Table of Contents
Our Goal
During the period when the patient uses PANDAGE on the wound, an aptamer-equipped chip detects the source of infection at the wound site, while engineered probiotics in the hydrogel secrete growth factors to promote the patient’s wound healing.
I. Cloning
The sequence ordered from IDT will be cloned into pMG36E for subsequent transformation. First, digest the IDT plasmid using BsaAI and NruI-HF. After performing DNA electrophoresis on the digestion product, purify the gel. Then, use SalI and Acc65I to cut the purified linear gene and pMG36E to create sticky ends. Finally, perform ligation between the linear gene and the cut pMG36E.
Analysis
The OD600 peak for MG1363 was approximately 1.2, which did not reach the detection limit of the machine, resulting in a growth curve that aligned well with the CFU data. The growth of MG1363 was not as robust as that of E. coli (DH5α), with its exponential phase occurring between 30 and 40 hours.
II. Signal Peptide and Chaperone Protein
A signal peptide is an amino acid sequence present in newly synthesized proteins. Typically, during protein synthesis, the signal sequence is recognized and bound by specific molecules, determining the direction and pathway of protein transport. Chaperone proteins, on the other hand, use ATP for molecular assembly and assist in protein folding, reducing the likelihood of protein degradation during synthesis. Therefore, in the context of vector sequence engineering design, the collaborative role of signal peptides and chaperone proteins is indispensable.
Lane 1: ladder, Lane 2: pUC57, Lane 3: pUCIDT-AMP (PelB-rhGM-CSF), Lane 4: pUCIDT-AMP (Usp45-rhGM-CSF), Lane 5: pKJE7, Lane 6: pKJE7+pUC57, Lane 7: pKJE7+pUCIDT-AMP (PelB-rhGM-CSF), Lane 8:pKJE7(Induced), Lane 9: pKJE7+pUC57(Induced), Lane 10: pKJE7+ pUCIDT-AMP (PelB-rhGM-CSF)(Induced), Lane 12: hGM-CSF control, Lane 13: ladder
Lane 1: ladder, Lane 2: pUC57, Lane 3: pUCIDT-AMP (PelB-rhGM-CSF), Lane 4: pUCIDT-AMP (Usp45-rhGM-CSF), Lane 5: pKJE7, Lane 6: pKJE7+pUC57, Lane 7: pKJE7+pUCIDT-AMP (PelB-rhGM-CSF), Lane 8:pKJE7(Induced), Lane 9: pKJE7+pUC57(Induced), Lane 10: pKJE7+ pUCIDT-AMP (PelB-rhGM-CSF)(Induced), Lane 12: hGM-CSF control, Lane 14: ladder
Based on Figure B-I, results were observed in all samples containing the GM-CSF gene, indicating that the protein was successfully folded.
According to Figure B-II, after lysing the cells, the pellet was extracted to observe unfolded proteins. However, due to the sample’s high viscosity, it was difficult to accurately interpret the results.
From these results, we were unable to conclusively demonstrate the importance of chaperone proteins, as the presence of chaperone proteins did not directly affect protein folding. However, we speculate that chaperone proteins may influence the success rate of folding, thereby increasing protein yield. Therefore, to confirm this aspect, further validation using ELISA would be required.
Additionally, to verify whether the signal peptide successfully facilitated the secretion of GM-CSF from the bacteria, it would be necessary to concentrate the bacterial supernatant using a concentrator before running SDS-PAGE, and also confirm with ELISA. However, due to limited funding and time constraints, this part of the experiment cannot be completed as scheduled.
III. Observation of L. lactis Growth at 37°C
Within the hydrogel, L. lactis will be close to the wound, affected by the surface temperature of the skin. L. lactis may not be able to grow at its optimal temperature but will also not be exposed to the high temperatures of body heat. To determine the growth pattern and curve of L. lactis at the maximum temperature of 37°C, we attempted to establish the extreme conditions for our product.
L. lactis can survive for about 5-10 hours in a 37°C environment, indicating that relatively high temperatures do indeed have a negative impact on the survival of our target probiotic. Further research revealed that there is another strain of L. lactis that is more suitable for growth at 37°C, which could be explored for adjustments in the future.
According to the data presented, Erythromycin exhibits significantly lower bactericidal activity compared to Ampicillin. This difference is particularly evident under the resistance selection conditions for the pMG36E plasmid, which involves an Erythromycin concentration of 200 µg/mL.
IV. AMP Effection
Detect the bactericidal and antibacterial effects of AMP. Introduce AMP into the culture tubes of E. coli and L. lactis during their exponential phase (at this point, it’s 0 minutes). Subsequently, take samples under 38hr to perform CFU counts and observe how AMP effects the growth curve.
After AMP treatment, it was observed that both AMP concentrations of 25 μg/mL and 50 μg/mL had a significant bactericidal effect compared to the untreated control at the 30-minute mark.
V. Probiotic Growth Curve
To reduce the frequency of bandage changes, the survival time of the probiotics is also a critical consideration. Ensuring that our probiotics can continuously provide growth factors is essential. Thus, we first tested the growth curve of L. lactis at room temperature and estimated that the probiotics could survive for approximately 5-6 days under natural conditions.
With this result in mind, we then explored the growth behavior of the probiotics within the hydrogel. It was found that L. lactis could indeed survive in the hydrogel, though its growth was slightly inhibited compared to natural conditions. Nevertheless, the bacteria exhibited good growth, with a survival time of approximately 6 days. This confirms that the probiotic can serve as a viable dressing, offering a long-term and sustained treatment tool for wound care.
After AMP treatment, it was observed that both AMP concentrations of 25 μg/mL and 50 μg/mL had a significant bactericidal effect compared to the untreated control at the 30-minute mark.
VI. Overall product explanation
To ensure that the chondroitin 4-sulfate (CS) hydrogel maintains its specific shape after gel formation, the liquid hydrogel is injected into a cylindrical mold, which is 3D-printed using high-precision White V4 resin (produced by Formlabs Form 3+). The hydrogel is then solidified at 60°C, and hBD-3 (a type of antimicrobial peptide, AMP) is added in deionized water. The mixture is incubated at 4°C. A lid is placed on the top of the mold to keep the hydrogel moist.
Since CS hydrogel is negatively charged, controlling drug release from the hydrogel via an external electric field is crucial. To achieve this, a conductive material is wrapped around the mold, leaving a central hole for wires to pass through the CS hydrogel, creating a concentric electric field.
We also designed holes on the back of the mold so that when the electric field exerts pressure on the hydrogel, the encapsulated drug is released through these holes.
We select foam dressings as a base material to pandagel since it can provide excellent absorption of wound exudate, helping to maintain an optimal moisture balance, which is critical for wound healing. Additionally, this kind of dressings are also soft and flexible, making them comfortable for patients while conforming to the wound area.
Lastly, foam dressings are available in various sizes and shapes, making them adaptable to different wound types and body areas.
On either side of the foam dressings, screen-printed gold electrodes are placed. In the central circular area (0.11 cm^2) of the gold electrode (working electrode), a thiolated, modified anti-LPS aptamer is attached. Upon binding with its biomarker, the aptamer generates a detectable change in resistance, which is then transmitted for further signal processing.
The CS hydrogel, printed and formed into a gel, is placed in the center of the screen-printed gold electrode. Once the system detects the presence of a harmful pathogen via the aptamer-based sensor, the electric field is activated, which triggers the release of the AMP from the hydrogel.
The wires are connected to the Working electrode, Reference electrode, and Counter electrode, respectively, to collect data on the corresponding resistance changes. These connections allow for accurate monitoring and measurement of electrochemical reactions within the system.
The complete Pandage assembly includes an Arduino circuit board capable of receiving and processing signals, a mold to contain and stabilize the CS hydrogel, a sticky substrate for securing the device, and electrochemical sensing electrodes. The fully assembled Pandage device is demonstrated on a person’s hand (with gloves).
Pandage device from different perspectives：
