Overview
According to our Project Description, we are committed to developing a powerful smart bandage that helps people manage wounds more conveniently and safely, preventing potential health and public health risks caused by wound-related issues. To achieve this goal, we have designed several functional modules, including real-time monitoring, automated drug release, and accelerated wound healing. These modules are built using both electromechanical hardware and biological components, which are integrated into a unified and coordinated system.
In this product, we use Aptamer sequences as biosensors to detect harmful bacterial infections and immediately transmit the signals to the control system. This system works in conjunction with an electro-controlled hydrogel to enable precise drug release. At the same time, the hydrogel contains engineered topical probiotics that not only maintain the microbial balance at the wound site**[1][2]** but also continuously secrete biologically active hGM-CSF growth factors. This activates the immune system and accelerates tissue repair, addressing wound-related problems at their core**[3][4]**.

This innovative approach and multi-component integration enable our smart bandage to automatically and precisely monitor wound conditions compared to traditional clinical methods[n]. It also regulates the complex microbial environment at the wound site on a micro-ecological level, achieving simple and safe wound management. More importantly, beyond the biological engineering system, we have connected biological components with traditional electromechanical devices, allowing the smart system to perform more personalized and precise adjustments. Additionally, if our designed app can integrate with a future positioning system, it can further promote the concept of a smart city, helping to solve public health issues. In this era of biotechnology, actively integrating it with other engineering fields will help us build an increasingly refined technological environment.

Product Design
This smart bandage connects to a mobile application via Bluetooth, allowing patients, their families, or doctors to monitor the wound condition at any time. It also provides a more effective wound care experience through real-time monitoring and an automated management system, helping patients avoid unnecessary anxiety and wasting medical resources. It is suitable for groups with a need for rapid wound healing or a high risk of infection, such as athletes, cleaners, and chronic wound patients. In the future, we hope to integrate big data and location systems to further advance public health management in smart cities. This design not only addresses individual wound care but also aims to contribute to public health on a macro level by reducing the risk of infection spread.


Real-time detection
In our functional design, we first introduced Aptamer technology to detect harmful bacteria. Aptamers are short strands of DNA or RNA that exhibit specificity once correctly folded. Compared to traditional antibody technologies, aptamers not only have lower costs but also offer greater stability. After reviewing relevant literature, we found that the cost and stability issues associated with antibodies in wound management cannot be overlooked, leading us to select aptamers as the core biological detection components. Aptamers can rapidly and accurately bind to specific biomarkers, and with the assistance of thiol groups and gold atoms, they can instantly transmit signals to the smart system for processing. Based on the monitoring results, the system automatically and electronically controls the release of appropriate antimicrobial peptides (AMP), as detailed in the Dry Lab section[6][19].

Intelligent automated drug delivery
Antimicrobial peptides (AMPs) are naturally occurring short peptides, typically consisting of 12 to 50 amino acids, with significant antibacterial, antiviral, and antifungal properties. The structures of these peptides usually contain positive charges and hydrophobic regions, which enable them to interact effectively with the cell membranes of microorganisms.[18]
Compared to antibiotics, AMPs can effectively inhibit harmful bacteria without causing irreversible damage to the surrounding microbiome due to their less aggressive action. This aspect is a key finding from our literature review: the balance of skin symbiotic bacteria is crucial for wound healing and maintaining health[1][2]. If antibiotics are used to wipe out bacteria all at once, it can slow down the healing process and decrease the skin’s immune response to diseases[7][8][9].
As the problem of antibiotic overuse becomes increasingly serious, AMPs are being considered as potential alternative therapies and are actively being researched for the development of new antimicrobial drugs. Furthermore, the antimicrobial properties of AMPs show good potential for clinical applications in wound healing, infection prevention, and inflammatory diseases.
In our smart bandage, the chitosan hydrogel contains sections made of another type of hydrogel, Chondroitin Sulfate (CS) hydrogel, in which the AMPs will be stored. Due to the acidic groups present, CS hydrogel carries a negative charge, encapsulating the positively charged AMPs. However, when stimulated by a specific strength of positive electric field released from the smart system, the corresponding concentration of AMPs will be released and diffuse into the chitosan hydrogel, achieving the effect of drug delivery and sterilization[10].
The use of topical probiotics
To address the issue of wound healing, we have chosen probiotics as the foundational biomaterial in our product, cultivated within the chitosan hydrogel of the bandage. Recent literature indicates that topical probiotics can stabilize and restore the skin’s microbiome, and there are successful applications of this approach in many commercially available cosmetics and anti-acne products[1][2]. However, the direct use of growth factors for treatment poses challenges due to high costs, short half-lives, and potential side effects, which limit their clinical application[4].
To overcome these challenges, we have engineered probiotics that can continuously and stably release active recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF, BBa_K5361002). This approach not only maintains the skin’s microbiome balance but also accelerates wound healing, reduces the frequency of dressing changes, and avoids high costs and the risk of secondary infections.
In selecting the probiotic strains, we chose Lactococcus lactis MG1363, which has been used in clinical phase II trials for wound care[11]. We believe this strain will provide greater safety assurance compared to other probiotics that are still in earlier stages of research.
Growth factors function and selection
Regarding the selection of growth factors, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) plays a vital role in activating neutrophils and macrophages, enhancing their ability to engulf pathogens and thereby improving overall immune system function. For accelerating wound healing, hGM-CSF binds to specific receptors, promoting cell proliferation and differentiation, including epithelial cell migration, keratinocyte proliferation, and phenotypic transformation of fibroblasts. Additionally, the immune-activating properties of hGM-CSF can stimulate the upregulation of another growth factor, vascular endothelial growth factor (VEGF), which facilitates vascular regeneration. These mechanisms are key to its role in promoting faster wound healing[3][4].
In terms of safety, hGM-CSF does not exhibit significant side effects, with the most common reactions being pain, swelling, redness, and warmth at the injection site. Compared to other growth factors, such as platelet-derived growth factor (PDGF), which has been associated with cancer risk, hGM-CSF is considered to have a higher safety profile[3][4].
Experimental Design

We have designed an experimental workflow to help validate the concept for subsequent product analysis and assembly. In the following section, we will first introduce the functions of each component, followed by a detailed explanation of the experimental procedures represented by the letter codes (A-J) in the flowchart.
Enhance active protein yield
As a human protein, hGM-CSF faces significant challenges in being efficiently translated and correctly folded within the limited prokaryotic system of Lactococcus lactis. If we directly insert the gene encoding the protein, it is highly likely that the expression efficiency would be low, and the protein would misfold, leading to aggregation as inclusion bodies inside the cell and eventually being degraded[12]. To address this issue, we first optimized the codons for compatibility with L. lactis and co-expressed it with the chaperone protein complex pKJE7 (BBa_K5361007). These chaperone proteins assist in proper protein folding through several key mechanisms, crucial for maintaining cell function. First, they bind to the hydrophobic regions of the nascent polypeptide during synthesis, preventing misfolding or unnecessary aggregation. Then, they provide an environment that promotes correct folding, allowing the heterologous protein to fold into its correct conformation in a stable and isolated environment[13].
Add signal peptide sequence
Even if the hGM-CSF polypeptide can be produced within Lactococcus lactis and folded correctly with the help of chaperone proteins, as a heterologous protein, it would still face challenges being secreted into the hydrogel surrounding the cell, let alone diffusing to the wound to promote healing. To address this issue, we artificially added a signal peptide sequence to the N-terminus of the protein-coding region[14][15][16]. After rhGM-CSF is transcribed, translated, and correctly folded, the exposed signal peptide sequence will assist the bacteria’s physiological system in recognizing and secreting hGM-CSF into the hydrogel, allowing it to ultimately diffuse to the wound site and take effect.
Select suitable vector
Since we aim to amplify and store the plasmid in E. coli DH5α while also ensuring efficient expression in Lactococcus lactis in the future, we need an appropriate plasmid that meets these requirements. The pMG36E (BBa_K5361900) plasmid perfectly aligns with our expectations. It is specifically designed for bacterial expression, featuring a strong promoter and a high-copy-number origin of replication (ori). This plasmid can efficiently replicate and express genes in a broad range of hosts, including E. coli and L. lactis, and it carries erythromycin resistance.

Splice the gene into pMG36E(BBa_K5361900)
Motivation:
Prepare vectors containing the target gene to facilitate subsequent transformation into the target bacterial strains.
Objective:
Successfully ligate the PelB-rhGM-CSF (BBa_K5361001) or Usp45-rhGM-CSF (BBa_K5361004) gene from the pUCIDT (amp) Golden Gate plasmid (BBa_K5361005 or BBa_K5361006) into the pMG36E (BBa_K5361900) expression vector, enabling the expression of the target protein in bacteria.
Method:
- Amplify and preserve the plasmid using DH5α bacteria.
- Perform gene ligation using restriction enzymes, transferring the target gene to the expression vector pMG36E (BBa_K5361900).
- Verify the success of the gene transfer using electrophoresis and gel purification techniques.

Transform the designated plasmid into E. coli or L. lactis
Motivation:
Enabling bacteria to carry the target gene and express the growth factor for subsequent protein expression experiment.
Objective:
Transform the ligated target vector(pUCIDT (amp) Golden Gate plasmid (BBa_K5361005 or BBa_K5361006), pKJE7 (BBa_K5361007), and pMG36E (BBa_K5361900)) containing the gene of interest into E. coli and Lactococcus lactis.
Method:
- Transform the plasmid into DH5α for amplification and preservation.
- Transform the plasmid into the target bacterial strains (E. coli and L. lactis).
- Verify successful transformation via plasmid extraction and electrophoresis.
Induce protein expression
motivation:
The chaperone proteins require induction to be expressed, ensuring that the growth factor properly folds and reaches the correct location.
Objective:
Ensure that the inducer functions correctly, allowing the target protein to be produced in the expected location for successful sampling and subsequent analysis.
Method:
- Cultivate the preserved bacterial strains to a healthy state for protein induction.
- Add L-Arabinose to induce chaperone protein secretion.
- Separate the cells to obtain different types of protein samples.
Growth curve of E. coli and L. lactis
Motivation:
Measure and plot the growth curves of E. coli and L. lactis to quantitatively understand the relationship between bacterial concentration and time. This will aid in experimental design and data analysis for the final product hardware.
Objective:
Accurately plot the growth curves of these bacteria under optimal growth conditions, providing a clear relationship between time and concentration, which will enhance the predictability of both experiments and product performance.
Method:
- After growing the bacteria to a healthy state, dilute them into a new culture tube to start fresh.
- Measure CFU (colony-forming units) at fixed intervals to determine bacterial concentration at each time point.
- Use the data points to allow the Dry Lab team to establish a reliable predictive model.
Prepare aptamer sensor
Motivation:
Aptamers can specifically detect biomarkers, in this case, LPS (lipopolysaccharide), and generate changes in resistance values as feedback signals to the engineering system.
Objective:
To properly fold the aptamer into the correct conformation to exhibit its specificity, reduce the thiol groups for stable connection with gold electrodes, and ensure the signal can be transmitted into the engineering system for computation.
Method:
- Dissolve the aptamer in an appropriate buffer to promote correct folding.
- Reduce the thiol groups and connect them to gold atoms.
- Apply bacterial solution for a simple electrode reaction test.
SDS PAGE protein analysis
Motivation:
To confirm the location and overall folding status of the growth factors produced by different genotypes using previously prepared samples.
Objective:
To clearly observe the presence and location of hGM-CSF on the gel during electrophoresis, aiding in accurate analysis.
Method:
- Prepare the samples from Process C and categorize them based on the same position with different genotypes.
- Run the gel with samples from the same category, using pure hGM-CSF as a control.
- Determine the presence and solubility of the protein by observing the bands.
ELISA to verify the specificity of hGM-CSF
Motivation:
ELISA allows us to determine whether hGM-CSF is properly folded and has recognition sites that can be detected by antibodies, helping us assess its quality.
Objective:
To maintain the high sensitivity of the ELISA and minimize errors, enabling us to accurately determine the protein concentration under the given conditions.
Method:
- Use an ELISA kit.
- Construct a standard calibration curve.
- Use colorimetric methods to determine the concentration.
Add Your Heading Text Here
Motivation:
After protein production, it must undergo a series of processes to successfully express its activity. TF-1 cells require GM-CSF for growth; therefore, we use the biological activity assay of GM-CSF as a means to observe whether the secreted protein is correctly folded and active.
Objective:
To use TF-1 cells to detect the activity of GM-CSF in the secretions of successfully transformed L. lactis, confirming that the GM-CSF we produced indeed has therapeutic functionality.
Method:
- Culture TF-1 cells for passage.
- Collect the secretions from L. lactis and administer them to TF-1 cells.
- Use crystal violet to assess cell viability.
Hydrogel preparation
Motivation:
The formulation of hydrogels can provide L. lactis with a suitable living environment, allowing it to continuously express the target gene and promote wound healing and health.[17]
Objective:
To create a mechanically and chemically stable hydrogel that is suitable for the survival of L. lactis.
Method:
- Develop different hydrogel formulations.
- Test the basic properties of each formulation, such as water release and toughness.
- Evaluate the results of different formulations and select the optimal solution.
Refrerence
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