Identification
Cycle 1: Construction of Anchoring Peptide Expression Vector
Design
The anchoring peptide gene was inserted into the plasmid pET-30a to create a fusion protein expression vector (Fig 1).
Build
The linearized vector and the inserted fragment were cloned and transformed into DH5α via heat shock. Positive clones were screened by PCR and Sanger sequencing (Shanghai Sangon Inc.). The plasmid extracted from DH5α was then transformed into BL21(DE3). After induction, the anchoring peptide-GFP fusion protein was purified using a nickel column. The anchoring peptide-GFP fusion protein was cleaved with TEV protease (at 30°C for 4 hours), yielding the His-tagged anchoring peptide and GFP without the His-tag. The cleavage mixture was then passed through a His gravity column (Takara, Beijing, China) to collect the anchoring peptide.
Test
Using the BCA assay and SDS-PAGE analysis, we found that the concentration of the anchoring peptide was low and the sample was impure.
Learn
In the original anchoring peptide vector, the TEV cleavage site was located after the 6xHis tag. TEV protease was used to cleave the TEV site on the fusion protein, leaving the His-tag on the anchoring peptide while GFP lacked the tag, allowing for separation and purification via a nickel column. However, since the TEV protease also contains a His tag, and the His-tagged anchoring peptide strongly adhered to the nickel column and was difficult to elute, it was discovered that TEV protease mixed with the purified anchoring peptide. Additionally, there was significant loss of the anchoring peptide during the process.
Cycle 2: Reversing the TEV Site and His-tag Order
Design
The vector was redesigned by swapping the positions of the TEV site and the 6xHis tag (Fig 1).
Build
Primers were redesigned for PCR to obtain the modified plasmid. The new plasmid was transformed into BL21(DE3), and after induction, the anchoring peptide-GFP fusion protein was purified using a nickel column. TEV protease was used to cleave the anchoring peptide-GFP fusion protein at 30°C for 4 hours.This yielded His-tagged GFP and anchoring peptide without the His-tag. The cleavage mixture was then passed through a His gravity column (Takara, Beijing, China) to collect the anchoring peptide.
Test
The newly constructed vector significantly increased the concentration of the anchoring peptide and eliminated TEV protease contamination.
Learn
The concentration of the anchoring peptide increased to meet the requirements of the subsequent experiments.
PET Biodegradation
Cycle 1: Enhancement of GC linker and Reordering of Component Assembly
Design
Taking into account the necessity for robust and efficient assembly, we have adopted a strategy that involves the separate assembly of functionally synthesized components. We have selected the pET-30a vector as the framework for this section. This vector offers well-documented advantages such as high expression levels, a strong promoter for efficient transcription, and a multiple cloning site that facilitates the insertion of multiple genes.
Construct
The plasmid BBa_K5487005 encompasses key elements: the membrane protein Lpp-OmpA and the green fluorescent protein sfGFP, which serve as a test for the efficiency of membrane protein linkage. This plasmid was constructed using homologous recombination seamless cloning, a method that efficiently inserts multiple genes into a vector with minimal effort and time. The process involves the amplification of the target genes with specific primers, followed by an overlap extension PCR. The direct transformation into competent E. coli cells DH5α allows for the in vivo recombination and integration of the genes into the plasmid. The plasmid BBa_K5487003 contains three critical proteins, connected in a sequential order: Lpp-OmpA, MHETase, and Z1-PETase, designed for the degradation of PET plastics. Subsequently, these plasmids were introduced into E. coli DH5α using the heat shock method, a widely used technique for transforming E. coli with foreign DNA. This optimization can lead to a higher yield of transformed cells, affecting the overall efficiency of the transformation process.
Testing
After transformation, E. coli DH5α was cultured overnight in LB medium containing kanamycin resistance, and plasmid extraction and sequencing revealed that the segments MHETase and Z1-PETase did not successfully integrate within the pET-30a framework.
Learning and Experimentation
Through literature review and consultation with peers, we discovered that several articles have incorporated multiple rigid GC linkers to enhance the efficiency of segment-to-segment connections. This insight has prompted us to reconsider our approach and explore the potential benefits of incorporating such linkers into our construct design.
Cycle 2: Chassis Organism Replacement
Design
Building upon the lessons learned from the first cycle, we recognized the necessity to enhance the functionality and expression of our key components, Lpp-OmpA, MHETase, and Z1-PETase. To achieve this, we strategically incorporated multiple rigid GC linkers within these elements to reinforce their structural stability and improve their interaction dynamics. Furthermore, we experimented with the sequence of MHETase and Z1-PETase to optimize their arrangement for enhanced expression and functionality.
Construct
With the revised order of Lpp-OmpA, Z1-PETase, and MHETase, we re-engineered the plasmid. We then decided to introduce it into E. coli BL21, a chassis organism known for its robust protein expression capabilities. This choice was made after considering the organism's compatibility with our construct and its potential to improve the overall performance of the system.
Testing
The transformed E. coli BL21 was cultured under conditions optimized for protein expression. Both the growth rate and the expression levels of the target proteins were closely monitored. The results were analyzed to assess the impact of the chassis organism on the construct's performance.
Learning and Experimentation
Upon reviewing the data from the tests, we observed that the growth rate and protein expression efficiency in E. coli BL21 were significantly improved compared to our initial trials with E. coli MG1655. This led us to conclude that E. coli BL21 might be a more suitable chassis organism for our construct. The decision to use BL21 was further supported by its well-documented history of successful use in protein expression studies and its ability to handle the metabolic demands of our complex construct.
In light of these findings, we will continue to refine our construct within the E. coli BL21 chassis, focusing on further optimization of the rigid GC linkers and the component arrangement. We will also explore additional genetic modifications and environmental factors that could potentially enhance the expression and functionality of our system. This iterative process will involve a combination of computational modeling, laboratory experimentation, and data analysis to fine-tune our approach and achieve the desired outcomes.
Cycle 3: Optimization of Degradation Conditions
Design
Following the successful expression of the target proteins in E. coli BL21 during the second cycle, we have now established a baseline for PET degradation under the initial conditions of 50°C in PBS buffer. With the detection of TPA, a key degradation product, we are poised to optimize the degradation process further. To enhance the efficiency and specificity of PET degradation, we will systematically vary the bacterial concentration (OD600), buffer pH, and IPTG induction concentration to identify the optimal conditions that maximize the degradation efficiency.
Construct
The plasmid containing the optimized sequence of Lpp-OmpA, Z1-PETase, and MHETase, along with the rigid GC linkers, remains unchanged. We will utilize this construct for all degradation experiments to ensure consistency and to isolate the effects of the varying conditions on the degradation process.
Testing
A series of degradation experiments will be conducted under the following conditions:
Bacterial Concentration (OD600): Varying the initial bacterial concentration to 1, 5, 20, and 50 to determine the optimal cell density for PET degradation.
Buffer pH: Buffers of different pH (7/8/9/10) were used to assess the impact of pH on the enzymatic activity and degradation rate.
IPTG Concentration: Testing different concentrations of IPTG to induce protein expression and identify the level that yields the highest degradation efficiency without causing undue stress to the bacterial cells.
Each condition will be tested in triplicate to ensure the reliability of the results. The degradation efficiency will be quantified by measuring the amount of TPA produced after 24 hours of incubation at 50°C.
Cycle 4: Enhancing MHETase Catalytic Efficiency
Design
In pursuit of higher catalytic efficiency for MHETase, we are considering employing virtual mutagenesis technology to predict the structural impact of every possible mutation within each residue of MHETase. This approach will allow us to simulate and analyze the effects of these mutations on the enzyme's structure and function. Subsequently, we will conduct molecular docking of all single-point mutations with the MHET molecule to evaluate their binding affinity. Through this screening process, we aim to identify mutations that exhibit a higher affinity for MHET compared to the wild-type MHETase. The promising candidates will then be subjected to molecular dynamics simulations to further confirm their stability and improved binding characteristics.
Construct
Leveraging homologous recombination techniques, we will construct several variants of MHETase based on the identified mutation hotspots. Specifically, we will create the following mutants: K397A, R71N, R273A, S305T, and N89A. Each of these variants will be tested for their ability to produce TPA, a key intermediate in the degradation pathway of PET, to determine which mutation(s) result in the most significant enhancement of catalytic efficiency. The goal is to select the variant(s) that not only maintain the enzyme's structural integrity but also exhibit improved catalytic properties, thereby accelerating the PET degradation process.
Upcycling
Cycle 1 Feasibility Assessment of Upcycling System
Design
Under the catalysis of terephthalate 1,2-dioxygenase (TPADO) and 1,4-dicarboxylic acid dehydrogenase (DCDDH), terephthalic acid (TPA) can be converted into protocatechuic acid (PCA). We utilized the Escherichia coli strain MG1655 (DE3) for the expression of target proteins and employed ultrasonication to lyse the cell suspension, thereby obtaining a crude enzyme solution for substrate conversion. This approach aims to verify the feasibility of the proposed conversion pathway and to explore the optimal conditions regarding time, temperature, and the concentration of key components in the system.
Build
A 10 mL transformation system was established using 100 µL of crude enzyme solution prepared from a cell suspension with an optical density (OD600) of 80. A 5 mL solution of 2.4 mM TPA in dimethyl sulfoxide (DMSO) was added, and the final volume was adjusted to 10 mL with 50 mM glycine buffer (pH 8.5). The reaction mixture was incubated at 30°C for 24 hours to facilitate the degradation process.
Test
High-performance liquid chromatography (HPLC) analysis revealed that the target product, PCA, was not detected (Fig 2).
Learn
During the optimization process, it was determined that excessive DMSO exerts toxic effects on the cells, 30°C is not the optimal temperature for degradation, the current culture medium is insufficient to support the reaction, and a high concentration of enzymes is critical for effective transformation.
Cycle 2 Replacement of E. coli strain
Design
Initially, the E. coli MG1655 (DE3) strain was utilized for protein expression; however, low expression levels were observed. Consequently, the E. coli BL21 (DE3) strain was utilized for enhanced protein expression. A novel transformation system was established, incorporating altered temperature conditions, transformation protocols, and bacterial suspension densities to upgrade the degradation of TPA.
Build
Following a 24-hour induction period, the E. coli BL21 (DE3) strain was resuspended in 3 mL of M9 medium to an OD600 of 65 and lysed using ultrasonication to obtain a crude enzyme solution. Subsequently, 100 µL of a 0.12 M TPA solution in DMSO was added, and the mixture was adjusted to a final volume of 10 mL with a pH 8.5, 50 mM glycine buffer. The degradation process was carried out at 20°C with orbital shaking for 24 hours.
Test
HPLC analysis results demonstrate the successful detection of the target PCA (Fig 3).
Learn
The E. coli BL21 (DE3) strain exhibits superior protein expression capabilities compared to the E. coli MG1655 (DE3) strain.
Cycle 3 Optimization of Cell Membrane Permeability System
Design
To achieve whole-cell catalysis, we focused on the transmembrane transport of TPA. Enhancing cell membrane permeability can improve the overall yield of the upcycling pathway. Therefore, increasing membrane permeability has become our primary strategy.
Build
We decided to use n-butanol to facilitate transmembrane transport. Through experimental investigation, we aim to determine the optimal timing and concentration of n-butanol addition within the system. Initially, n-butanol was added to E. coli cultures in TB medium at an OD600 of 0.3 to a final concentration of 10 mM, which resulted in suboptimal enzyme expression. Consequently, the timing and concentration of n-butanol addition were adjusted. E. coli was cultured in TB medium for 24 hours, and following cell harvest, 1% (v/v) n-butanol was introduced to the 10 mL upcycling system.
Test
It was confirmed that the BL21 (DE3) strain, after 24 hours of cultivation in TB medium, followed by cell harvest and the addition of 1% n-butanol to the 10 mL degradation system, could successfully mediate the transmembrane process of TPA without toxic effects on protein expression, leading to its degradation into PCA (Fig 4).
Learn
The presence of n-butanol in the degradation system can exert toxic effects on E. coli during the logarithmic growth phase. On the other hand, an appropriate concentration of n-butanol can enhance the permeability of the E. coli cell membrane, thereby facilitating the transmembrane transport of TPA. This achieves the effect of whole-cell catalysis and lays the foundation for the subsequent conversion of PCA into vanillin.
Cycle 4 Detection of Vanillin
Design
In order to achieve whole-cell catalysis and facilitate the transmembrane transport of terephthalic acid without cell lysis, thereby obtaining the desired product, we opted to incorporate n-butanol into the degradation system to enhance the permeability of the cell membrane. This approach is based on the synergistic action of catechol O-methyltransferase, carboxylic acid reductase, and phosphopantetheinyl transferase, which can convert PCA into the high-value product vanillin. Building upon the validation from the first cycle, we further verified the feasibility of the second step in the multi-enzyme cascade.
Build
The E. coli BL21 (DE3) strain was resuspended in 3 mL of M9 medium to an OD600 of 65, and n-butanol was added to a final concentration of 1% (v/v) after the cells were fully induced for 24 hours. Subsequently, 100 µL of a 0.12 M TPA solution in DMSO was added, and the mixture was adjusted to a final volume of 10 mL with a pH 8.5, 50 mM glycine buffer. The degradation process was carried out at 20°C with orbital shaking for 24 hours.
Test
The successful detection of vanillin confirmed the complete feasibility of the pathway (Fig 5).
Learn
During our investigation, we identified the optimal transformation system.