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.