operate inside a laminar flow hood). 12.5 µl PCR MIX, 1 µl upstream primer, 1 µl downstream primer, 0.5 µl template, 10 µl ddH2O. Gently centrifuge to mix the reaction system.

Set up the PCR program: (Usually use high-fidelity enzymes from Shenggong, or set according to the instructions for different enzymes)

| Temperature | Time | Cycles |
| --- | --- | --- |
| 98℃ | 3：00 | 1x |
| 95℃ | 0：15 | 30x |
| X℃ | 0：10 | 30x |
| 72℃ | Y | 30x |
| 72℃ | 5: 00 |  |
| 12℃ | ∞ |  |

X should be set lower than the lowest Tm value of the two primers by 5-6. For example, if the Tm values of the two primers are 80 and 72, it should be set to 65.

## **2. Nucleic Acid Gel Electrophoresis**

Prepare the nucleic acid gel: Remember the ratio 1:10:100 (0.6g agarose: 6 µl nucleic acid dye: 60 ml 1x TAE buffer).

After adding agarose and TAE buffer, mix well and heat in a microwave until the solution is clear and transparent without any agarose lumps (use a larger conical flask to prevent boiling over). After heating, add the nucleic acid dye (highly toxic, handle with care), mix well. Place the plastic plate in the groove, then pour the solution, insert the comb (remove it vertically without damaging the wells). Do not add all at once to prevent the gel from being too thick.

Add 7 µl marker, 25 µl PCR product. Electrophoresis at 150V for 20 minutes.

## **3. Gel Recovery**

Remove the electrophoresed nucleic acid gel from the glass plate, place it under ultraviolet light, and remember to take protective measures. After taking a photo for preservation, cut off a small piece of the gel and place it into a 1.5 mL EP tube.

1. Cut the single DNA band from the agarose gel (try to remove excess parts) and place it into a clean centrifuge tube, weigh it.
2. Note: When using a gel cutter (OSE-GC) to cut the gel, align the cutter with the DNA band in the agarose gel and press down to cut. After cutting, push the center rod to push the gel piece into a clean centrifuge tube. Depending on the width of the gel well, you can perform single or continuous cutting.
3. Add 3 times the volume of gel dissolution liquid PE (if the gel weight is 0.1g, its volume can be considered as 100 µl, then add 300 µl gel dissolution liquid PE. When using a gel cutter to cut 1% agarose gel, the weight of a single piece is about 0.06 g, the actual weight of the gel piece is related to the gel concentration and thickness), dissolve at room temperature (15-25°C) for 5-10 minutes, during which gently invert the centrifuge tube up and down to ensure the gel piece is fully dissolved. (If the volume of the gel piece is too large, it can be cut into smaller pieces beforehand).
4. Note: For large fragments greater than 5 kb or gels with a concentration greater than 1.5%, it is recommended to heat dissolve at 50°C for 5-10 minutes; after the gel piece is completely dissolved, it is best to lower the solution temperature to room temperature before applying it to the column, as the adsorption column has a stronger DNA binding capacity at room temperature.
5. Add the solution from step 2 to an adsorption column CA5 (place the adsorption column into a collection tube), let it sit at room temperature for 2 minutes, centrifuge at 12,000 rpm (approximately 13,400 ×g) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA5 back into the collection tube.
6. Note: The volume of the adsorption column is 800 µl, if the sample volume is greater than 800 µl, it can be added in batches.
7. Add 600 µl wash solution PW (check if anhydrous ethanol has been added before use) to the adsorption column CA5, centrifuge at 12,000 rpm (approximately 13,400 ×g) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA5 back into the collection tube. Note: If the DNA being recovered is for salt-sensitive experiments, such as blunt-end ligation experiments or direct sequencing, it is recommended to let the PW sit for 2-5 minutes before centrifugation.
8. Repeat step 4.
9. Place the adsorption column CA5 back into the collection tube, centrifuge at 12,000 rpm (approximately 13,400 ×g) for 2 minutes to remove as much wash solution as possible. Let the adsorption column CA5 sit at room temperature for a few minutes to thoroughly dry to prevent residual wash solution from affecting the next step of the experiment.
10. Note: Residual ethanol in the wash solution can affect subsequent enzymatic reactions (restriction enzyme digestion, PCR, etc.).
11. Place the adsorption column CA5 into a clean centrifuge tube, add an appropriate amount of elution buffer TB to the center of the adsorption membrane and let it sit at room temperature for 2 minutes. Centrifuge at 12,000 rpm (approximately 13,400 ×g) for 2 minutes to collect the DNA solution. Note: The elution volume should not be less than 30 µl, as a smaller volume can affect the recovery efficiency. The pH value of the elution buffer has a significant impact on the elution efficiency. If sequencing is to be performed later, ddH2O should be used as the elution buffer, and its pH value should be maintained between 7.0-8.5. A pH value below 7.0 will reduce the elution efficiency; and the DNA product should be stored at -20°C to prevent DNA degradation. DNA can also be eluted with a buffer (10 mM Tris-Cl, pH 8.0).

## **4. Homologous Recombination**

To ligate the target gene to the vector, adjust the reaction system to ensure both components are at the same concentration. For example, when the vector and gene concentrations are equal, prepare the system as follows: maintain a 1:2 molar ratio of linearized vector to insert fragment, dilute with ddH₂O to 5 µl, then add 5 µl of homologous recombination enzyme. Incubate at 50°C for 30 minutes.

## **5. Transformation**

For general verification of whether the vector is successfully constructed, use DH5α, and for induced expression, use BL21(DE3) competent cells.

If commercial ones are not available, they can be prepared on-site:

1. Cultivate the original E. coli stock culture. Using a toothpick, transfer a single colony from the biosafety cabinet into 20 mL of LB medium (without antibiotics). Incubate at 37°C and 180 rpm for 5 hours until the liquid phase becomes turbid (measured OD value reaches 0.3–0.6).
2. Transfer 1 ml of the bacterial liquid into a 1.5 ml EP tube, centrifuge at 12,000 rpm for 1 minute at 4°C (use a refrigerated centrifuge), discard the supernatant (prepare as many centrifuged bacterial liquids as needed for the competent cells).
3. Add 1 ml of ice-bathed CaCl2 (calcium chloride concentration of 0.1 mol/l, i.e., dissolve 1.11 g of calcium chloride in 100 ml of sterile water) to each tube, gently mix, and ice bath for 30 minutes. (Be gentle, handle the bacterial liquid on ice)
4. Centrifuge at 12,000 rpm for 1 minute, invert to remove residual liquid, then add 100 µl of 0.1 mol/l calcium chloride, the competent cells are prepared, store at -80°C.
5. Add 50 ng of plasmid to the competent cells. If using PCR products, add 10 µl after homologous recombination. Ice-bathe for 30 minutes (thaw the competent cells on ice; maintain ice-bathed conditions throughout the procedure; ensure pipette tips remain below the liquid surface without touching the walls).
6. (Preheat the water bath in advance) Heat shock at 42°C for 60-90 seconds.
7. Quickly ice bath for 2-3 minutes (must be fast and the ice bath time should not be too long, usually take 2 and a half minutes as a compromise).
8. Add 1000 µl of LB medium, culture at 37°C at 180 rpm for 1 hour to recover.
9. Centrifuge at 12,000 rpm for 1 minute, discard the supernatant 800 µl, gently mix.
10. Take the entire resuspended bacterial suspension and spread it on LB medium with the corresponding antibiotics, spread evenly with a glass rod, seal (to prevent water loss), and culture upside down for 12-16 hours. (If it is a plasmid obtained through the PCR process, spread it all).

The entire transformation process must be carried out in a biosafety cabinet and an alcohol lamp must be ignited, the pipette tips, culture medium, and EP tubes must be sterilized.

---

# **Expression**

## **1. Bacterial Culture and Target Protein Expression**

1. **Colony Picking:** Inoculate 100ul glycerol stock culture into 20 mL of LB liquid medium with the appropriate antibiotic, incubate overnight at 37°C.
2. **Expression:**Add 500 µL antibiotic to 500 mL MR medium, then inoculate with 5 mL (1%) bacterial culture. Incubate at 37°C,for 2-3 hours until OD600 reaches 0.6.
3. **Induction:**Once the OD600 reaches the target range, induce protein expression according to the medium used:
   * For MR Medium: Add 400 µL of 0.5M IPTG (to a final concentration of 0.4 mM).Add 3.75 mL of 200 mg/mL L-arabinose (to a final concentration of 1.5 mg/mL). Incubate at 30°C, 200 rpm for 20 hours.
   * For TB Medium: Add 5400 µL of 0.5M IPTG (to a final concentration of 0.54 mM). Incubate at 20°C, 200 rpm for 20 hours.

### **Synthesis of PEA**

1. **Colony Picking:** Inoculate 100ul glycerol stock culture into 20 mL of LB liquid medium with the appropriate antibiotic, incubate overnight at 37°C.
2. **Expression:**Add 500 µL antibiotic to 500 mL MR medium, then inoculate with 5 mL (1%) bacterial culture. Incubate at 37°C,for 2-3 hours until OD600 reaches 0.6.
3. **Induction:**Add 400 µL(final concentration:0.4mM) of 0.5M IPTG(IPTG supplementation was required only for the PEA02-BL2112 (DE3) group), 3.75ml (final concentration:1.5mg/ml) of 200mg/ml L-arabinose, 25ul of 200 mg/mL Thiamine, 2.5 ml of a 400mg/mL solution of each monomer(sodium(RS)-3-hydroxybutyrate and 3-Aminopropanoic acid, monomer supplementation was required only for the PEA01-BL2112 (DE3) group), and incubate at 30°C, 200 rpm for 96 hours.

## **2. Bacterial Culture Harvesting and Pellet Resuspension**

1. Take four 50 mL centrifuge tubes, and add 50 mL of bacterial culture to each tube, balancing the tubes appropriately.
2. Centrifuge at 3500 rpm, 4℃ for 15 minutes.
3. Discard the supernatant, and resuspend the bacterial pellet in each tube with 5-10 mL of PBS buffer. Combine the resuspended cultures into a single centrifuge tube, and store at -20°C.
4. Depending on the total volume of the bacterial culture (e.g., 400 mL), repeat the above steps as necessary.

---

# **Verification**

## **1. Bacterial Lysis**

1. Transfer 20-50 mL bacterial culture to a 50 mL beaker.
2. Place the beaker in a larger beaker filled with ice, keeping the ice 1-2 cm below the small beaker's rim to prevent water backflow.
3. Sonicate at 170-190W, 1s on/1.5s off, for 15 minutes (avoid excessive agitation or foaming).
4. Stop when the liquid turns clear and less viscous; if not, extend by 5-10 minutes.
5. Centrifuge lysate at 12,000 rpm for 1.5 hours.
6. Collect supernatant into a 50 mL centrifuge tube.

## **2. Nickel Column Purification**

1. Remove nickel column from 4°C, drain ethanol solution.
2. Equilibrate column with 1× PBS buffer three times.
3. Equilibrate with 10 mM imidazole solution.
4. Add 10-15 mL supernatant to column, invert to mix, incubate on ice for 45 minutes.
5. Attach tubing, secure column, and collect flow-through.
6. Wash with 30 mM imidazole buffer at 2-3 drops/sec.
7. Test impurities with Coomassie Brilliant Blue; continue washing if blue.
8. Elute protein with 300 mM imidazole, incubate 5 minutes, and collect in 2 mL tubes.
9. Repeat elution if strong fluorescence; collect 15-20 mL, store at 4°C.
10. Wash column with 1× PBS, store in 20% ethanol at 4°C.

## **3. Coomassie Brilliant Blue Assay for Protein Concentration**

1. **Standard Curve Preparation:**Mix 1 mL Coomassie Brilliant Blue solution with 20 µL protein standard. After 1.5 minutes, measure absorbance at OD595 to create the standard curve.
2. **Sample Measurement:**Mix 1 mL Coomassie Brilliant Blue solution with 20 µL protein sample. After 1.5 minutes, measure OD595, and use the standard curve to determine protein concentration.

## **4. SDS-PAGE**

### **4.1 Preparation of polyacrylamide gels**

1. Installation of the gel board model: Wash the glass board, dry it and set it aside. When making glue, choose the right glass plate, assemble the glue plate model, put the glass plate into the rack after alignment, and the clamps on both sides are tightened, so that the short glass is leaning out and the long glass is leaning in.
2. According to the following table formula configuration of the separation of the glue solution, mixing with a light hand shaking, carefully inject the mixture into the prepared glass plate gap, leaving enough space for the concentration of the glue (~ 2.5cm), gently in the top layer of the addition of 0.5ml of deionized water to cover the (liquid sealing should be slow, to avoid deformation of the glue is washed out), in order to stop the inhibition of the air oxygen on the coagulation. Just add water can be seen between the water and the gel liquid interface, and then gradually disappear, soon appeared again interface, which indicates that the gel has been polymerized. And then leave a few moments to make the polymerization complete, the whole process takes about 30 minutes (25 ℃ room temperature).

Separation gel concentration | Gel volume | Volume of each component required(in ml) | | | | ||  |  | H2O | 30%Acr-Bis（29：1） | SDS-PAGE Separating Gel Buffer(4×） | 10%APS | TEMED |
| 6% | 5ml | 2.75 | 1.0 | 1.25 | 0.05 | 0.004 |
| 8% | 5ml | 2.42 | 1.33 | 1.25 | 0.05 | 0.003 |
| 10% | 5ml | 2.08 | 1.67 | 1.25 | 0.05 | 0.004 |
| 12% | 5ml | 1.75 | 2.0 | 1.25 | 0.05 | 0.002 |
| 15% | 5ml | 1.25 | 2.5 | 1.25 | 0.05 | 0.002 |

3. The preparation of 5% concentrated gel: the first has been polymerized to separate the upper layer of the gel water suction, and then use filter paper to absorb the residual water. Prepare the concentrated gel solution according to the following formula(Take preparing a piece of glue, about 2 mL, as an example). Mix it and inject it into the upper end of the separation gel, insert the comb and be careful to avoid air bubbles.

| Reagent | Volume(mL) | Purpose |
| --- | --- | --- |
| H₂0 | 1.4 | Solvent |
| 30% Acrylamide/Bis Solution (29:1) | 0.33 | Gel matrix formation |
| 0.5 M Tris-HCI (pH 6.8) | 0.25 | Provides stacking pH environment |
| 10% (w/v) SDS | 0.02 | Denaturant and charge provider |
| 10% (w/v) Ammonium Persulfate (APS) | 0.02 | Polymerization initiator (free-radical source) |
| TEMED | 0.002 | Polymerization catalyst |

4. After the polymerization of concentrated gel is complete, put the gel template into the electrophoresis tank and fix it, with the small glass plate facing inward and the large glass plate facing outward, add 1× electrophoresis buffer to both the upper and lower tanks, and carefully pull out the comb, check for leakage, and remove away air bubbles at the bottom of the gel between the two glass plates.

### **4.2 Sample Preparation and Loading**

1. While the gel concentrate is being polymerized, mix the protein sample with an equal volume of 4× sample buffer, denature in a water or metal bath at 95°C for 10 min, and cool to room temperature for use.
2. Using a micro-syringe, add the standard solution and denatured protein sample (typically 10–20 μL) to the bottom of the sample well.

### **4.3 Electrophoresis and Staining**

1. Connect the power supply. Use a constant voltage of 80-100V during the concentration gel stage. When the bromophenol blue band enters the separation gel, increase the voltage to 120-150V.
2. When the bromophenol blue band migrates to approximately 0.5 cm from the bottom of the gel, turn off the power supply to stop electrophoresis.
3. Carefully pry open the glass plate, remove the gel, and trim off the concentrating gel. Place the gel into a container, add sufficient Coomassie Brilliant Blue staining solution, and slowly agitate on a shaker for 1-2 hours.
4. Drain the staining solution (reusable).
5. Add sufficient decolorizing solution and decolorize on a shaker, replacing the solution several times until the background is transparent and protein bands are clearly visible.

## **5. Preparation of Bacterial Lyophilized Powder Using a Freeze Dryer**

1. After 96 hours of induction, centrifuge to collect the bacterial cells. Discard the supernatant completely. Resuspend the bacterial pellet in pre-chilled 1x PBS buffer, gently pipette to ensure uniformity, and prepare a high-concentration bacterial suspension.
2. In a laminar flow hood, aliquot the bacterial suspension into sterile lyophilization vials at 0.5–1.0 mL per vial. Avoid touching the vial walls during aliquoting.
3. Immediately transfer the aliquoted vials (opening facing upward) to a -80°C ultra-low temperature freezer. Freeze for at least 4 hours or overnight to ensure complete solidification of the samples.
4. Pre-cool the freeze dryer to below -50°C.
5. Swiftly remove pre-frozen samples from the freezer and immediately transfer them to the freeze dryer's cold trap shelf. This process must be rapid to prevent sample thawing.
6. Cover with the glass dome and activate the vacuum pump. Freeze dry at -50°C for 24 to 36 hours.
7. Upon completion of freeze-drying, promptly remove the samples from the freeze dryer. Immediately seal the vial opening with a sterile rubber stopper in a laminar flow hood, then firmly press the aluminum cap to achieve a vacuum seal. Store the sealed bacterial lyophilized powder sample tubes at 4°C in the dark and label them accordingly.

## **6. Purification of PEA**

1. Mix the freeze-dried bacterial cells with an appropriate amount of chloroform. Incubate at 45°C with shaking for 3-4 hours to fully extract PEA from the cells.
2. Filter the chloroform extract through a 0.45 μm membrane filter to remove bacterial debris and other insoluble impurities. Collect the clear filtrate.
3. Place the filtered, clear chloroform extract in a fume hood. Allow the chloroform to evaporate naturally under airflow until the solution concentrates to approximately 1/10 of its original volume or until viscous material precipitates.
4. Add 100 μl of chloroform to resuspend the precipitate, then add 1 ml of methanol. Centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant. After the methanol has completely evaporated, resuspend the pellet in 500μl of PBS buffer.

## **7. Nile Red Staining**

1. Thoroughly resuspend the purified PEA precipitate in 1 mL of PBS buffer by pipetting. Perform a serial dilution of this PEA suspension using PBS buffer to prepare sample suspensions with optical densities (OD₆₀₀) of 1.0, 0.5, and 0.3 at 600 nm.
2. Transfer 1 mL of each PEA sample suspension (OD=1.0, 0.5, 0.3) into separate 1.5 mL centrifuge tubes.
3. Precisely add 3 μL of Nile Red stock solution to each tube.
4. Immediately vortex to mix thoroughly, maintaining complete protection from light throughout (e.g., wrap tubes in aluminum foil).
5. Place the light-protected tubes on a shaker and incubate at 25°C under dark conditions at 200 rpm for 20 minutes to ensure complete dye binding to PEA.
6. After incubation, centrifuge tubes at 12,000 rpm for 1 minute to precipitate PEA. Carefully discard the supernatant (containing unbound free dye).
7. Add 1 mL of pre-chilled PBS buffer to each tube containing the precipitate. Gently resuspend the pellet by pipetting or vortexing to ensure complete and uniform dispersion.
8. Transfer the resuspended sample to a cuvette for detection using a fluorometer. Set the excitation wavelength (Ex) to 530 nm and measure the fluorescence emission intensity at 610 nm (emission wavelength Em). Simultaneously measure the fluorescence value of 1 mL PBS buffer alone as a blank control.

## **8. FTIR**

Sample Preparation: Take approximately 1–2 mg of purified and dried PEA sample (white powder or film form). Ensure the sample is dry with no moisture or solvent residues.

## **9. TEM**

### **Sample Preparation Before Submission:**

1. Take a sample approximately the size of a mung bean and place it in a 1.5 mL centrifuge tube.
2. Add an appropriate amount of 2.5% glutaraldehyde solution and gently pipette to mix.
3. Take 500 μL of the above preliminarily fixed sample suspension and add it to a centrifuge tube containing 9.5 mL of PBS. Securely cap the tube and gently invert it 10 times to ensure thorough mixing.
4. Tightly wrap the tube cap and body junction with sealing film to prevent leakage during transport.
5. Place the sealed tube upright in a dedicated foam box or sturdy cardboard box. Surround the box with pre-frozen ice packs to maintain a transport temperature of approximately 4°C.

## **10. Purification of K5C via Inverse Transition Cycle (ITC)**

1. After sonication of cells, centrifuge the lysate at 4°C and 10,000 rpm for 30 minutes. Collect the supernatant.
2. Add NaCl to the supernatant to a final concentration of 2 M.
3. Incubate the solution at 35°C for 1 hour to induce the K5C phase transition.
4. Centrifuge at 10,000 rpm, 35°C for 15 minutes. Retain the supernatant for analysis and collect the K5C-containing pellet.
5. Resuspend the pellet in pre-chilled buffer (200 mM PB buffer, pH 7.0) and incubate on ice for 30 minutes to solubilize K5C.
6. Incubate the solubilized mixture at 35°C for 1 hour, then centrifuge at 10,000 rpm and 35°C for 15 minutes. Resuspend the pellet in pre-chilled buffer and incubate on ice for 1 hour to dissolve.
7. After final dissolution, centrifuge at 4°C and 10,000 rpm for 15 minutes. Collect the supernatant containing purified K5C; the pellet may be retained for subsequent analysis.

## **11. Bionic Mineralization for Preparing K5C@Fe₃O₄ Carrier**

1. Prepare precursor solution A (2 mL): Dissolve 0.16 g FeCl₃ and 0.65 g FeCl₂·4H₂O in ultrapure water, then mix with purified K5C protein to a final concentration of 900 μg/mL.
2. Prepare precursor solution B (5 mL): 3.0 M NaOH solution.
3. Slowly add solution B to solution A under gentle shaking (100 rpm), ensuring the pipette tip remains below the liquid surface to avoid localized high pH.
4. Incubate the mixture by shaking at 35°C and 180 rpm for 30 minutes. Observe the solution darkening, indicating Fe₃O₄ nanoparticle formation.
5. Attach a strong magnet to the outer wall of the reaction vessel and separate the K5C@Fe₃O₄ nanoparticles via magnetic force.
6. Discard the supernatant and wash the nanoparticles with an excess of buffer (200 mM PB buffer, pH 7.0). Repeat the magnetic separation and washing steps at least twice to remove unbound components.
7. Suspend the final K5C@Fe₃O₄ nanoparticles in suitable buffer (200 mM PB buffer, pH 7.0) and store at 4°C for later use.

## **12. Covalent Immobilization of Enzymes and PET Degradation Detection**

1. In a 1 mL reaction system, add 250 μL of SpyTag-TfCa-SnoopCatcher enzyme solution to 500 μL of pre-prepared bioactive magnetic bead suspension. Incubate at 37°C with shaking at 180 rpm for 4 hours to achieve covalent immobilization of TfCa.
2. After immobilization, wash with PB buffer (200 mM, pH 7.0). Use a strong magnet to adhere the beads to the tube wall, then carefully discard the supernatant. Wash the beads three times to remove unbound enzyme.
3. Add 250 μL of SnoopCatcher-Tfh enzyme solution to the magnetic beads and incubate for 4 hours under the same conditions (37°C, 180 rpm) to immobilize the second enzyme.
4. After the second immobilization, wash the beads three times with PB buffer. Centrifuge at 12,000 rpm for 1 minute and carefully discard the supernatant.
5. Resuspend the final immobilized enzyme complex in glycine buffer (pH 9.0) to a final volume of 1 mL.
6. Add 10 mg of solid PET microplastics to the reaction mixture and incubate with shaking at 40°C for 24 hours.
7. After incubation, centrifuge at 12,000 rpm for 2 minutes and collect the supernatant for subsequent product analysis.

## **13. HPLC**

Prior to HPLC analysis, samples require thermal treatment. Heat the sample solution at 90°C for 15 minutes to promote compound dissolution and reaction. Following thermal treatment, centrifuge at 12,000 rpm for 20 minutes to precipitate insoluble material. Carefully collect the supernatant and perform high-performance liquid chromatography (HPLC) analysis using the Waters E2695 system to quantitatively determine compound concentrations.

### **13.1 HPLC analysis**

A Waters E2695 chromatographic system, equipped with a Shim-pack GIS column, was employed in this study for HPLC analysis. The mobile phase was comprised of a mixture of methanol and acetonitrile, flowing at a rate of 0.5 mL/min, with detection at a wavelength of 254 nm. The elution protocol commenced with a gradient elution using 95% formic acid and 5% from 0 to 2 minutes. This was followed by a linear gradient elution, increasing the acetonitrile concentration to 20% while maintaining 80% formic acid from 2 to 14 minutes. The system was then re-equilibrated to the initial conditions of 95% formic acid and 5% acetonitrile over the 14 to 15-minute interval, employing a gradient pump mode throughout the entire chromatographic run. The total peak areas of MHET, terephthalic acid (TPA) and PCA were utilized in determining the quantity of product from each PET hydrolysis reaction.

### **13.2 Standard Curve of HPLC analysis**

Prepare stock solutions of tetradecanoylphorbol acetate (TPA), p-coumaric acid (PCA) and methyl 4-hydroxy-3-methoxyphenyl ether (MHET) at a concentration of 10 mg/mL, with TPA specifically dissolved in dimethyl sulfoxide (DMSO). For the construction of a standard curve, dilute these stock solutions to prepare a series of mixed solutions with final concentrations of 0.05, 0.1, 0.25, 0.5, 1, and 2 mM. Subject the mixed solutions to thermal treatment at 90°C for 15 minutes to facilitate compound solubilization and reaction. Post-thermal treatment, centrifuge the solutions at 12,000 rpm for 20 minutes to pellet any insoluble material. Carefully collect the supernatant and proceed with high-performance liquid chromatography (HPLC) analysis using a Waters E2695 system to quantify the compounds and establish the standard curve.

## **14. Ethylene glycol（EG）detection**

### **14.1 Preparation of Gold Nanoparticles**

1. Mix 1 mL of chloroauric acid with 49 mL water, then heat to boiling or until bubbles form.
2. Add 2 mL of 1% trisodium citrate (pre-prepared) all at once. Insert a stirring bar and heat for 30 minutes.
3. Turn off heat and stir for 15 minutes.
4. Store at 4°C.

### **14.2 EG Detection**

1. Prepare a series of EG solutions from the 1 M stock with final concentrations of 0, 10, 20, 30, 40, 50, 100, and 150 mM.
2. Mix 0.8 mL of the gold nanoparticle solution with each prepared EG solution.
3. Record the color and spectral data using a sensor and a UV-vis spectrophotometer.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Results

* [1. Biodegradation of PET](#PETBiodegradation)
  + [1.1 Plasmid Construction & Protein Expression](#PETPlasmidProtein)
  + [1.2 Optimization of Immobilized System](#PETImmobilizedOpt)
  + [1.3 Conclusion & Outlook](#PETConclusion)
* [2. Upcycling of EG to PEAs](#EGtoPEAs)
  + [2.1 Engineering EG as Carbon Source](#EGCarbonSource)
  + [2.2 Detection of EG](#EGDetection)
  + [2.3 Synthesis & Detection of PEAs](#PEASynthesis)
  + [2.4 Conclusion & Outlook](#EGConclusion)
* [3. Upcycling of TPA to PCA](#TPAtoPCA)
  + [3.1 Plasmid Construction & Enzyme Optimization](#TPAEnzymeOpt)
  + [3.2 HPLC Analysis of TPA-to-PCA Conversion](#TPAHPLCAnalysis)
  + [3.3 Conclusion & Outlook](#TPAConclusion)

# **1. PET Biodegradation**

## **1.1 Plasmid Construction & Protein Expression**

To address the challenges associated with the degradation of PET by free enzymes, namely the accumulation of intermediate products and difficulties in post-reaction recovery, we prepared a functionalized magnetic carrier, K5C@Fe3O4, via a one-step biomimetic mineralization method. This carrier was designed for the sequential immobilization of SpyTag-TfCa-SnoopTag and SnoopCatcher-Tfh. Towards this end, we constructed three plasmids: piGEM25\_01 for the expression of K5C, piGEM25\_02 for SpyTag-TfCa-SnoopTag, and piGEM25\_03 for SnoopCatcher-Tfh. The results from PCR (Figure 1D) and subsequent sequencing confirmed the successful construction of all three plasmids. We first expressed K5C and successfully purified it by leveraging the thermosensitive property of its ELP (Elastin-like polypeptide) tag via the Inverse Transition Cycling (ITC) method. Subsequently, the SpyTag-TfCa-SnoopTag and SnoopCatcher-Tfh proteins were also expressed. The SDS-PAGE analysis, shown in Figures 1E and 1F, confirmed the soluble expression of each protein.

![Plasmid profile of piGEM25_01(A), piGEM25_02(B), piGEM25_03(C), 1% agarose gel electrophoresis results of PCR resutls(D), 15% SDS-PAGE resutls (E and F)](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p1.webp)

Figure 1: Plasmid profile of piGEM25\_01(A), piGEM25\_02(B), piGEM25\_03(C), 1% agarose gel electrophoresis results of PCR resutls(D), 15% SDS-PAGE resutls (E and F). In panel D, M: Trans 5K DNA Marker; Lane 1: pET-30(a); Lanes 2-3: pET-30(a)-SpyTag-Tfca-SnoopTag; Lane 4: pET-30(a)-SnoopCacther-Tfh; Lane 5: pET-30(a). In panel E, M: protein marker; Lanes 1-4 depict the purification of ELP-SC via ITC: Lane 1: Insoluble pellet after lysis of cells expressing ELP-SC; Lane 2: Product after the first ITC purification cycle; Lane 3: Highly purified ELP-SC after the second cycle; Lane 4: Soluble impurities removed by centrifugation. In panel F, Lane 2: Crude lysate of cells expressing SpyTag-TfCa-SnoopTag; Lane 4 : Crude lysate of cells expressing SnoopCatcher-Tfh.

## **1.2 Optimization of the Immobilized Dual-Enzyme System**

To enhance the immobilization efficiency of SpyTag-TfCa-SnoopTag, the preparation conditions for the magnetic carrier were optimized. Figure 2A reveals that 3.0 M NaOH is the optimal concentration for the mineralization reaction. In this study, the concentrations of the products, terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET), were determined using High-Performance Liquid Chromatography (HPLC). A comparison was made between two strategies: sequential immobilization (immobilizing TfCa first, followed by Tfh) and co-immobilization (mixing both enzymes with the carrier in a single step). Figure 2B indicates that the sequential immobilization method yielded significantly higher degradation efficiency, with the concentration of the sole product, TPA, successfully detected at 1.2 mM. To further evaluate the recyclability of the system, the magnetic particles were recovered post-reaction and reused in a new round of PET degradation. As shown in Figure 2C, the system retained 61% of its initial degradation activity after 10 cycles of reaction and recovery, demonstrating its excellent operational stability and potential for repeated use.

![Biochemical characterization of the immobilized dual-enzyme system](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p2.webp)

Figure 2: Biochemical characterization of the immobilized dual-enzyme system. (A) Effect of NaOH concentration during biomineralization on the activity of the subsequently immobilized enzyme, determined by HPLC measurement of TPA produced from MHET degradation. (B) Comparison of PET degradation activity between two immobilization strategies: Group A, sequential immobilization (TfCa then Tfh); Group B, co-immobilization (all components mixed at once). (C) Reusability assessment of the immobilized system over 10 reaction cycles. The relative activity was measured after each cycle, with the activity of the first cycle defined as 100%. Error bars in (B) and (C) represent the standard deviation from three independent experiments.

## **1.3 Conclusion & Outlook**

By integrating ELP-based thermosensitive purification, biomimetic mineralization, and specific self-assembling covalent protein immobilization, this study successfully constructed a magnetically recyclable, immobilized dual-enzyme system for the efficient degradation of PET. The sequential immobilization strategy was identified as the optimal assembly method for this system. In the future, the system's performance can be further enhanced by optimizing the dual-enzyme ratio and modifying the structure of the linker peptides.

---

# **2. Upcycling of EG to PEAs**

To further upgrade and utilize the EG produced in the previous step, we conducted the following experiments. This diagram systematically illustrates the complete synthetic pathway designed in this study, converting ethylene glycol (EG)—a byproduct of PET degradation—into high-value polyethylene succinate (PEA).

![Complete metabolic pathway for the conversion of ethylene glycol (EG) to poly(ester amide) (PEA) in engineered E. coli](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p3-1.webp)

Figure 3: Complete metabolic pathway for the conversion of ethylene glycol (EG) to poly(ester amide) (PEA) in engineered E. coli. fucO, lactaldehyde reductase; aldA, aldehyde dehydrogenase A; glcDEF, glycolate dehydrogenase; glcB, malate synthase G; gcl, glyoxylate carboligase; hyi, hydroxypyruvate isomerase; glxK, glycerate 2-kinase 2; glxR, tartronate semialdehyde reductase 2; garR, Glyoxylate Reductase; phaA: acetyl-CoA acetyltransferase; phaB: acetoacetyl-CoA reductase; phaC: polyhydroxyalkanoate synthase; panD: aspartate decarboxylase; act: β-alanine CoA-transferase; pct540: propionate CoA-transferase mutant; EG, Ethylene glycol; GLA, glycolaldehyde; GA, Glycolate; GLO, Glyoxylate; TSA, (2R)-tartronate semialdehyde; (OH)-PYR, hydroxypyruvate; GLR, D-glycerate; 2PG, 2-phospho-D-glycerate; MAL, malate

## **2.1 Engineering E. coli for EG Utilization and Detection**

Since wild-type E. coli cannot utilize EG as a carbon source, to enable BL21(DE3) to efficiently utilize EG, we first established a positive baseline using the previously reported four-gene combination gcl, hyi, glxR, glxK (constructed as piGEM25\_05). Building upon this, based on the minimal critical node recommendations proposed by modeling, we retained gcl and glxK, introduced the isoenzyme garR to replace glxR, and evaluated the extended value of the upstream fucO and aldA. Three expression combinations were constructed in parallel for comparison: piGEM25\_05 (gcl, hyi, glxR, glxK, literature baseline control), piGEM25\_04 (gcl, garR, glxK, model-driven minimal combination), piGEM25\_06 (fucO, aldA, upstream extension validation module). The rationale and logic for gene selection are detailed in the Model section. The plasmid map is shown in (Figure 4A-C). PCR amplification and electrophoresis validation confirmed that the target band size matched expectations (Figure 4D). Sequencing results further confirmed the successful construction of this recombinant plasmid.

![Plasmid profile of piGEM25_04(A), piGEM25_05(B), piGEM25_06(C), 1% agarose gel electrophoresis results of PCR resutls (D)](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p4-1.webp)

Figure 4: Plasmid profile of piGEM25\_04(A), piGEM25\_05(B), piGEM25\_06(C), 1% agarose gel electrophoresis results of PCR resutls (D) . In panel D, M: Trans 5K DNA Marker; Lane 1:pCDFDuet-1(linearized); Lane 2:gcl; Lane 3:garR; Lane 4:glxR; Lane 5:glxK; Lane 6:hyi; Lane 7:PRSFDuet-1(linearized); Lane 8: fuco; Lane 9:aldA;

In this experiment, the functional differences in ethylene glycol (EG) metabolism among three distinct gene combinations were systematically compared. As shown in Figure 5, both engineered strains EG04-BL21(DE3) (expressing piGEM25\_04) and EG05-BL21(DE3) (expressing piGEM25\_05) effectively utilized EG as the sole carbon source for growth. Specifically, after 72 hours of cultivation, EG04-BL21(DE3) reached an OD600 of 0.218 with an EG degradation rate of 28%; EG05-BL21(DE3) exhibited an OD600 of 0.24 with a 24% EG degradation rate. While both groups demonstrated comparable metabolic efficiency, the three-gene combination employed in EG04-BL21(DE3) achieved streamlined optimization of the metabolic pathway. Furthermore, the co-transformed strain EG04-06-BL21(DE3) (simultaneously expressing piGEM25\_04 and piGEM25\_06) and the blank control EV-BL21(DE3) failed to grow in medium where EG served as the sole carbon source.

![Rational engineering of BL21 (DE3) chassis cells utilizing ethylene glycol as the sole carbon source](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p5-1.webp)

Figure 5: Rational engineering of BL21 (DE3) chassis cells utilizing ethylene glycol as the sole carbon source.Error bars indicate standard error (n = 3)

## **2.2 Ethylene glycol（EG）detection**

### 2.2.1 UV-Vis Spectral measurement results

To conduct real-time monitoring of the concentration during the EG assimilation process, we have developed an EG detection system for this purpose，The changes in the spectra were detected using a UV-Vis spectrophotometer. As shown in Figure 6A, the increase in EG concentration led to an enhancement in the intensity of the characteristic absorption peak (SPR peak) of the gold nanoparticles at 525 nm, but no spectral shift was observed. Figure 6B shows that the addition of different concentrations of EG to the gold nanoparticle solution resulted in color changes that were indistinguishable to the naked eye. These indicates that the size and shape of the gold nanoparticles did not change significantly during the experiment, and no notable particle aggregation occurred. This further confirms that the change in peak intensity is primarily attributed to the alteration of the dielectric environment (refractive index), rather than changes in the intrinsic properties of the nanoparticles. As shown in the Figure 6C, there was a linear relationship between A525 value and EG concentration in the range of 0-150mM, with a linear regression correlation coefficient of 0.988. This result indicated that UV-Vis absorption method is promising for the quantification analysis of EG concentration.

![The UV-Vis spectra obtained in the presence of different concentrations of EG](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p6.webp)

Figure 6A: The UV-Vis spectra obtained in the presence of different concentrations of EG.

![Color changes of the gold nanoparticle solution with increasing EG concentration (0, 10, 30, 50, 100 mM)](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p7.webp)

Figure 6B: Color changes of the gold nanoparticle solution with increasing EG concentration (0, 10, 30, 50, 100 mM).

![Linear fitting curve of absorbance at 525 nm versus EG concentration detected by UV-vis spectrophotometer](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p8.webp)

Figure 6C: Linear fitting curve of absorbance at 525 nm versus EG concentration detected by UV-vis spectrophotometer.

### 2.2.2 Based on the detection results from the AS7341 sensor

The prepared 800 µL AuNPs solution was mixed with 1200 µL of EG standards at varying concentrations in an LB medium system and introduced into a cuvette for sensor detection. The AS7341 sensor was activated to sequentially illuminate the sample with six LEDs aligned opposite the light-transmitting hole of the cuvette holder. Light passing through the sample was captured by the AS7341 spectral sensor, which acquired absorption spectral data at a wavelength of 525 nm. Subsequently, using the spectral data, a calibration plot was constructed to establish a linear relationship between the absorbance at 525 nm and EG concentration. As shown in Figure 7, the A525 value exhibited a linear correlation with EG concentration over the range of 0–150 mM, with a linear regression correlation coefficient of 0.9734, indicating that the method we designed here was accurate and feasible for the detection of EG.

![the linear fitting curve of the absorbance value at 525 nm versus the EG concentration](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p9.webp)

Figure 7: the linear fitting curve of the absorbance value at 525 nm versus the EG concentration.

## **2.3 Synthesis & Detection of PEAs**

### 2.3.1 Design and Construction of the PEA Synthesis Pathway

After resolving the issue of wild-type E. coli's inability to utilize EG as a carbon source, we attempted to achieve the conversion of EG into biodegradable polyester amide (PEAs) within E. coli. This required first constructing a synthetic pathway from natural amino acids to PEAs in BL12 (DE3). We designed and constructed the piGEM25\_07 plasmid to synthesize polymer monomers (sodium RS-3-hydroxybutyrate and 3-aminopropionic acid) (Figure 8A) and the piGEM25\_08 plasmid to activate and polymerize these monomers (Figure 8B). As shown in Figure 8C, PCR results and subsequent sequencing confirmed the successful construction of both recombinant plasmids. Expression of the target protein was then investigated. SDS-PAGE results in Figure 8D demonstrate the successful soluble expression of the key enzyme gene.

![Plasmid profile of piGEM25_07(A), piGEM25_08(B), 1% agarose gel electrophoresis results of PCR resutls (C)and 10% SDS-PAGE resutls (D)](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p10-1.webp)

Figure 8: Plasmid profile of piGEM25\_07(A), piGEM25\_08(B), 1% agarose gel electrophoresis results of PCR resutls (C)and 10% SDS-PAGE resutls (D). In panel C, M: Trans 5K DNA Marker; Lanes 1:pGro7; Lanes 2:phaC; Lanes 3:Pct540; Lanes 4:Act; Lanes 5:pTrc99a; Lanes 6:phaA; Lanes 7:phaB; Lanes 8: panD; In panel D, M: protein marker ; Lanes 1 and 2 is the supernatant and pellets of the lysed E. coli cell expressing piGEM25\_08 Proteins(Control group, without LA) ; Lanes 3 and 4 is the supernatant and pellets of the lysed E. coli cell expressing piGEM25\_08 Proteins (Experimental group, with LA induction) ; Lanes 5 and 6 is the supernatant and pellets of the lysed E. coli cell expressing piGEM25\_07 and piGEM25\_08 Proteins (Control group, without IPTG and LA) ; Lanes 7 and 8 is the supernatant and pellets of the lysed E. coli cell expressing piGEM25\_07 and piGEM25\_08 Proteins (Experimental group, with IPTG and LA induction);

### 2.3.2 Synthesis of PEA and Identification by Nile Red Staining

To validate whether the PEA synthesis pathway constructed in BL12 (DE3) can synthesize PEA, we engineered two strains: PEA01-BL12 (DE3) (transformed with only plasmid piGEM25\_08, requiring exogenous monomer addition for PEA synthesis) and PEA02-BL12 (DE3) (co-transformed with piGEM25\_07 and piGEM25\_08). A control group, EV-BL21(DE3) (transformed with piGEM25\_08 alone, without inducer), was established. After 72 hours of cultivation, PEA01-BL12 (DE3), PEA02-BL12 (DE3), and EV-BL21(DE3) were cultured for 72 hours. After incubation, samples with different OD600 values (OD600=0.3, 0.5, 1.0) to characterize the presence of PEAs within E. coli cells. As shown by the bacterial cell colors in Figure 9A-C, the control strain EV-BL21(DE3) was not stained by Nile red, while PEA01-BL12 (DE3) and PEA02-BL12 (DE3) were stained. The stained PEA02-BL12 (DE3) sample was then analyzed using a fluorescence spectrophotometer with excitation at 530 nm and emission measured at 610 nm. As shown in Figure 9D, compared to the control group, PEA02-BL12 (DE3) exhibited a strong fluorescence emission peak at 610 nm after Nile Red staining. The Nile Red staining experiment demonstrates that the de novo synthesis pathway for PEA constructed in BL21(DE3) successfully achieved PEA synthesis.

![Nile Red staining results of the control group(A), PEA01-BL12 (DE3)(B), and PEA02-BL12 (DE3)(C) and fluorescence spectrum analysis(D)](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p11-1.webp)

Figure 9: Nile Red staining results of the control group(A), PEA01-BL12 (DE3)(B), and PEA02-BL12 (DE3)(C) and fluorescence spectrum analysis(D). In panel A, Control group (EV-BL21(DE3)) bacterial samples at OD600 = 0.3, 0.5, and 1.0 (left to right). Cells were cultured in MR medium with 0.4 mM IPTG induction, 30°C for 96h. In panel B, PEA01-BL21(DE3) samples at OD600 = 0.3, 0.5, and 1.0 (left to right). Cultured in MR medium with 1.5mg/ml LA induction, 30°C for 96h. In panel C, PEA02-BL21(DE3) samples at OD600 = 0.3, 0.5, and 1.0 (left to right). Cultured in MR medium with 0.4 mM IPTG and 1.5mg/ml LA induction, 30°C for 96h, supplemented with 3HB and 3AP monomers. In panel D, fluorescence emission spectra of Nile Red-stained samples (excitation at 530 nm, emission measured at 610 nm). All samples were prepared by adding 3 μL of Nile Red stock solution to 1 mL of PEA suspension, followed by vortex mixing and incubation in the dark at 25 °C with shaking at 200 rpm for 20 min. After staining, samples were centrifuged (12,000 rpm, 1 min) to remove unbound dye, resuspended in PBS, and analyzed by fluorometry.

We further extracted intracellular polyesteramides (PEAs) and observed them under fluorescence microscopy after staining with Nile Red. As shown in Figure 10, compared to the control group without inducer addition, the PEAs extracted from the experimental group exhibited specific staining with Nile Red and emitted significant fluorescence under the microscope. This further validated our successful synthesis of PEAs using the whole-cell system.

![Fluorescence microscopy of intracellular PEAs stained with Nile Red](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p11-5.webp)

Figure 10: Fluorescence microscopy of intracellular PEAs stained with Nile Red. The experimental group （A）shows strong fluorescence, whereas the negative control （B）exhibits minimal background signal.

## **2.4 Conclusion & Outlook**

In this study, bioinformatics analysis identified key metabolic genes involved in EG utilization by Escherichia coli. Compared to the previously reported four-gene combination gcl–hyi–glxR–glxK, the gcl-garR-glxK combination maintains (and slightly enhances) EG utilization efficiency while reducing one gene module. Subsequent efforts may optimize the expression levels of these three genes and employ bioinformatics methods to identify more efficient metabolic pathways, thereby enhancing EG utilization in E. coli. Furthermore, to ultimately achieve efficient carbon flux conversion into intracellular polyesteramides (PEAs), we constructed a non-native metabolic pathway within E. coli, successfully achieving intracellular PEAs synthesis. However, current yields remain low. Future work will focus on optimizing gene expression within the pathway and refining extraction steps to enhance PEAs yield and extraction efficiency. Additionally, our project has successfully developed a simple, convenient, and specific EG detection method. The entire process requires no complex pretreatment; simply mixing the sample with the sensing solution yields results within minutes, significantly outperforming traditional chromatographic methods. We believe that in the near future, optical detection, signal processing, and display functions could even be integrated into a portable device to better serve societal needs.

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# **3. Upcycling of TPA to PCA**

Having addressed the valorization of EG, one of the PET degradation products, we turned our attention to upcycling the other major byproduct, terephthalic acid (TPA). This effort was motivated by a 2024 project from our university's UESTC-iGEM team, which encountered very low vanillin yields when converting TPA—one of the two main products of PET hydrolysis—into vanillin. We hypothesized that a key limiting factor was the insufficient concentration of protocatechuic acid (PCA), a precursor to vanillin. To address this, our project focused on optimizing the expression conditions for the enzymes in the TPA-to-PCA pathway. We first designed plasmid piGEM25\_09 to express TphA1, TphA2, TphA3, and TphB, which collectively catalyze the conversion of TPA to PCA. In a key departure from the previous project, our expression protocol included the supplementation of FeCl₃, L-cysteine, and ZnSO₄. The SDS-PAGE analysis in Figure 11B confirmed the successful soluble expression of these target proteins in the E. coli BL21(DE3) host. We then measured the concentration of the resulting PCA using HPLC and investigated the effect of cell density on TPA conversion efficiency. As shown in Figure 12D, at an OD600 of 60, the PCA titer reached 1250 μM.

![15% SDS-PAGE results of the pellets and supernant of empty vector and the lysed E. coli cell harbouring piGEM25_09 plasmid](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p12.webp)

Figure 11: 15% SDS-PAGE results of the pellets and supernant of empty vector (panel B: lane 1 and lane 2) and the lysed E. coli cell harbouring piGEM25\_09 plasmid (panel B: lane 3 and lane 4).

## **3.2 HPLC Analysis of TPA-to-PCA Conversion**

![HPLC analysis for the bioconversion of TPA to PCA](https://static.igem.wiki/teams/5979/uestcpic/wetlab/result/p13.webp)

Figure 12: HPLC analysis for the bioconversion of TPA to PCA. Standard curves obtained from HPLC for (A) mono-(2-hydroxyethyl) terephthalate (MHET), (B) terephthalic acid (TPA), and (C) protocatechuic acid (PCA). (D) Effect of bacterial optical density (OD₆₀₀) on the final concentration of PCA produced from TPA by the engineered E. coli.

## **3.3 Conclusion & Outlook**

We have successfully upcycled TPA into PCA using engineered E. coli that co-expresses multiple enzymes. Through the optimization of expression conditions for TphA1, TphA2, TphA3, and TphB, a substantial increase in PCA yield was achieved. Looking ahead, our future work will focus on addressing the challenge of TPA transmembrane transport to further elevate PCA production.

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