Wet Lab Overview

Research Background and Problem Statement

With the escalating global plastic pollution crisis, biodegradation, particularly enzymatic degradation, has emerged as a highly promising green solution. The application of computational biology approaches, such as artificial intelligence and molecular dynamics simulations, for constructing predictive models can efficiently screen potential plastic-degrading enzymes from vast protein sequence datasets, thereby significantly reducing the experimental validation cycle and associated costs. However, the accuracy and reliability of computational models ultimately necessitate validation through authentic experimental biological data.

The objective of our research team is to develop a predictive model for plastic-degrading enzymes. To ensure the accuracy of the model's predictions, it is imperative to utilize a set of experimentally validated enzyme sequences, known to possess plastic-degrading capabilities, as a test set for the model. Currently, although relevant sequences are available in databases, their functional annotations exhibit variable accuracy, and direct usage may introduce bias. Therefore, experimentally validating the function of selected enzyme sequences is a crucial prerequisite for providing a high-reliability test set for the model.

Experimental Objectives and Hypotheses

Experimental Objectives

This experiment aims to empirically validate, using molecular biology and biochemistry methods, whether five selected plastic-degrading enzyme sequences from the UniProt database can be successfully expressed in Escherichia coli and exhibit degrading activity. This will provide a experimentally confirmed, high-quality test set for our developed predictive model.

Experimental Hypotheses

We hypothesize that the five selected plastic-degrading enzyme sequences will:

• Successfully be inserted into the pET22b expression vector and transformed into Escherichia coli BL21(DE3).
• Undergo inducible soluble expression in Escherichia coli BL21(DE3).
• Possess hydrolase activity towards plastic analogs (4pNPA) upon expression of the recombinant proteins.

If all the above hypotheses are validated, these five sequences will be confirmed as positive candidates and can serve as a gold standard test set for model evaluation.

Materials and Methods

Materials

Gene Sequences

Five coding sequences of plastic-degrading enzymes selected from the UniProt database (designated as: PLA01, PET01, PET02, PET03, PET04).

Expression System

• Expression Vector: pET22b(+) plasmid (featuring a His-Tag and pelB signal peptide for periplasmic targeting).
• Host Strain: Escherichia coli BL21(DE3).

Methods

Plasmid Construction and Transformation

The five target sequences will be synthesized and cloned into the pET22b expression vector by a service provider to construct recombinant expression plasmids. The recombinant plasmids will be transformed into Escherichia coli BL21(DE3) chemically competent cells via heat shock.

Figure 1. Schematic representation of the recombinant pET22b(+) expression vectors. The constructs were designed for the soluble secretory expression of five target proteins in E. coli. Key features, indicated in the diagram, include a T7 promoter for inducible expression, a pelB signal peptide sequence to direct protein secretion, and an ampicillin resistance gene (Amp^R) for selection. The codon-optimized target genes were inserted into the multiple cloning site (MCS), resulting in a C-terminal hexa-histidine tag (His-tag) fusion for subsequent affinity purification. Detailed information regarding the exogenous sequences inserted into each plasmid is provided in Table 1-1.

Table 1-1 Information on exogenous genes inserted into recombinant plasmids.

Clone Verification

• Colony PCR: Single colonies were picked for PCR amplification. Amplification products were analyzed by electrophoresis on a 1% agarose gel at 85 V for 90 min. The size of the inserted fragment was verified using a gel imaging system.
• Sanger Sequencing: PCR-positive clones were sent for DNA sequencing to ensure the complete accuracy of the gene sequence and the absence of frameshift mutations.

Protein Expression and Induction

• Sequencing-confirmed positive clones were inoculated into LB medium (supplemented with Ampicillin) and incubated at 37°C until reaching the logarithmic growth phase (OD₆₀₀ ≈ 0.6-0.8).
• Expression was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.5 mM, followed by incubation at 20°C for 16 hours.

Functional Validation

Enzyme Activity Assay

Bacterial cells were collected after induced expression and lysed by sonication on ice using a Sonicator (Qsonica, Newtown, CT, USA). An intermittent duty cycle (5s on / 10s off) was employed during sonication to minimize thermal effects. Following lysis, cell debris was removed by centrifugation at 10,000 ×g for 5 min at 4 °C. The resulting supernatant was collected as the crude enzyme extract. Enzyme activity was assessed using 4pNPA as a substrate in a 96-well plate, following the method described by Shi et al. (2021). The hydrolysis of 4pNPA produces 4-nitrophenol (4NP). The release rate of 4NP was measured at a wavelength of 405 nm using a multimode microplate reader (CLARIOstar Plus, BMG LABTECH GmbH, Ortenberg, Germany) to characterize the enzyme's degradation activity.

Western Blot Validation

Induced bacterial cultures were centrifuged at 10,000 ×g for 5 min at 4 °C to pellet bacterial cells and obtain the supernatant. Equal volumes of samples (15 μL per well) were subjected to SDS-PAGE electrophoresis (140 V constant, 55 min) and subsequently transferred to a membrane at 100 V for 1 h. Primary antibody incubation was performed using a mouse anti-His tag monoclonal antibody (1:1000 dilution), followed by incubation with a HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution). This immunoassay was designed to specifically detect the expression and distribution of the target protein both intracellularly and extracellularly. Protein bands were visualized using the Odyssey infrared laser scanning imaging system (Model: 9142, LI-COR Biosciences, Lincoln, NE, USA).

Data Analysis Plan

Clone Verification Data Analysis

Gel Electrophoresis Analysis

Clones exhibiting PCR product band sizes consistent with the expected size of the target gene on gel electrophoresis will be designated as primary positive clones. The colony PCR results for the modified plasmids PLA_01, PET_01, PET_02, PET_03, and PET_04 amplified in Escherichia coli BL21(DE3) are shown in the figure below:

Figure 2. Agarose gel electrophoresis of colony PCR products

Sequencing Result Analysis

The chromatogram files returned from Sanger sequencing were aligned and compared with the original target sequence. With 100% sequence identity and correct reading frame, the recombinant engineered strains were deemed successfully constructed.

Protein Functional Data Analysis

Enzyme Activity Assay Analysis

Absorbance values at 405 nm were recorded over time (6 min intervals). Bacterial strains transformed with the empty vector plasmid (pET22b) served as the negative control. The enzyme activity rate (ΔA₄₀₅/min) was calculated. A significantly higher enzyme activity rate in the experimental groups compared to the negative control group (e.g., by an order of magnitude or through a t-test with p-value < 0.05) indicated successful expression of active target enzymes by the engineered strains.

Figure 3. Enzymatic activity assay of plastic-degrading enzymes using 4pNPA as a substrate

(a) Comparison of the hydrolytic activity of crude enzyme extracts from Escherichia coli BL21(DE3) expressing five distinct recombinant plasmids (PLA01, PET01, PET02, PET03, PET04) against the substrate 4pNPA. The strain transformed with the empty vector plasmid (pET22b) served as the negative control (EV). Reactions were conducted at 37°C, and the release rate of 4-nitrophenol (4NP) was monitored at a wavelength of 405 nm

(b) Each data point represents the mean of eight technical replicates (n=8), and the error bars indicate the standard deviation (Mean ± SD).

Western Blot Analysis

The presence of a specific hybridization band at the expected molecular weight indicates successful expression of the target protein. Band intensity can be used for semi-quantitative assessment of expression levels.

Figure 5. WB results of the bacterial cells and supernatant

Conclusion

The wet-lab validation of the five plastic-degrading enzyme sequences has been successfully completed. PCR and sequencing results confirmed the successful construction of the recombinant plasmids. 4pNPA enzyme activity assays demonstrated that all five engineered strains produced catalytically active target proteins. Western Blot analysis further provided supplementary evidence for the protein expression of three sequences (PLA01, PET01, PET02).

Based on all the data, we conclude that these five plastic-degrading enzyme sequences are bona fide, expressible, and functionally active positive sequences. Therefore, they fully meet the requirements for a high-standard test set to evaluate the accuracy of plastic-degrading enzyme prediction models, and will be used for subsequent model reliability validation and performance assessment.

References

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