In recent years, the pollution caused by microplastics in the ocean has garnered global attention. Especially in coastal areas, where human activity is frequent, the accumulation of microplastics has severely impacted the health and stability of marine ecosystems. Among the various types of plastics, PET (polyethylene terephthalate) is one of the most common, widely used in everyday items such as beverage bottles and food containers. However, the degradation and recycling of PET have always posed significant challenges, as traditional physical and chemical methods have proven ineffective in addressing this issue.

Fortunately, scientists have discovered a potential solution from nature. Researchers in Japan found a bacterium (Ideonella sakaiensis) capable of degrading PET and isolated an enzyme from it called PETase. This enzyme has the ability to break down hcPET (high crystallinity PET), effectively dismantling its long-chain molecules, enabling degradation.

To apply this discovery in practice and further enhance the activity of PETase, we employed a surface display technique to present PETase on the surface of E. coli bacteria. This approach significantly boosted the activity of the whole-cell catalyst, making it more suitable for real-world application. Importantly, to anchor the engineered bacterial strains in open waters and to address potential bio-safety concerns, we adopted the Part (http://parts.igem.org/Part:BBa_K4380000 ) from the 2022 Vilnius-Lithuania. Using cellulose-binding domains, we fixed the engineered strains onto bacterial cellulose surfaces. Through this method, we successfully developed a hardware system capable of effectively treating PET in open water environments.

The promotion and application of this technology undeniably offer a powerful tool in addressing the issue of marine microplastic pollution. We hope that with continuous research and innovation, we can make even more significant contributions to the protection of marine environments.

In the first-generation microplastic processor, we conducted an extensive review of relevant literature and designed the genetic circuitry depicted above. This circuitry comprises elements such as the T7 promoter, ribosome binding site (RBS), PETase gene, and terminator. In this configuration, engineered bacteria are capable of continuously expressing PETase, which subsequently enables the degradation of microplastics present in the environment. This approach aligns with the overarching goals of environmental protection and sustainable development.

To study the expression and function of PETase in Escherichia coli Rosetta, we synthesized the PETase gene (Azenta life science, USA) and cloned it into the pET23b vector, allowing the target protein to be continuously expressed in E. coli Rosetta without the need for IPTG induction. The recombinant vector was transformed into E. coli Rosetta cells, and positive clones were selected on LB agar plates containing ampicillin. Positive clones were verified by DNA sequencing (Generalbiol, China). Positive clones were cultured overnight in LB medium, and cell pellets were collected by centrifugation and resuspended in Tris-HCl (pH 7.4). Cells were then lysed by sonication (150 W, sonication for 1s, interval 3s, for a total of 20 minutes) to obtain cell lysate. The total protein concentration in the lysate was measured using a Bradford reagent kit (Beyotime, China).

Enzyme activity was measured using para-nitrophenyl butyrate (pNPB) as a substrate. A 100 mM pNPB stock solution (Merck, Germany) was prepared in acetonitrile. PETase activity was tested in a 100 μL reaction system: 1 mM pNPB, 50 mM Tris-HCl buffer (pH 7.4), 20 μL crude enzyme solution. The reaction was carried out at 30°C for 30 min. The release of para-nitrophenol ester from PETase cleavage of pNPB was measured at 405 nm using a microplate reader. The content of pNPB was calculated based on a standard curve. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of para-nitrophenol ester per minute. The specific activity of PETase enzyme is defined as U/mg.

By varying the substrate pNPB concentration, we observed an increase in PETase activity. However, at a substrate concentration of 1000 μM, the enzyme activity growth rate plateaued, suggesting that the engineered bacterial strain successfully expressed PETase. Further analysis of the enzyme kinetics curve using Graphpad Prism 8.0 revealed a maximum reaction rate (Vmax) of 0.92 U/min and a Km value of 329.5 μM, calculated using the Michaelis-Menten equation (Figure 2A). Additionally, we explored the impact of temperature and pH on the microplastic processor's performance. The results indicated that the processor effectively operated within a temperature range of 25°C to 45°C, with optimal efficiency observed at 40°C. For pH, the processor functioned well within the pH range of 7.4 to 9.8, with peak efficiency at pH 9.2. This finding suggests that the microplastic processor can operate effectively in slightly alkaline environments, making it suitable for deployment in seawater environments without the need for additional alkaline conditions (see Figure 2B and 2C).

However, in our first-generation microplastic processor, we faced a cumbersome challenge: the need to disrupt bacteria to release PETase enzyme. This process was overly complex and inefficient. To enable the direct application of the microplastic processor in Escherichia coli, we conducted an extensive literature review. Ultimately, we decided to employ surface display technology to present the PETase enzyme on the surface of Escherichia coli. This approach allows engineered Escherichia coli to efficiently and conveniently access PETase.

Cell surface display technology, also known as microbial surface display technology, is a biotechnological approach that enables the expression and localization of target proteins on the surface of microbial cells. This technology allows researchers to manipulate and alter proteins directly on the surface of living cells, making them more amenable to studies of biochemical and biophysical properties, enzyme catalysis, and cellular engineering. At the core of this technique is the utilization of naturally occurring signal peptides or carrier proteins capable of directing proteins to the cell surface. Typically, these proteins transport the target protein to the cell surface through specific pathways. In the field of genetic engineering, we can link the gene sequence of the target protein with the gene sequence of these signal peptides or carrier proteins through genetic engineering methods, creating fusion genes. These fusion genes are then introduced into microbes, allowing for the expression and localization of the fusion protein on the cell surface. Ice Nucleation Protein (INP) is a commonly used protein carrier in cell surface display technology, and it can display the target protein in the periplasmic space of bacteria.

In the design of the second-generation microplastic processor, we constructed the following gene circuitry. This circuitry comprises the T7 promoter, ribosome binding site (RBS), INP-PETase gene, and terminator. In this design paradigm, we successfully achieved continuous expression of INP-PETase by the engineered bacteria. INP is capable of anchoring itself to the cell membrane's surface, thereby presenting PETase outside the cell, enabling it to efficiently degrade microplastics present in the environment.

The ice nucleation protein encoding gene INP was fused upstream of the PETase gene. The recombinant plasmid based on PET23b was transformed into E. coli Rosetta. Positive clones were selected in LB medium contaning 100 μg/mL ampicillin antibiotic under controlled conditions (37°C, 180 rpm).

To explore the catalytic activity of whole cell catalysts. The bacterial culture of engineered E. coli and control group (wild type E. coli) were harvested, adjusted to an OD600 of 1, and subjected to centrifugation (10,000 rpm, 1 min) to collect cell pellets. These pellets were then resuspended in 1 mL of PBS (pH 7.0). In a 1 mL bacterial suspension, 1 mM pNPB was added, and the reaction was allowed to incubate at 30°C for 30 minutes. Subsequently, the supernatant was collected by centrifugation, and the absorbance at 405nm was measured using a microplate reader. A standard curve was generated using para-nitrophenol ester concentrations ranging from 0 to 1 mM dissolved in PBS.

To further investigate the effective components of the whole-cell catalyst, we cultured E. coli Rosetta overexpressing INP-PETase overnight. From this culture, we separated extracellular and intracellular components through centrifugation. The cell pellet underwent a series of processes, including washing, sonication, and additional centrifugation, to segregate intracellular components and cell membranes. We then extracted cell membrane proteins using a Tris-HCl solution with Triton X-100, followed by centrifugation to remove insoluble fractions. The protein concentrations of all three components (extracellular, intracellular, and cell membrane) were determined using a Bradford reagent kit.

The findings revealed that E. coli/INP-PETase is capable of degrading 1 mM p-nitrophenol butyrate (pNPB), resulting in the production of 0.3 mM p-nitrophenol ester. In contrast, both the wild-type Escherichia coli Rosetta and E. coli/PETase yield negligible amounts of para-nitrophenol esters. These experimental outcomes demonstrate that the catalytic efficiency of PETase can be substantially enhanced through the application of surface display technology (Figure 4). On the othre hand, we found that there was almost no PETase activity outside the cell, but in the cell contents, the activity of PETase was about 0.15U/mg, and the activity of PETase on the cell membrane was the highest, reaching 0.35U/mg. Thus, through the surface display, the main active components of PETase are located in the cell membrane and bacterial contents (Figure 5).

However, biosafety experts have cautioned us that if we intend to release this strain into lakes or oceans, we would face significant biosafety concerns. It is not advisable to introduce a genetically modified organism into an open environment without effective control mechanisms in place. We need to consider strategies for immobilizing the engineered bacterial strain in open water bodies to address these biosafety concerns and ensure environmental stability.

We found 2022 Vilnius-Lithuania igem team representation of a cellulose binding protein (CBD, http://parts.igem.org/Part:BBa_K4380000), We decided to use part of it as a binding protein for a microbial fixation technology based on bacterial cellulose (Figure 6). We also received the selfless help of the team at Squirrel-Beijing-I, who donated their bacterial cellulose to help us verify the feasibility of their fixation technology.

We designed the third generation of microplastic processors. The first module is the anchoring and degradation module, which continuously expresses INP-PETase inside the engineered bacteria（Figure 7）. The second module is the anchoring module, which continuously expresses INP-CBD and can anchor the engineered bacteria on the cellulose membrane to prevent the engineered bacteria from flowing into the water and causing biological pollution (Figure 8).

To enhance the adhesion of Escherichia coli to cellulose membranes, we have devised a plan to introduce INP-CBD onto the foundation of the INP-PETase engineered strain, utilizing a polycistronic approach to achieve this project (Figure 7). Prior to embarking on this endeavor, we first conducted individual tests to assess the functionality of INP-CBD. We initiated the process by synthesizing the INP-CBD gene sequence, followed by cloning it into the pET23b vector. To ensure a robust fusion, we incorporated a proline-based 17 x helixl peptide (AEAAAKEAAAKEAAAKA) as a connector between INP and CBD. Subsequently, the resulting recombinant plasmid was named pET23b/INP-CBD and was introduced into Escherichia coli Rosetta. Positive clones were selected using the selective antibiotic ampicillin at a concentration of 100 μg/ml.

Subsequently, the adhesion effect of the recombinant strain to bacterial cellulose was tested. The sterile bacterial cellulose membrane (BC membrane) was cut into 5 cm x 5 cm pieces. The recombinant E. coli was cultured overnight in 5 mL LB medium containing 100 μg/mL ampicillin. The following day, bacterial pellets were collected by centrifugation, washed with PBS (pH 7.4), and the recombinant E. coli was resuspended to adjust the bacterial OD600 = 0.5. The recombinant E. coli suspension was then incubated with the BC membrane for 1 hour. Afterwards, the membrane was gently rinsed with 10 mL PBS to remove non-specifically adhered cells, and the rinse solution was collected. The rinsing solution was subsequently diluted in a gradient and spread onto LB Agar plates containing ampicillin.. For comparison, wild-type E. coli was processed in the same way. After 24 hours of incubation, we calculated the CFU/mL for each sample. By comparing the non-specific adhesion of the INP-CBD fusion protein E. coli with that of the wild type on the bacterial cellulose membrane, we evaluated the adhesive effect of INP-CBD.

After the recombinant Escherichia coli strain was incubated with bacterial cellulose membrane, the CFU value of the washing solution was counted. The wild type strains without INP-CBD fusion had the least adhesion to bacterial cellulose membrane. The average CFU of flushing solution is 1.2 × 10 ^ 6CFU / mL. The average CFU of the rinse solution of recombinant Escherichia coli strain was 7.6 × 10 ^ 5 CFU / mL (Figure 9). T-test was used for comparison. The p values obtained were all less than 0.05, indicating that there was a statistically significant difference in the adhesion ability of the two strains. This significantly enhanced adhesion confirmed the successful display of CBD on the surface of Escherichia coli and its function of binding to cellulose membrane.

We have successfully constructed the PETase overexpression recombinant plasmid in the pET23b vector and produced PETase in E. coli Rosetta. Through enzymatic kinetics studies, we determined that the crude enzyme solution has a maximum catalytic rate of 0.92 U/min, with a Km value of 325.5 μM. To enhance efficiency, we employed INP, a surface display protein, to deliver PETase into the periplasmic space of E. coli. Experimental data conclusively show that this display technique significantly boosts the performance of whole-cell catalysts. Lastly, to evaluate the potential application of the engineered strain in open waters, we tested the adhesion capability of the INP-CBD engineered E. coli to bacterial cellulose.

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Dou, Jian-lin, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417.

Morag, Ely, et al. "Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum." Applied and environmental microbiology 61.5 (1995): 1980-1986.