1.1 Purpose

The objective of this part of the experiment was to enable Escherichia coli to produce large quantities of bacterial cellulose (BC). Literature indicates that the synthesis of BC is a complex process regulated by multiple enzymes. Among them, the bcsA gene encodes the catalytic subunit of cellulose synthase. This protein is the "core factory" for cellulose synthesis, directly using UDP-glucose (UDPG) as a substrate to catalyze the β-1,4 polymerization of glucose monomers, forming long cellulose chains. The bcsB gene encodes a cyclic di-GMP-binding protein. By binding the signaling molecule c-di-GMP, this protein activates the catalytic activity of BcsA, acting as a regulatory switch in the cellulose synthesis process. BcsA and BcsB are the core of the multi-enzyme complex for cellulose synthesis. Therefore, we chose to prioritize their expression.

Recombinant Strain Construction

Based on the codon preference of Escherichia coli, the cellulose synthase encoding genes bcsAB were codon-optimized, and the restriction sites for EcoRI, XbaI, SpeI, PstI, and NotI were eliminated (sequence verified by SnapGene® analysis). The optimized gene was synthesized by Genewiz (South Plainfield, NJ, USA) and cloned into the pSB1A3 vector (BioBricks™ standard RFC#10). A polycistronic expression cassette was constructed using the J23100 constitutive promoter (BBa_J23100) and the B0034 ribosomal binding site (BBa_B0034), which was then ligated into the vector via the Golden Gate assembly method. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and plated on LB agar plates (pH 7.0) containing 100 μg/mL ampicillin (Amp⁺), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of LB/Amp⁺ liquid medium, then cultured at 37°C and 220 rpm until the OD₆₀₀ reached 0.6-0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). Sequencing verification was performed by Qingke Biotechnology (Beijing, China). The sequence-verified engineered strain was inoculated into 50 mL of LB/Amp⁺ medium and cultured at 37°C and 200 rpm to the mid-log phase (OD₆₀₀≈0.5). The OD₆₀₀ was measured every 30 min using a GenStar® GS-2000 spectrophotometer (Beijing) with a wavelength accuracy of ±0.5 nm, using uninoculated medium as a blank control. The growth curve was plotted using OriginPro 2023 software, with data presented as the mean ± standard deviation (SD) of three independent experiments. Wild-type E. coli BL21 was used as a blank control.

Cellulose Yield Analysis

To quantify the amount of cellulose synthesized, the bcsAB co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium (containing 100 μg/mL ampicillin). After incubation with shaking at 30°C and 180 rpm for different durations, 100 mL of the culture was centrifuged at 8,000 × g for 5 min. A 20 mL aliquot of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-cooled PBS (pH 7.4) and subjected to sonication on an ice bath (Biosafer1000, Saifei) with the following parameters: power 70 W, 1 s on, 3 s off, for a total processing time of 20 min. The lysate was centrifuged at 15,000 × g for 20 min to remove cell membranes and impurities, and 20 mL of the supernatant was collected as the intracellular sample. Both intracellular and extracellular samples (20 mL each) were mixed with 20 mL of 4 M NaOH and incubated at 80°C for 2 h to dissolve non-cellulosic components and promote cellulose precipitation. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose precipitate, which was then repeatedly washed with distilled water until the pH reached 7.0 to remove residual NaOH. The washed cellulose was dried at 60°C to a constant weight, and the cellulose content was calculated by weighing the dried mass. The cellulose synthesis of the wild-type E. coli BL21 control strain was measured in the same manner.

2.1 Purpose

To determine the optimal time window for cellulose production by the engineered strain BL21-bcsAB, we characterized its production kinetics. This aims to provide crucial data to support subsequent process optimization and scale-up production.

2.2 Methods

Strain Inoculation and Culture

The bcsAB co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium containing 100 μg/mL ampicillin and cultured with shaking at 30°C and 180 rpm.

Sample Collection

After culturing for different durations, 100 mL of the bacterial culture was centrifuged at 8,000 × g for 5 min. A 20 mL aliquot of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-cooled PBS and sonicated on an ice bath. The lysate was then centrifuged at 15,000 × g for 20 min, and 20 mL of the supernatant was collected as the intracellular sample.

Sample Processing and Cellulose Collection

Both intracellular and extracellular samples were mixed with 20 mL of 4 M NaOH. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose precipitate. The precipitate was repeatedly washed with distilled water until the pH reached 7.0, then dried at 60°C to a constant weight. The mass of the dried cellulose precipitate was weighed to calculate the cellulose content in the corresponding sample.

3.1 Purpose

The expression of foreign genes imposes a metabolic burden on the host, potentially affecting its growth rate. To eliminate the interference of such growth differences on the comparison of total yield, this experiment aims to specifically compare the cellulose synthesis capability of a unit number of engineered bacteria versus control bacteria by normalizing the cell density (OD₆₀₀=1).

3.2 Methods

• Culture and Pre-treatment: The BL21-bcsAB strain and the BL21 strain were cultured at 30°C and 180 rpm for 12 h. The cultures were then adjusted to an OD₆₀₀=1, and 2 mL of each culture was centrifuged at 10,000 × g for 10 min at 4°C to collect the bacterial pellets.
• Congo Red Incubation: The pellets were resuspended in a 1% tryptone solution containing 40 mg/mL Congo red and incubated with shaking at 37°C and 180 rpm for 2 h.
• Separation and Detection: The incubated samples were centrifuged at 13,000 × g for 30 min at 4°C, and the supernatant was collected. The absorbance of the supernatant was measured at 490 nm using a microplate reader (Note: The figure caption indicates 400 nm, but the experimental method is followed). The total amount of Congo red bound by the bacteria was calculated by subtracting the amount of unbound Congo red (in the supernatant) from the initial amount added.

3.3 Results

The experimental results (Figure 5) showed that compared to the BL21 control group (absorbance approx. 0.6), the supernatant absorbance of the BL21-bcsAB engineered group was significantly lower (approx. 0.2), with the difference being highly statistically significant (p < 0.0001). This indicates that, for the same number of cells, the engineered bacteria bound significantly more Congo red.

4.1 Purpose

Post-cesarean section surgery is often accompanied by a severe inflammatory response in the body, which is particularly intense at the suture site. Therefore, we aim to add an anti-inflammatory substance—curcumin—to our surgical suture to help reduce the body's inflammatory response. This experiment aims to construct a curcumin synthesis system (co-expressing DCS and CURS1 genes) in an E. coli strain, verify its functionality in producing curcumin, and quantitatively compare the curcumin production capacity of the engineered strain with that of the wild-type strain.

4.2 Methods

Recombinant Strain Construction

The DCS and CURS sequences were synthesized by a biological company (Generalbial, China) and codon-optimized for E. coli. The restriction sites for EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with the RFC#10 standard and for cloning compatibility with the pET28a(m) vector. The genes were cloned into the pET28a(m) vector via NdeI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli DH5α and BL21. Positive clones were screened on LB agar plates containing 100 μg/mL kanamycin (Kana) and sent for sequencing verification to a biological company (Qingke Biotechnology, China) to obtain the recombinant engineered strain.

Curcumin Synthesis Analysis

The recombinant strain was inoculated and expanded in LB medium containing 100 μg/mL kanamycin at 37°C and 150 rpm. When the OD₆₀₀ reached 0.6, protein expression was induced with 0.5 mM IPTG. The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min).

The protein concentration was determined using a Bradford assay kit (Beyotime, P0006).

To analyze curcumin production, the reaction was conducted in 200 μL of 100 mM PBS (pH 7.4). The reaction system contained 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L crude enzyme lysate. The reaction was incubated at 37°C for 1 h. The sample was then extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and redissolved in 200 μL of methanol. The absorbance at 420 nm was measured using a spectrophotometer, with methanol used as a blank for baseline correction. The sample solution was placed in a cuvette to measure its absorbance. The curcumin concentration was calculated based on a standard curve prepared with curcumin standards.

Purpose

This experiment aims to investigate the response characteristics of the heterologously expressed curcumin synthesis pathway (DCS-CURS) in the engineered E. coli strain to varying substrate concentrations, thereby determining whether the system exhibits typical enzymatic reaction kinetics (e.g., Michaelis-Menten kinetics).

Methods

The DCS and CURS co-expressing engineered strain was cultured in LB medium containing 100 µg/mL kanamycin. When the OD₆₀₀ reached 0.6, protein expression was induced by adding 0.5 mM IPTG.

The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min). The protein concentration was determined using a Bradford assay kit.

To analyze curcumin production, the reaction was conducted in 200 µL of 100 mM PBS (pH 7.4). The reaction system contained 100 µM feruloyl-CoA, varying concentrations of malonyl-CoA (creating a substrate concentration gradient), and 40 mg/L crude enzyme lysate.

The reaction was incubated at 37°C for 1 h. The sample was then extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and redissolved in 200 µL of methanol.

The absorbance at 420 nm was measured using a spectrophotometer, with methanol used as a blank for baseline correction. The curcumin concentration was calculated based on a standard curve prepared with curcumin standards, and from this, the rate of curcumin synthesis at different substrate concentrations was determined.

Purpose

Cesarean sections involve significant blood loss and a high risk of postoperative bacterial infections. We therefore aim to incorporate antibacterial and hemostatic substances into surgical sutures. Chitosan, a high-molecular-weight polymer, possesses excellent film-forming properties and a positive charge, allowing it to rapidly form a protective barrier on the wound surface. It disrupts bacterial cell membranes via electrostatic interactions and promotes platelet aggregation, making it particularly suitable for scenarios requiring rapid hemostasis, such as C-sections. Its degradation product, chitooligosaccharide (COS), is a low-molecular-weight, water-soluble molecule that can penetrate deep into tissues. It enhances antibacterial effects by inhibiting bacterial DNA replication and actively accelerates wound healing by activating fibroblasts and endothelial cells.

Chitosan can be obtained by processing shrimp and crab shell waste, while COS requires the enzymatic catalysis of chitosan by chitosanase. Therefore, we plan to construct a chitosanase-producing engineered bacterium for bulk enzyme production. However, traditional processes involving cell disruption for enzyme extraction are costly and inefficient. This experiment aims to design and validate the effectiveness of an innovative cell surface display strategy. This strategy involves constructing an engineered bacterium that directly expresses chitosanase on its cell surface, enabling the catalysis of chitosan to COS without cell lysis. We will compare the catalytic activity of the surface-displaying engineered strain (BL21-INP-CHI) with that of a crude enzyme lysate (CHI) to demonstrate that this engineering approach enhances catalytic efficiency.

Methods

Construction of Recombinant Strain for Surface Display of CHI1

The chitosanase gene (CHI1) from Bacillus thuringiensis and the ice nucleation protein gene (INP) were synthesized to create an INP-CHI1 fusion fragment (Generalbial, China). As previously described, the sequence was codon-optimized for E. coli and designed to comply with the BioBricks™ RFC#10 standard. Using the J23100 constitutive promoter (BBa_J23100) and the B0034 ribosomal binding site (BBa_B0034) as cis-acting elements, the fragment was cloned into the pSB1A3 vector via EcoRI and XbaI restriction sites to generate pSB1A3-INP-CHI1. The control plasmid, pSB1A3-CHI1, was obtained via inverse PCR using a BKL Kit. The recombinant plasmids were transformed into E. coli DH5α. Positive clones were screened on LB agar plates containing 100 µg/mL ampicillin and verified by sequencing (Qingke Biotechnology, China). The sequence-verified plasmid, pSB1A3-INP-CHI1, was then extracted and transformed into E. coli BL21. Single colonies were picked and cultured in 5 mL of LB liquid medium (containing 100 µg/mL ampicillin) at 37°C and 180 rpm until the OD₆₀₀ reached 0.6. A 1 mL aliquot of the culture was preserved with 25% (v/v) glycerol and stored at -80°C for future use.

Catalytic Activity Test of the Strain

A standard enzyme activity assay was employed to evaluate the catalytic performance of both the BL21-INP-CHI engineered strain and the CHI crude enzyme lysate. For the assay, 5 mL of PBS-resuspended BL21-INP-CHI cells (OD₆₀₀=0.6) or an equal volume of CHI crude enzyme lysate (obtained by sonicating cells at 70W power, 1s on, 3s off for 20 min, followed by centrifugation at 10,000 × g for 30 min) were incubated with 1 mL of 1% chitosan substrate in a PBS buffer system (pH 7.0) at 37°C for 30 min. After the reaction, the amount of reducing sugar produced was measured using a Biosharp reducing sugar assay kit and quantified against a glucose standard curve (0-100 µg/mL). One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per minute at 37°C. Each experiment was performed in triplicate, and the data are presented as mean ± standard deviation.

Purpose

Effect of Temperature on Enzyme Activity: To determine the enzyme activity profile of the chitosanase-expressing engineered strain BL21-INP-CHI at different temperatures, thereby identifying the optimal temperature for maximal enzyme activity to guide efficient catalysis.

Effect of pH on Enzyme Activity: To investigate the impact of different pH conditions on the activity of the chitosanase produced by BL21-INP-CHI, thereby determining the optimal pH environment to optimize the reaction system.

Methods

In the chitosanase activity assay, 1 mL of a PBS suspension of BL21-INP-CHI or crude enzyme lysate was mixed with 1 mL of 1% commercial chitosan (Cat. No. C105799, Innochem) and incubated for 30 min at various temperatures: 25, 37, 45, 50, and 65°C. To investigate the effect of pH, the reaction was conducted at 37°C in PBS buffers with pH values of 4, 5, 6, 7, and 8. The total reducing sugar content was measured using a reducing sugar assay kit (Cat. No. BL1789B, Biosharp). One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute, calculated based on a glucose standard curve.

Figure1. Characterization of the engineered strain.

Purpose

To enhance the sustainability of our project, this experiment aimed to develop an environmentally friendly and efficient microbial fermentation method for extracting chitin from shrimp shell waste. This process is intended to provide a green source of raw material for the subsequent production of chitosan and chitooligosaccharides.

Methods

Strain Culture: A stab culture of B. subtilis was picked and inoculated into 5 mL of LB liquid medium, then incubated with shaking at 30°C and 180 rpm for 12 h until the cell density reached an OD₆₀₀=0.6. A 1 mL aliquot of the culture was mixed with 25% (v/v) glycerol as a cryoprotectant and stored at -80°C for future use. A 2% inoculum of the B. subtilis culture was transferred to 50 mL of LB liquid medium and incubated at 30°C and 180 rpm for 8 h until OD₆₀₀=0.6, to be used subsequently.

Similarly, a stab culture of Acetobacter sp. was picked and inoculated into 5 mL of LB liquid medium (containing 2% ethanol), then incubated with shaking at 30°C and 180 rpm for 24 h until OD₆₀₀=0.6. It was then cryopreserved at -80°C for future use. A 5% inoculum was transferred to 50 mL of LB liquid medium (containing 4% ethanol) and incubated at 30°C and 180 rpm for 16 h, to be used subsequently.

The engineered E. coli for chitooligosaccharide production was inoculated at a 1:100 ratio into 50 mL of LB liquid medium (containing 100 μg/mL ampicillin), and cultured at 37°C and 180 rpm until OD₆₀₀=0.6, to be used subsequently.

Shrimp and Crab Shell Pre-treatment: Shrimp shells were washed, pulverized using a blender, passed through a 60-mesh sieve, and dried at 60°C for 48 h for later use. The fermentation medium was prepared according to the formula (shrimp shell powder 50 g/L, glucose 50 g/L, yeast extract 5 g/L, KH₂PO₄ 10 g/L). The pH was adjusted to 7.2 using 1 M NaOH or HCl. The medium was then dispensed into 250 mL Erlenmeyer flasks (100 mL per flask), sterilized by autoclaving at 121°C for 20 min, and cooled for later use.

Yield Determination: A total of 3 groups were set up. In the experimental group, 10 mL of the B. subtilis culture was inoculated into 100 mL of the fermentation medium and fermented at 30°C and 180 rpm for 3 days. Subsequently, ethanol was added to a final concentration of 6%, along with 10 mL of the Acetobacter sp. culture, and fermentation was continued at 30°C and 180 rpm for 3 days. The residue was collected by centrifugation (13,000 × g, 10 min), washed with distilled water until neutral, and dried at 60°C for 48 h to obtain chitin. The chitin yield was calculated by comparing its mass to the initial mass of the shrimp shells. For the two control groups, the B. subtilis culture or the Acetobacter sp. culture was replaced with an equal volume of PBS, respectively. Finally, the chitin yields of the 3 groups were compared.

Purpose

This experiment aims to evaluate the performance of our BL21-INP-CHI whole-cell catalyst in a practical application scenario. Specifically, it tests its efficiency in hydrolyzing the crude chitosan—prepared from shrimp shells using the aforementioned method—at different time points, and compares its performance against a traditional crude enzyme lysate.

Methods

A stab culture of B. subtilis was picked and inoculated into 5 mL of LB liquid medium, then incubated with shaking at 30°C and 180 rpm for 12 h until the cell density reached an OD₆₀₀=0.6. A 1 mL aliquot of the culture was mixed with 25% (v/v) glycerol as a cryoprotectant and stored at -80°C for future use. A 2% inoculum of the B. subtilis culture was transferred to 50 mL of LB liquid medium and incubated at 30°C and 180 rpm for 8 h until OD₆₀₀=0.6, to be used subsequently.

Similarly, a stab culture of Acetobacter sp. was picked and inoculated into 5 mL of LB liquid medium (containing 2% ethanol), then incubated with shaking at 30°C and 180 rpm for 24 h until OD₆₀₀=0.6. It was then cryopreserved at -80°C for future use. A 5% inoculum was transferred to 50 mL of LB liquid medium (containing 4% ethanol) and incubated at 30°C and 180 rpm for 16 h, to be used subsequently.

The engineered E. coli for chitooligosaccharide production was inoculated at a 1:100 ratio into 50 mL of LB liquid medium (containing 100 μg/mL ampicillin), and cultured at 37°C and 180 rpm until OD₆₀₀=0.6, to be used subsequently. The shrimp shells were washed, pulverized using a blender, passed through a 60-mesh sieve, and dried at 60°C for 48 h for later use.

The fermentation medium was prepared according to the formula (shrimp shell powder 50 g/L, glucose 50 g/L, yeast extract 5 g/L, KH₂PO₄ 10 g/L). The pH was adjusted to 7.2 using 1 M NaOH or HCl. The medium was then dispensed into 250 mL Erlenmeyer flasks (100 mL per flask), sterilized by autoclaving at 121°C for 20 min, and cooled for later use.

In 100 mL of the fermentation medium, 10 mL of the B. subtilis culture was inoculated and fermented at 30°C and 180 rpm for 3 days. Subsequently, ethanol was added to a final concentration of 6%, along with 10 mL of the Acetobacter sp. culture, and fermentation was continued at 30°C and 180 rpm for 3 days. The residue was collected by centrifugation (13,000 × g, 10 min), washed with distilled water until neutral, and dried at 60°C for 48 h to obtain chitin. The chitin yield was calculated by comparing its mass to the initial mass of the shrimp shells.

1 g of chitin was mixed with 10 mL of 10% (v/v) hydrogen peroxide and reacted at 60°C for 2 hours to obtain chitosan. It was then dissolved in 0.1 M acetic acid to prepare a 1% solution. Subsequently, the fermentation broth of the engineered strain E. coli BL21-pSB1A3-INP-CHI1 was centrifuged (10,000 × g, 2 min), and the cell pellet was washed twice with PBS (pH = 7). Different groups were set up by mixing 1 mL of the cell suspension with 1 mL of 1% chitosan solution and reacting at 50°C for different durations. After centrifugation at 13,000 × g for 15 min, the supernatant was collected and filtered through a 0.22 μm membrane to obtain the chitooligosaccharide sample. A 100 μL aliquot of the sample was taken, and the total reducing sugar content was measured using a reducing sugar content assay kit (BL1789B, biosharp). One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute.

Figure 3. Determination of the hydrolysis rate of crude chitosan by the engineered chitosanase bacteria.

Purpose

This experiment aims to prepare our final surgical suture (ReGenstitch) and evaluate its basic appearance and performance in a simulated practical use scenario, comparing it with commercially available surgical sutures to understand its application potential.

Methods

Preparation of the Surgical Suture: 1 gram of chitosan was dissolved in 80 mL of 1% glacial acetic acid with continuous stirring for 8 hours at room temperature (25°C). 5 grams of bacterial cellulose was dissolved in 20 mL of 1% glacial acetic acid with continuous stirring for 2 hours at room temperature (25°C). The chitosan solution was gradually added to the bacterial cellulose solution under continuous stirring. Then, glycerol (as a plasticizer, added at 20% of the dry weight of CS and BC) was added, and stirring continued for another hour to obtain 6 identical mixtures. TPs (thermoplastic starch) were dispersed into the mixtures to final concentrations of 0% (CBT0), 2% (CBT2), 4% (CBT4), 6% (CBT6), 8% (CBT8), and 10% (CBT10) (w/w, based on the dry weight of CS and BC in the mixture). Each portion was processed with continuous stirring for 8 hours. The mixtures were poured into circular plates with a radius of 10 cm (50 mL per plate). Then, all plates were subjected to a drying process.

Comparison with Existing Sutures: Commercially available absorbable PGA sutures (JinHuan, SH, China) and non-absorbable polyamide sutures (ChengHe, NB, China) were selected for appearance comparison with our final suture. Furthermore, a simulated suturing test was performed on store-bought pork with skin to mimic the practical application scenario of surgical sutures.

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