Team:NWU-CHINA-A/Proof Of Concept
The purpose of our project is to design a broad-spectrum antibacterial and biodegradable adhesive bandage. There are five parts about it:(figure 1)
Figure 1 five parts about the adhesive bandage
PhaP-AMP
In the process of screening antimicrobial peptides, we preferred to select human antimicrobial peptides to reduce the risk of application in the human body. At the same time, in the process of reading the literatures, we found a series of synthetic antimicrobial peptides with small molecular weight and strong antibacterial activity. Finally, we determined that HD5, HD5d5, HBD3, DCD1L, P5, P6.2 expressed in with PhaP.
In the process of verifying the expression of the protein, we found that PhaP-HD5 and PhaP-HD5d5 can be stably and efficiently expressed in E. coli Coincidentally, both of them are human defensins (or derivatives). Human defensin is one of the antimicrobial peptides, which is harmless to human body. So we initially thought that HD5 and HD5d5 are the antimicrobial peptides we wanted.
We proved that the transformed E. coli can express PhaP-AMP successfully(figure 2), PhaP-HD5 and PhaP-HD5d5 are the most expressed complexs among them.
Figure 2 the expression of PhaP-AMP
PHA
In order to verify the adhesion between PHA and PhaP-AMP, we did the adhesion experiment between PHA and PhaP.
First, we used electrospinning technology to make PHA films. Secondly the films was immersed in the supernatant after bacterial sonication for 2 hours. Thirdly, we washed the films gently with lysis buffer, then put them into a centrifuge tube and added SDS-PAGE buffer, heat at 90℃ until the thin membrane softens. In order to make the adhesion between PhaP-AMPs and PHA better, we used nano-level PHA for verification. Added PHA and supernatant in a ratio of 1:20, stirred two hours by the magnetic stirrer, then centrifuged at 8000rpm for 2min, rinsed twice with lysate, and added SDS-PAGE buffer.
We took the liquid from the above product for SDS-PAGE, as we can see, PhaP-HD5d5 combines the most products with PHA，whether it is electrospinning or nanoparticles.
Figure 3 Combination of PHA and PhaP-AMP
Finally, we chose HD5d5 as our target antimicrobial peptide.
PHAs are considered as one of the most promising "green plastics". Due to the COVID-19, we were unable to characterize the biodegradability of PHAs in the laboratory, but by consulting the literature, we learned that:PHAs are biodegradable and biocompatible, and can be degraded into H2O and CO2 by many microorganisms in the natural environment[1]. The biodegradability of PHAs allows it to be quickly decomposed in the natural environment without causing environmental problems. Today, with serious medical waste pollution, the biodegradability of PHAs is conducive to environmental protection and is also the mainstream direction of future social material development.
Electrospinning, as the most promising process method for preparing polymer nanofibers, has received widespread attention from academia and industry. The nanofiber cloth prepared by electrospinning has a porous structure of fibers. Its smaller pore size can block contact between bacteria and wound surface. More importantly, the nanofibers prepared by electrospinning technology can structurally mimic the extracellar matrix (ECM) secreted by cells and accelerate wound healing. Electrospinning is a very good technique for making adhesive bandages.
Figure 4 the micrograph of electrospinning
Non-woven fabric
During the product planning process, our team discussed the outer materials of the adhesive bandage. Non-woven fabric is composed of directional or random fibers. It is called cloth because of its appearance and certain properties.
Non-woven fabric has the characteristics of moisture-proof, breathable, flexible, light weight, non-combustible, easy to decompose, non-toxic and non-irritating, rich in color, low price, and recyclable.
Bacterial Mucin
PHA is the material for making the central island of adhesive bandage, which has antibacterial activity and plays a major bacteriostatic role. The outer layer of the adhesive bandage is non-woven. In order to connect the non-woven fabric and PHA, we chose the bacterial mucin. It consists of two types of proteins, adhesion protein and cohesive protein. Cohesion protein can be self-assembled into fibers, while adhesion protein is responsible for interfacial bonding. The combination of two types proteins forms the basis of bioadhesives.
The fibrillar structures of adhesion protein have intrinsic advantages for interfacial underwater adhesion. These advantages include tolerance to environmental deterioration, self-healing arising from self-polymerization, and large fiber surface areas. [2] So we can use bacterial mucin to connect non-woven fabric and PHA, to increase the water resistance of our adhesive bandage.
Glucose starvation suicide switch
Glucose starvation activates the catabolic response using cyclic-AMP (cAMP) receptor protein (CRP), one of the best-studied TF’s. Low glucose environments cause the cAMP-CRP complex to bind target DNA and regulate hundreds of gene targets[3].
We used PT-α crp to control the downstream gene. It is sensitive to glucose starvation. When glucose is deficient in the environment, glucose starvation induces the overexpression of autolysin gene in the T-α crp promoter(acmA).
Figure 5 PT-α crp mediated by glucose starvation turns on downstream autolysin
gene
References
[1]Polyak P, Dohovits E, Nagy GN, Vertessy BG, Voros G, Pukanszky B. Enzymatic degradation of poly-[(R)-3-hydroxybutyrate]: mechanismm, kinetics, consequences [J]. Int J Macromol, 2018, 112: 156-162
[2]Zhong C, Gurry T, Cheng AA, Downey J, Deng Z, Stultz CM, Lu TK. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nat Nanotechnol. 2014 Oct;9(10):858-66. doi: 10.1038/nnano.2014.199. Epub 2014 Sep 21. PMID: 25240674; PMCID: PMC4191913.
[3]Bothfeld W, Kapov G, Tyo KEJ. A Glucose-Sensing Toggle Switch for Autonomous, High Productivity Genetic Control. ACS Synth Biol. 2017 Jul 21;6(7):1296-1304. doi: 10.1021/acssynbio.6b00257. Epub 2017 Mar 30. PMID: 28274123.