Elastin-based silver-binding proteins with antibacterial capabilities
Aim: To develop novel elastin-like materials with antibacterial capabilities. Materials & methods: Artificial proteins bearing AG3 silver-binding motifs (GPG-AG3) were constructed using genetic engineering. GPG-AG3 materials were prepared as GPG-AG3 protein aggregates as well as chemically crosslinked spin-coated thin films. Both GPG-AG3 protein aggregates and thin films were incubated in silver nitrate solution and characterized using electron microscopy. Results & discussion: The GPG-AG3 substrates prepared in this work have the ability to nucleate silver under physiological conditions. When tested against gram-negative Escherichia coli bacterial culture, silver-coated GPG-AG3 materials were able to inhibit bacterial growth, confirming their antibacterial properties. Conclusion: Antibacterial artificial protein materials were successfully developed, demonstrating promise for use as wound dressings and biomedical implant coatings.
KEYWORDS: AG3 silver-binding domain n antibacterial activity n biomimetic synthesis of silver n elastin-like artificial protein
Truong Thi Hong Anh1, Ma Xing1, Duc Huynh Tien Le2, Ayae Sugawara‑Narutaki2 & Eileen Fong*1
1School of Materials Science & Engineering, Nanyang Technological University, Singapore 2Department of Chemical System Engineering, University of Tokyo, Tokyo, Japan�*Author for correspondence: [email protected]
environmentally friendly synthesis conditions [15,16]. There have been efforts to decorate polymeric substrates with silver-binding pep-tides [17,18] and these have shown that the silver crystallites nucleated on these substrates were antibacterial in nature [17]. However, prepara-tion of these materials often required tedious processing steps. New synthesis methods that can lead to the efficient synthesis of safe and functional silver-based materials are desired.
Here, the use of elastin-based artificial pro-teins incorporating silver-binding domains for the preparation of antibacterial materials is explored. Elastin is a major extracellular matrix protein, found commonly in connective tissues, such as the heart, lung, blood vessels and skin. Crosslinked elastin provides elasticity and resil-ience to tissues, allowing them to stretch and deform [19]. More importantly, elastin-like pro-teins have been shown to support cell growth [20] and wound healing [21], making them ideal candidates for wound dressings and dermal substitute applications [22–24].
Many elastin-like proteins reported in the lit-erature have the ability to undergo self-assembly to form aggregates or fibrils, in response to a change in salt concentration [25], pH [26] or temperature [27,28]. Similarly, the ‘double hydro-phobic’ elastin-like artificial protein framework (GPG) reported in this work has the ability to undergo self-assembly to form protein aggre-gates or short amyloids triggered by an increase in temperature [Le DHT, Hanamura R, Pham D-H et al.
Wound infection remains a serious problem in the clinic due to difficulty in countering the multitude of fast-evolving pathogens in the environment. Silver is one of the most effec-tive antibacterial agents due to its broad range of antimicrobial activities against bacteria, viruses and other eukaryotic micro-organisms [1]. Recently, silver nanoparticles have been explored as antibacterial agents due to their large surface area to volume ratios and physi-cochemical properties [2]. However, silver nanoparticles have the propensity to aggregate in solution, leading to subsequent loss of the bactericidal effect of silver over time [3]. More importantly, long-term exposure to silver, espe-cially uncoated silver nanoparticles, may have significant cytotoxic effects in humans [4–6]. As a result, researchers have developed meth-ods to stabilize silver nanoparticles to reduce their overall cellular toxicity [7,8]. To date, much of the attention has been devoted to produc-ing stable silver-based materials using chemical routes such as nanofibrous alkylthiol deriva-tives or polyvinylpyrolidone polymers coupled with silver nanoparticles [9–13]. Nevertheless, these approaches require lengthy synthesis steps and the products often have inadequate biocompatibility.
A scaffold approach is preferred for the prepa-ration of silver materials due to a reduced cel-lular uptake of silver [14]. With the discovery of silver-binding peptides, biomimetic approaches have been employed because of their mild and
SPECIAL FOCUS y Advanced nanobiomaterials for tissue engineering and regenerative medicine
ReseaRch aRtcle Anh, Xing, Le, Sugawara-Narutaki & Fong
Self-assembly of elastin-mimetic double hydrophobic
polypeptides, Submitted]. In this work, the GPG proteins were further engineered to incorpo-rate the silver-binding AG3 sequence at the C-terminus of the protein (GPG-AG3) (Figure 1). We predict that the GPG-AG3 proteins self-assemble in solution and serve as templates for silver nucleation. The low transition tempera-ture of GPG (15°C) is advantageous because it allows the entire synthesis process to occur readily at room temperature.
A histidine tag was incorporated to facili-tate protein purification since GPG protein aggregation was irreversible and purification by inverse transition cycling would not be possible [Le DHT, Hanamura R, Pham D-H et al. Self-assembly of
mitted]. In addition, lysine residues were also incorporated within the amino acid sequence to act as crosslinking sites for the preparation of crosslinked thin films.
In this study, we showed that GPG-AG3 artificial proteins could be used to synthesize silver nanoparticles. In addition, GPG-AG3 artificial proteins could also be chemically crosslinked to yield thin film scaffolds for silver nucleation. In both cases, antibacterial proper-ties were observed when tested against gram-negative Escherichia coli bacteria. In summary, this work demonstrates a simple approach for the preparation of antibacterial materials with potential applications in tissue engineering and regenerative medicine.
Materials & methods�n Chemicals & reagents
The plasmid pET22b-GPG encoding the elastin-like GPG sequence was obtained from the Sugawara-Narutaki laboratory at the University of Tokyo (Tokyo, Japan). Oligo-nucleotides were purchased from 1st Base Asia (Singapore). E. coli bacterial strains DH5a and BL21(DE3) were purchased from Invit-rogen (CA, USA). Reagents used for cloning, including restriction enzymes, T4 DNA ligases and DNA ladders, were purchased from New England Biolabs Inc. (MA, USA). Plasmid
DNA purification miniprep kit was obtained from Qiagen (CA, USA). Gel DNA Recovery Kit was obtained from Zymo Research (CA, USA). Urea and chemicals used for protein purif ication were from Merck (NJ, USA). Other chemicals were purchased from Sigma (MO, USA).
�n Construction of the plasmid containing GPG-AG3First, AG3 dsDNA fragment was constructed by DNA annealing. Brief ly, both forward (5´-GCCGGTCGACGCTTATTCTTCTG-GTGCTCCTCCTATGCCTCCTTTTCTC-GAGGCGG-3´) and reverse (5 -́CCGCCTC-GAGAAAAGGAGGCATAGGAGGAGCAC-CAGAAGAATAAGCGTCGACCGGC-3´) AG3 oligonucleotides were dissolved in water containing 10 mM Tris buffer, 100 mM NaCl and 100 mM MgCl
2 (pH 8). The solution was
heated to 100°C for 5 min and cooled over-night at rate of 1°C/min to room temperature. The AG3 oligonucleotides were annealed and digested with XhoI and SalI, separated on 1.5% DNA agarose gel and subsequently recovered using Zymo Gel DNA Recovery Kit. The digested AG3 DNA fragments were re-ligated into the plasmid pET22b-GPG using the XhoI site and transformed into E. Coli strain DH5a. Colonies were screened and verified by sequenc-ing. The verified pET22b-GPG-AG3 plasmid was subsequently transformed into E. coli strain BL21 (DE3) cells using heat shock.
�n Expression & purification of GPG-AG3Overnight cultures of BL21 (DE3) cells con-taining the pET22b-GPG-AG3 plasmids were innoculated in 1 l of terrific broth containing 50 µg/ml ampicillin. Protein expression was achieved via leaky expression. The follow-ing day, cells were harvested and centrifuged at 8000 rpm for 30 min at 4°C. The cell pel-let was resuspended in denaturing lysis luffer (8 M urea, 100 mM sodium phosphate mono-basic and 10 mM Tris-Cl; pH 8.0) at 10 ml/g wet cell mass and sonicated (40% output for
Figure 1. Amino acid sequence of the GPG-AG3 artificial protein. The GPG‑AG3 artificial protein comprises P for coacervation, G and silver‑binding AG3 domains. The protein also contains lysine residues for crosslinking and a 6× histidine tag for protein purification using Ni‑nitrilotriacetic acid. G: Self‑assembling glycine‑rich domains; P: Repetitive pentapeptide proline‑rich domains.
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5 × 3 s pulses) for 10 min on ice. The solu-tion was then centrifuged at 18,000 rpm for 1 h and the supernatant was mixed with Ni-nitriloacetic acid slurry for 1 h. The mixture was loaded onto a Ni-nitriloacetic acid column, and the column was washed three times with wash buffer (8 M urea, 100 mM sodium phos-phate monobasic and 10 mM Tris-Cl; pH 6.5). Bound proteins were eluted twice using elution buffer (8 M urea, 100 mM sodium phosphate monobasic and 10 mM Tris-Cl) at pH 5.5 and pH 4.0, consecutively. Eluted proteins were dia-lyzed (8 kDa molecular weight cutoff) against deionized water (pH 4.5) and subsequently lyo-philized. The purity of eluted GPG-AG3 pro-teins was verified using 15% SDS-PAGE electro-phoresis and 4800 Plus MALDI TOF/TOF™ (AB Sciex, MA, USA).
�n Preparation of crosslinked GPG-AG3 thin filmsLyophilized protein was dissolved in dimethyl sulfoxide (5 wt%) at room temperature. The protein solution was mixed homogeneously with disuccinimidyl suberate at a stoichometric ratio of 1:1. The protein mixture was pipetted onto cleaned 12 mm round glass coverslips and spin-coated at 3000 rpm to yield uniform thin films. The spin-coated films were left to dry overnight at room temperature to allow complete
crosslinking. The films were washed thoroughly with double-distilled H
2O (ddH
2O; at least five
times) to remove excess dimethyl sulfoxide and disuccinimidyl suberate.
�n Biomimetic synthesis of silver using GPG-AG3Lyophilized GPG-AG3 protein was first dis-solved in ddH
2O at 4°C to a final protein con-
centration of 100 µM in phosphate-buffered saline (PBS; pH 7.4, 2 mM NaCl). Silver nitrate was subsequently added to the protein mixture to achieve a final concentration of 0.2 mM (two Ag atoms/GPG-AG3 molecule). The solution was left at room temperature. After 3 days, samples were centrifuged and the pellets were collected. This process was repeated twice with 10% sodium dodecyl sulfate washes and then five times with ddH
2O washes.
Similarly, crosslinked GPG-AG3 films were incubated in 1 mM AgNO
3, PBS (pH 7.4,
2 mM NaCl) at room temperature for 3 days. Films were also washed vigorously in ddH
2O to
remove contaminants such as NaCl and AgCl.
�n Characterization of GPG-AG3 proteinsUV–visible spectroscopyThe lower critical transition temperature (LCST) of GPG-AG3 was determined using
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Figure 2. Verification of GPG-AG3 purity and molecular weight using SDS-PAGE and MALDI-TOF. (A) The presence of purified GPG‑AG3 protein was verified using 15% SDS‑PAGE gel electrophoresis. (B) MALDI‑TOF spectra of purified GPG‑AG3 protein dissolved in water. The theoretical molecular weight of GPG‑AG3 was calculated to be 18.2 kDa.
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UV–visible (UV–VIS) spectroscopy. First, lyo-philized GPG-AG3 protein was dissolved in PBS (pH 7.4) at 4°C (final concentration of 20 µM). The protein solution was pipetted into a 1-cm quartz cell and placed in the Peltier ETCR-762 thermostated single cell holder (Jasco, OK, USA). Samples were heated at 0.1°C/min and the optical transmittance at 350 nm was monitored using Jasco V-670 UV–VIS spectrophotometer, referenced against PBS. The LCST was deter-mined to be the temperature at which a 10% reduction in the original transmittance of the solution was observed.
The presence of silver was confirmed using UV–VIS spectrometer 2501PC (Shimadzu Corp., Kyoto, Japan; at wavelengths between 300 and 900 nm).
Field emission scanning electron microscopySamples were dropped onto carbon tape and left to dry at room temperature. Subsequently, samples were visualized using a field emission scanning microscope (6340F; Jeol Ltd, Tokyo, Japan), using an accelerating voltage of 5 kV and an emission current of 12 mA. The par-ticle size was measured manually using ImageJ software [101].
Transmission electron microscopyFor high-resolution transmission electron microscopy (HRTEM), samples were dropped onto copper grids coated with carbon and left to dry at room temperature. HRTEM samples were visualized using an accelerating voltage of 200 kV and an emission current of 105 mA.
Antibacterial assays2×YT agar Approximately 107 colony-forming units (CFU) of gram-negative E. coli bacteria were cultured on 2×YT agar plates precoated with the silver nanoparticles containing varying concentrations of silver nanoparticles. Plates were incubated over-night at 37°C before images were acquired. Sepa-rately, silver-coated GPG-AG3 thin films were placed in direct contact with E. coli at 107 CFU precoated onto the surfaces of 2×YT agar plates.
Liquid 2×YT mediaE. coli bacteria culture was grown to log phase (i.e., optical density at 600 nm 0.6–0.8) and sub-sequently reinnoculated at 1:100 dilution into 2×YT media supplemented with varying concen-trations of silver nanoparticles. Separately, silver-coated GPG-AG3 thin films were immersed in 5 ml of 2×YT media containing similar con-centrations of E. coli. In both cases, the growth curves were constructed by measuring the optical density at 600 nm at regular time intervals for up to 24 h.
�n Statistical analysisFor all experimental data, results represented as means ± standard deviation of three inde-pendent experiments. The statistical signifi-cance of differences was estimated by student’s t-test. Differences were taken to be statistically significant at p ≤ 0.05.
Results & discussionThe purity and molecular weight of the GPG-AG3 artificial protein was confirmed by
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Figure 3. Characterization of GPG-AG3 using UV–visible and field emission scanning electron microscopy. (A) Light absorbance (l = 350 nm) of GPG‑AG3 in phosphate‑buffered saline (pH 7.4) with increasing temperature. (B) Field emission scanning electron microscopy images of GPG‑AG3 protein aggregates in phosphate‑buffered saline (pH 7.4) at room temperature.
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SDS-PAGE electrophoresis and MALDI-TOF, respectively (Figure 2). The typical lyophilized protein yield was 40 mg/l.
To understand the thermoresponsive behav-ior of GPG-AG3 in solution, we first deter-mined the LCST of the protein using UV–VIS spectro scopy. Figure 3A shows the light absorption (l = 350 nm) of GPG-AG3 in PBS (pH 7.4) with increasing temperature. The LCST was found to be approximately 15°C, suggesting that self-assembly of GPG-AG3 proteins occurs readily at room temperature. The GPG-AG3 protein aggregates were visualized using field emission scanning electron microscopy. Figure 3B shows a representative image of the GPG-AG3 protein aggregates formed in PBS (pH 7.4) at room tem-perature. The average diameter of the protein aggregates was estimated to be approximately 100 nm.
The ability of GPG-AG3 to nucleate silver biomimetically was further examined using UV–VIS spectroscopy. Figure 4 shows the UV–VIS spectra of samples under various conditions. Characteristic surface plasmon resonance peaks for silver at 420 nm [15] were observed in the presence of 100 µM GPG-AG3 in PBS (pH 7.4, 2 mM NaCl) after 3 days. In comparison, no such peaks were observed in the controls.
We also observed a peak at 420 nm for the control GPG protein lacking the AG3 silver-binding sequence, but the peak intensity was threefold lower (Figure 4). This was probably due to the nonspecific binding of silver ions to the histidine residues at the C-terminus of GPG-AG3 [29]. Taken together, our data suggest that the silver nucleation triggered by GPG-AG3 was due to the presence of the AG3 silver-bind-ing domain. We also noted that silver nucleation did not occur on GPG-AG3 when NaCl was omitted. This observation was consistent with reported literature, where chloride ions have been thought to play an important role in providing electrostatic stability to silver nanocrystallites nucleated on the AG3 peptides [30].
To further characterize the silver-coated GPG-AG3 protein aggregates, we subjected sam-ples to HRTEM analysis. Figure 5 shows a trans-mission electron microscopy image of silver-coated GPG-AG3 protein aggregates after day 3. The dark spots are the silver nanoparticles. As expected, GPG-AG3 served as ‘templates’ for the nucleation of silver in solution.
The silver particles could also be isolated by thorough washing with 10% sodium dodecyl sulfate and ddH
2O. Figure 6A shows HRTEM
image of the silver nanoparticles obtained after
the removal of GPG-AG3. Twinned structures were observed (Figure 6A; arrows). The selected area electron diffraction patterns of the sil-ver nanoparticles indicated that the crystals have a (111) face-centered cubic lattice struc-ture (Figure 6A; inset). Energy-dispersive x-ray spectro scopy analysis of the silver nanoparticles also confirmed the presence of elemental silver (Figure 6B). The copper, carbon and oxygen sig-nals are from the copper sample grid used for transmission electron microscopy ana lysis. The diameters of the silver nanoparticles ranged from 20 to 25 nm (Figure 6C).
Figure 4. UV spectroscopy of various samples after 3 days. Samples were incubated in 0.2 mM silver nitrate solution at room temperature for 3 days. A characteristic surface plasmon resonance peak at 420 nm indicates the presence of silver nanoparticles. PBS: Phosphate‑buffered saline.
Figure 5. High-resolution transmission electron microscopy image of silver nanoparticles nucleated on GPG-AG3 after 3 days. The dark spots are silver nanoparticles.
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In the case of GPG-AG3 thin films, a brown coloration was noted in films incubated in 1 mM silver nitrate solution after 3 days. A higher con-centration of AgNO
3 was used here to ensure
that Ag+ was present in excess given the larger sample dimensions of the GPG-AG3 thin films. Unfortunately, we were unable to obtain reli-able UV or visual light data due to the opacity of the films. Instead, samples were visualized using field emission scanning electron micro-scopy (Figure 7A). Silver particles that were mostly spherical in shape were nucleated on the surface of the GPG-AG3 thin films. The mean particle size of the silver found on GPG-AG3 films was 510 ± 205 nm. In addition, the presence of silver was confirmed via energy-dispersive x-ray spec-troscopy analysis (Figure 7B). The silicon and oxy-gen peaks were from glass coverslip and protein film, respectively. Silver-bound GPG-AG3 thin films were washed vigorously in water to solu-bilize any AgCl that may be precipitated on the films since we would not be able to distinguish
AgCl from silver particles using microscopy. Our energy-dispersive x-ray spectroscopy data indicated the presence of AgCl, even though the intensity of the AgCl peaks were threefold lower than that of silver (Figure 7B). This observation was consistent with a recent report by Currie et al., where inevitable traces of Cl contamina-tion were also observed on the surfaces of their silk–silver-binding protein films [31].
To determine if the silver particles synthesized in this work had antibacterial properties, we cul-tured gram-negative E. coli bacteria in the pres-ence of silver. Data from our antibacterial assay showed that E. coli bacteria (107 CFU) growth was completely inhibited on 2×YT agar plates precoated with 20 µg/ml of silver nanoparticles (Figure 8A). Similarly, 20 µg/ml of silver nano-particles was sufficient to completely inhibit the growth of E. coli in 2×YT liquid media (Figure 8B). In comparison, supplementing media with only GPG-AG3 proteins (100 µg/ml) had no inhibition effect on bacterial growth.
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Figure 6. High-resolution transmission electron microscopy analysis of silver nucleated using GPG-AG3. (A) Image of silver nanoparticles obtained after the removal of GPG‑AG3 proteins (arrows: twinned structures). The inset shows the selected area electron diffraction patterns of silver nanoparticles. (B) Energy‑dispersive x‑ray spectroscopy spectrum confirming the presence of elemental silver. (C) Distribution of diameters of the silver nanoparticles.
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Similar antibacterial properties were also noted for the silver-coated GPG-AG3 thin films (Figure 9A). A bacterial inhibition zone could be clearly observed along the periphery of silver-coated GPG-AG3 thin film (Figure 9A; arrow). By contrast, bacterial growth could be found along the edges of the controls. Similarly, E. coli bac-terial growth was also inhibited in the presence of silver-coated GPG-AG3 thin films in 2×YT liquid media (Figure 9B).
ConclusionIn this work, an elastin-like material (GPG-AG3) with antibacterial properties was developed. We showed that both GPG-AG3 protein aggregates
and crosslinked thin films were able to cause the biomimetic nucleation of silver. Silver particles formed on GPG-AG3 protein aggregates have diameters of 20–25 nm while silver particles formed on GPG-AG3 thin films were largely spherical and significantly larger (510 ± 205 nm). The striking differences in particle size could be attributed to the higher concentrations of AgNO
3 used for the thin films, as well as differ-
ences in the presentation of AG3 on the surface. Further investigation is ongoing to elucidate the mechanisms leading to such differences.
The antibacterial properties of the silver par-ticles formed on GPG-AG3 protein aggregates and on crosslinked thin film were demonstrated
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Figure 7. Field emission scanning electron microscopy analysis of silver nucleated on GPG-AG3 thin film. (A) Representative image of silver nanoparticles formed on the surface of crosslinked GPG‑AG3 thin films. (B) Energy‑dispersive x‑ray spectroscopy spectrum confirming the presence of elemental silver.
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Figure 8. Bacterial growth in the presence of silver nanoparticles. Bacteria growth on (A) 2×YT agar plates and (B) 2×YT liquid media; both supplemented with varying concentrations of silver nanoparticles. Error bars represent means ± standard deviation. AgNP: Silver nanoparticle.
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Executive summary
� Elastin‑like artificial proteins bearing silver‑binding AG3 domains (GPG‑AG3) were constructed, successfully expressed and purified.
� The GPG‑AG3 artificial proteins were able to undergo self‑assembly to form protein aggregates in the presence of NaCl at room temperature.
� GPG‑AG3 thin films were also prepared by chemical crosslinking and spin‑coating onto glass coverslips.
� Both GPG‑AG3 protein aggregates and thin films have the ability to bind silver particles in the presence of silver nitrate. The presence of silver was confirmed using UV–visible spectroscopy and visualized using electron microscopy.
� Silver particles formed on GPG‑AG3 protein aggregates have diameters between 20 and 25 nm in phosphate‑buffered saline solution, while particles formed on GPG‑AG3 averaged approximately 500 nm in diameter.
� Silver nanoparticles isolated from both GPG‑AG3 protein aggregates and silver‑coated thin films demonstrated antibacterial activities towards gram‑negative Escherichia coli.
using gram-negative E. coli bacteria. On solid 2×YT agar plates and in liquid media, E. coli growth was inhibited by 20 µg/ml of silver. Similarly, no E. coli growth could be observed on silver-coated GPG-AG3 thin films. This work demonstrates the potential for silver-coated elas-tin-based materials to be used for antibacterial functions in tissue engineering and regenerative medicine.
Future perspectiveThe increasing use of antibacterial materials has driven the biomedical field to devise new meth-ods to synthesize safer and more effective silver-based materials. While biomimetic methods are desired for their mild and environmentally friendly synthesis conditions, few studies have focused on understanding how various process parameters affect silver synthesis and the result-ing antibacterial properties. Our future goal
is to identify the important parameters that will lead to controlled synthesis of silver nano-particles with maximal antibacterial properties and minimal toxicity.
AcknowledgementsThe electron microscopy and XRD work were performed at the Facility for Analysis, Characterization, Testing and Simulation in Nanyang Technological University.
Financial & competing interests disclosureThe authors would like to thank the School of Material Sciences and Engineering at the Nanyang Technological University (Singapore) for funding. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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Figure 9. Bacterial growths in the presence of silver-coated GPG-AG3 thin films. Bacteria growth on (A) 2×YT agar plates and (B) 2×YT liquid media was completely inhibited in the presence of silver‑coated GPG‑AG3 thin films (arrow). Error bars represent means ± standard deviation.
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n� Reports the use of silk-based recombinant protein materials with antibacterial properties.
