This year, our project, ReGenStitch, aims to develop a new generation of multifunctional bioactive sutures designed to improve post-operative recovery. To achieve this goal, one of our team's core contributions is the comprehensive new functional characterization and application demonstration of the existing iGEM part, bcsB (BBa_K3520002).
We successfully applied bcsB in an efficient bacterial cellulose (BC) production system, which provides a high-strength, highly biocompatible structural backbone for our suture. Through a series of rigorous experiments, we have added valuable quantitative data to this part, including its yield in a co-expression system, its production kinetics, and the synthesis efficiency per cell. We believe this detailed characterization data will provide a solid and reliable reference for future iGEM teams wishing to efficiently produce bacterial cellulose in Escherichia coli.
One of the core parts used in our project is bcsB (BBa_K3520002), which encodes the regulatory subunit of bacterial cellulose synthase. In our ReGenStitch project, we demonstrated a new application for bcsB and performed an in-depth characterization of its function.
1. New Application in an Efficient Bacterial Cellulose Production System
To construct the core structure of the suture, we co-expressed bcsB with its catalytic subunit, bcsA, in Escherichia coli BL21(DE3), thereby establishing an efficient bacterial cellulose production system. The entire genetic circuit (Figure 1) is driven by the strong constitutive promoter J23100, ensuring continuous and high-level expression of both genes.
Figure 1. Genetic circuit design for bacterial cellulose production. This polycistronic structure endows engineered bacteria with the ability to produce bacterial cellulose by co-expressing bcsA and bcsB.
2. Adding New Quantitative Characterization Data
To provide richer and more practical data for this part, we quantitatively characterized its function in our co-expression system from three dimensions.
Functional Validation and Yield Analysis We first verified the successful construction of the recombinant plasmid containing bcsA (2262 bp) and bcsB (2406 bp) via colony PCR (Figure 2). Subsequently, we directly measured the cellulose yield using an alkali-treatment and dry-weight method. The results (Figure 3) showed that the cellulose yield of our engineered strain (BL21-bcsAB) was significantly higher than the control strain, which produced almost no cellulose (p < 0.05). This strongly proves that bcsB successfully performed its regulatory function in our system.
Figure 2. Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-bcsAB. The left panel shows the band for bcsA, and the right panel shows the band for bcsB, with sizes matching expectations.
Figure 3. Cellulose yield analysis of the engineered strain and control strain. The results indicate that the engineered strain co-expressing bcsA and bcsB can efficiently produce bacterial cellulose.
Production Kinetics Characterization To determine the optimal operational cycle of the system, we plotted a time course of cellulose production. The results (Figure 4) show that cellulose accumulation follows a typical S-shaped growth curve. The yield reaches a plateau at approximately 48 hours of cultivation, with a peak yield of about 970 mg/L. This kinetic data provides a key process parameter for determining the optimal fermentation and harvest time for future teams using this system for large-scale production.
Figure 4. Time-course curve of cellulose production by the BL21-bcsAB strain.
Characterization of Per-Cell Production Capacity
To verify whether our device fundamentally enhances the synthesis efficiency of individual cells, rather than indirectly increasing total yield by affecting strain growth rate, we employed a Congo Red binding assay. At a standardized cell density (OD₆₀₀=1), Congo Red specifically binds to cellulose. The results (Figure 5) show that the supernatant absorbance of the engineered strain group was significantly lower than that of the control group (p < 0.0001). This irrefutably proves that our device significantly enhanced the cellulose synthesis efficiency of individual cells.
Figure 5. Comparison of Congo Red absorbance by bacteria. Lower absorbance indicates higher per-cell cellulose production.
Summary
Through the experiments above, we have added detailed quantitative characterization data to the bcsB (BBa_K3520002) part and successfully demonstrated its critical role in constructing an efficient heterologous cellulose production system. This data not only validates its function but also provides valuable performance parameters and a practical foundation for future iGEM teams utilizing this part for related applications.
Our successful characterization and application of the bcsB part open up broad application prospects for the iGEM community and the wider scientific field. Based on this reliable core component for BC production, future innovations can be developed around the following directions:
Figure 6. Future application directions for the bacterial cellulose production device based on bcsB.
Advanced Functional Wound Dressings: By co-expressing functional proteins such as antimicrobial peptides or growth factors, this system can be used to easily develop composite biological dressings with active antibacterial and healing-promoting functions.
Tissue Engineering Scaffolds: The unique 3D nanofiber network of BC makes it an ideal biological scaffold for repairing skin, cartilage, bone, and even blood vessels.
Drug Delivery Carriers: The porous network structure of BC makes it an excellent platform for drug loading and controlled release. It can be used to load anticancer drugs or antibiotics for targeted therapy or chronic disease management.
Green Electronics and Biosensors: As a sustainable and biodegradable flexible material, BC can be composited with conductive polymers to serve as a substrate for flexible circuit boards or wearable biosensors, driving the development of sustainable electronics.
