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Converting a natural protein compartment into a nanofactory for the size-constrained synthesis of antimicrobial silver nanoparticles Tobias Wolfgang Giessen, and Pamela A Silver ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00117 • Publication Date (Web): 08 Jun 2016 Downloaded from http://pubs.acs.org on June 9, 2016

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ACS Synthetic Biology

Converting a natural protein compartment into a nanofactory for the size-constrained synthesis of antimicrobial silver nanoparticles Tobias W. Giessen†,‡ and Pamela A. Silver*,†,‡ †

Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA ‡

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA

KEYWORDS: encapsulin, nanocompartment, nanobiotechnology, inorganic nanomaterials, biogenic, silver nanoparticles.

ACS Paragon Plus Environment

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ABSTRACT

Engineered biological systems are used extensively for the production of high value and commodity organics. On the other hand, most inorganic nanomaterials are still synthesized via chemical routes. By engineering cellular compartments, functional nanoarchitectures can be produced under environmentally sustainable conditions. Encapsulins are a new class of microbial nanocompartments with promising applications in nanobiotechnology. Here, we engineer the Thermotoga maritima encapsulin EncTm to yield a designed compartment for the sizeconstrained synthesis of silver nanoparticles (Ag NPs). These Ag NPs exhibit uniform shape and size distributions as well as long-term stability. Ambient aqueous conditions can be used for Ag NP synthesis while no reducing agents or solvents need to be added. The antimicrobial activity of the synthesized protein-coated or shell free Ag NPs is superior to that of silver nitrate and citrate-capped Ag NPs. This study establishes encapsulins as an engineerable platform for the synthesis of biogenic functional nanomaterials.


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Engineered organisms are used with increasing success in diagnostics, therapeutics and for the production of high-value or commodity organics.1, 2 Initial steps have been taken to expand the scope of synthetic biology towards the synthesis of functional inorganic nanomaterials.3-5 Up till now, only a limited number of biomolecular nanostructures have been utilized to guide the fabrication of nanomaterials, most prominently virus capsids4 and ferritins.6 Initial proof of principle studies focused on using ferritins for the in vitro synthesis of inorganic nanophase materials, including iron sulfide, manganese oxide and uranium oxide.7 Subsequently, other protein nanocages were introduced for the synthesis of inorganic nanomaterials, exemplified by early studies using cowpea chlorotic mottle virus (CCMV) for the in vitro mineralization of polyoxometalate species.8 Protein cages are now being used as biocompatible systems for the production of inorganic-based imaging probes, nanoarchitectures with interesting optical and electronic properties as well as other applications in biomedicine, materials science and catalysis.4,

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Here, we establish a recently discovered class of proteinaceous microbial

nanocompartments called encapsulins as a new platform for the production of functional inorganic nanomaterials. Using biogenic protein-based nanostructures as templates and catalysts for the production of nanomaterials has a number of inherent advantages: The size-range of available biogenic nanostructures useful for nanobiotechnology ranges from very small nanocontainers (e.g. miniferritins, douter = 9 nm)10 to mesoscale structures (e.g. bacterial microcompartments, douter = up to 500 nm)11 allowing the synthesis of many different nanomaterials displaying specific properties only accessible at these scales. The defined size and shape of capsid- or rod-like biological nanoarchitectures allows size-constrained synthesis in their interior, leading to a homogeneous and monodisperse population of nanosized objects.4 Proteinaceous compartments can be easily


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modified with atomic precision via chemical and genetic routes giving rise to a large number of diverse and complex potential applications.12 The possibility to use both the interior and exterior surfaces of shell-like nanostructures allows for the potential synthesis of nanomaterials that integrate multiple functionalities.13, 14 And lastly, using biology for the synthesis of inorganics will decrease the amount of harmful chemicals and solvents used in inorganic syntheses as well as reduce the energy input needed. In nanobiotechnology, inorganic nanomaterials of a defined size and shape are synthesized by combining a natural or engineered compartment with a precursor metal salt. Targeted mineralization inside the nanocompartment is initiated by either directing precursor ions to the interior of a nanostructure via charge complementarity followed by the addition of a reducing agent,15 or by using genetic fusions of metal-binding peptides.16

Figure 1. Engineering of the T. maritima protein compartment. Schematic depiction of capsid engineering. The ferritin-like protein (Flp, orange) was removed from the wild-type encapsulin system and the AG4 peptide (red) was fused to the N-terminus of EncTm yielding EncTmAG4 which would then allow the size-constrained synthesis of silver nanoparticles in its interior when exposed to silver ions. Encapsulins are a recently discovered class of microbial nanocompartments. They form icosahedral shells of 60 (T = 1, douter = 22-24 nm) or 180 (T = 3, douter = 32-28 nm) subunits that contain 5 Å pores and have been shown to encapsulate specific cargo proteins.17 Their single shell-forming capsid protein possesses the HK97-like fold present in many tailed phages


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suggesting a common evolutionary origin.18 Encapsulin cargo is directed to the inside of the capsid via a short C-terminal targeting sequence. So far, all identified cargo proteins seem to be involved in redox processes including iron oxide precipitation19 and encapsulation of peroxidases.20 This led to the proposal that encapsulin systems might be involved in oxidative stress response. The fact that the N-terminus of the capsid protein protrudes towards the inside of the capsid while the C-terminus is presented on the outside makes it amenable to display genetically fused peptides on the inner and outer surfaces of the encapsulin compartment. In addition, the naturally-occurring targeting sequences can be used to encapsulate non-native cargo proteins.21 Those properties make encapsulins highly engineerable systems with many potential applications in diagnostics, drug delivery, photonics and catalysis. Here, we engineer the T. maritima encapsulin system and convert it from a natural iron oxideforming nanocompartment into a designed silver nanoparticle-synthesizing system.

Results and Discussion The T. maritima capsid protein (EncTm) can be engineered to contain internal silver precipitation sites. EncTm assembles into a 24 nm 60 subunit capsid encapsulating ferritin-like proteins (Flp) arranged in decameric complexes.17 We deleted Flp from the EncTm operon and genetically fused the DNA-encoded silver-precipitating peptide AG4 (NPSSLFRYLPSD)9, 16 to the N-terminus of EncTm resulting in the engineered capsid protein EncTmAG4. Based on our design the AG4 peptide will be displayed on the interior surface of the engineered compartment. The exact mechanism of Ag precipitation by AG4 is unknown, but the following model has been proposed: AG4 binds to preformed nuclei of elemental silver present in any Ag salt solution. Interaction with AG4 would result in a locally reducing environment around the bound nuclei,


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allowing accelerated reduction of ions at the peptide-metal interface. In addition, the surface energy of the crystal lattice would be lowered by AG4-binding leading for example to a lowered surface energy of the [111] face enabling faster growth at the re-entrant edges.16 When incubated with silver nitrate (AgNO3) silver precipitation will be selectively initiated inside the engineered EncTmAG4 compartment (Figure 1). The fixed dimensions of the capsid will constrain the size of the resulting silver nanoparticle (Ag NP) resulting in a monodisperse population of protein-coated particles. To make a prediction about the available space inside the engineered compartment based on the solution structure of the AG4 peptide (8-10 nm extended structure with C-terminal bend between the residues YLP)22 and the crystal structure of EncTm, we created a qualitative homology model of EncTmAG4 which showed that the space available for nanoparticle synthesis inside the engineered capsid is in the range of dinterior = 13-15 nm (Supplemental Figure S1). Biogenic silver nanoparticles in this size range would be particularly appealing for antimicrobial applications. Silver ions (Ag+) exhibit both bacteriostatic and bactericidal activity, based on the concentrations used, by directly damaging membranes and DNA and promoting the generation of reactive oxygen species (ROS).23 It has been established that leaching Ag+ is the main contributor to the antimicrobial effects of Ag NPs.24 However, Ag NPs smaller than 20 nm show an increase in antimicrobial activity that cannot be solely explained by the release of Ag ions, but likely involves size-, shape- and coating-dependent effects that lead to improved cell-particle contact, bioavailability and uptake.25


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Figure 2. Characterization of EncTmAG4 in vivo. (A) Relative cell viability of EncTm- and EncTmAG4-expressing E. coli in the presence of different concentrations of AgNO3. Cell viability was based on colony forming units (CFUs). (B) Left: ICP-MS analysis of washed cells after overnight exposure to 0, 50 and 100 µM AgNO3. Right: TEM micrographs of fixed cells, expressing EncTm or EncTmAG4 after overnight exposure to 50 µM AgNO3. Intracellular electron-dense material can only be seen in EncTmAG4 cells. Size-exclusion chromatogram of EncTmAG4. The inset shows the SDS-PAGE gel of the indicated fractions (red asterisk), a negative stain TEM micrograph and the size distribution of purified EncTmAG4 (determined by DLS). The engineered capsid protein affects the silver sensitivity of bacterial cells. Expression of EncTmAG4 in E. coli leads to an increase in silver resistance compared with a strain expressing wild-type (wt) EncTm as measured by CFU counting after over-night incubation (Figure 2A). This effect likely results from the removal of Ag+ by AG4-mediated precipitation of elemental silver lowering the local concentration of toxic Ag ions. The observed increase in cell survival in


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the presence of silver suggests that EncTmAG4 is active for Ag ion binding and precipitation. Using inductively coupled plasma mass spectrometry (ICP-MS) we showed that an EncTmAG4expressing strain retains significantly more silver than a control strain expressing wild-type EncTm when incubated with 50 and 100 µM AgNO3 (Figure 2B). Transmission electron microscopy (TEM) of fixed cells showed that extracellular silver precipitates are present in both strains. However, only the EncTmAG4-expressing strain showed additional intracellular electron-dense material, representing elemental silver reduced by AG4-containing capsids (Figure 2B). The engineered capsid protein promotes formation of nanocompartments. The engineered compartment was purified from EncTmAG4-expressing cells via polyethylene glycol (PEG) precipitation followed by size-exclusion chromatography (Figure 3A). TEM and dynamic light scattering (DLS) showed that EncTmAG4 was still able to form uniform spherical/icosahedral capsids with a diameter of 23.5 nm and a narrow size distribution (Figure 3A, inset, Supplemental Figure S2). This indicates that the engineered capsid forms stable nanocompartents very similar in size and monodispersity to wt EncTm.17 EncTmAG4 capsids synthesize Ag NPs from Ag ions in their interior. The purified engineered compartment (10 µM) was incubated with 20 mM AgNO3 in 50 mM Hepes buffer at 37°C for up to 24 h in the dark. No additional reducing agent was added. Progress of Ag NP formation was followed by recording the solution’s absorbance at 420 nm. Ag NPs exhibit a characteristic surface plasmon resonance absorption between 400 and 500 nm, depending on the size distribution of the particles in question.26 After 24 h, a prominent absorption band at 420 nm could be observed while no absorption was visible when the engineered compartment or AgNO3 was incubated alone in Hepes buffer (Figure 3B). The sigmoidal kinetic curve for Ag NP growth


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shown in Figure 3C suggests the involvement of an autocatalytic pathway where initial nucleation centers would form at AG4 sites which would in turn mediate the precipitation of more Ag ions.27 Incubating the reaction mixture longer than 24 h led to an increase in amorphous precipitate (Supplemental Figure S3).

Figure 3. In vitro characterization of the EncTmAG4 compartment. (A) Size-exclusion chromatogram of EncTmAG4. The inset shows the SDS-PAGE gel of the indicated fractions (red asterisk), a negative stain TEM micrograph and the size distribution of purified EncTmAG4 (determined by DLS). (B) UV-Vis spectrum showing the characteristic Ag NP surface plasmon resonance absorption band at 420 nm when EncTmAG4 is incubated with AgNO3 for 24 h. EncTmAG4 and AgNO3 in Hepes buffer as well as buffer alone (blank) were used as controls. (C) Time-course of silver nanoparticle formation. Assays were carried out in triplicate. (D) Stability of synthesized Ag NPs. UV-Vis spectra were recorded for samples stored in distilled


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water (left) or MH liquid medium (right) at 25°C in the dark for 1 and 30 days. Both proteincoated and shell-free Ag NPs were used. The synthesized Ag NPs show desirable stability under normal storage conditions. The propensity of the synthesized Ag NPs to dissolution was tested by monitoring the characteristic absorbance band at 420 nm over time. Dissolution or aggregation of Ag NPs would result in decreased absorption at 420 nm and a broadening of the absorption band, respectively. Even after 30 days at 25°C in the dark, no noticeable decrease in absorbance at 420 nm could be observed when protein-coated particles where stored in distilled water (Figure 3D). The same was observed for shell-free silver particles where protein was removed by sodium dodecyl sulfate (SDS) and proteinase K treatment. When particles were stored in Mueller Hinton (MH) liquid medium under the same conditions, a decrease in absorption and broadening of the absorption band was observed for both coated and uncoated Ag NPs (Figure 3D). The presence of high concentrations of starch and casein, both shown to be able to nucleate/reduce silver ions,28, 29 in MH medium could be responsible for the formation of larger aggregates consisting of multiple Ag NPs. They could also help the nucleation of new particles that would now be able to grow in an unconstrained manner using slowly dissolving silver ions from preformed Ag NPs. Both effects could explain the observed broadening of the absorption band. Given the fact that MH medium is a complex medium, other compounds or combinations thereof could also be responsible for the observed effects.


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Figure 4. TEM imaging, ICP-MS and size-distribution of EncTmAG4-synthesized Ag NPs. (A) TEM micrographs of Ag NPs without (left) and with (right) uranyl formate (UF) stain. In the UF-stained sample the protein shell surrounding the electron-dense core can be seen while only Ag cores are visible in the unstained sample. (B) Negative stain TEM micrograph showing an empty, partially filled and completely filled capsid. The inset shows a HR-TEM image of a Ag NP with a characteristic line spacing (d) of 0.25 nm. (C) ICP-MS analysis of assays containing EncTmAG4, EncTm and no capsids. After 24 h synthesized Ag NPs were collected via centrifugation, washed with distilled water and subjected to ICP-MS analysis. (D) Size distribution of Ag NPs after capsid removal as determined by DLS. The structure and composition of synthesized Ag NPs is consistent with the properties expected of particles synthesized in the interior of the engineered encapsulin compartment. In unstained TEM samples, electron-dense spherical particles of the expected size are clearly visible. When samples are stained using uranyl formate, the protein shell is visualized (Figure 4A, Supplemental Figure S4). Figure 4B shows completely filled (left), empty (center) and partially filled (right) capsids indicating that mineralization likely starts at one or a number of


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neighboring AG4 nucleation sites. Ag NP growth then continues until the available space inside the protein shell is filled. High-resolution (HR)-TEM of Ag NPs reveals a characteristic lattice spacing (111) of d = 0.25 nm (Figure 4B, inset, Supplemental Figure S5).30 After collecting synthesized Ag NPs via centrifugation followed by washing with distilled water, the resulting samples were subjected to ICP-MS analysis. The Ag content of EncTmAG4-containing samples was markedly higher compared with EncTm and buffer controls (Figure 4C). DLS analysis after the protein-coated Ag NPs were treated with SDS and proteinase K revealed an average Ag NP size of 13.5 nm and a narrow size distribution (Figure 4D). This is in good agreement with the size expected based on the capsid model of EncTmAG4 where the protein shell would limit the maximum size of internally synthesized Ag NPs to 13-15 nm (Supplemental Figure S1). This places the accessible NP size range of our encapsulin-based system in between those based on smaller protein cages like ferritins, which allow synthesis/encapsulation of particles between 58 nm,6 and T = 1, T = 3 or T = 7 virus-like particles which generally exhibit larger internal diameters (e.g. MS2 phage: 21 nm and P22 phage 54 nm).4 Considering that the physical, chemical and biological properties of NPs strongly depend on their size,31 having access to NPs around 15 nm will allow us to access properties of NPs unique to this particular size range. The narrow size distribution observed for our NPs is an advantage over many virus-like particle systems that lead to a broader size distribution of synthesized NPs due to their increased inner diameters.32 In addition, our monodisperse NPs will make it easier to engineer specific functionalities that strongly depend on monodispersity. To confirm the elemental composition of the synthesized NPs, dark-field scanning TEM (DF-STEM) with energy dispersive X-ray spectroscopy (EDS) was used (Figure 5A and B). Comparison of EDS spectra of particles with and without electron-dense cores shows that the particles are indeed Ag NPs (Figure 5C and D).


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The characteristic Ag Lα and Ag Lβ transitions are clearly visible in filled particles while no Ag signal could be observed in unfilled compartments. The sulfur and copper signals in both samples are caused by the Hepes-containing buffer and the EM grids, respectively.

Figure 5. Elemental composition of EncTmAG4-encapsulated Ag NPs. (A) Dark-field STEM (200 keV) image of a single Ag NP. (B) EDS intensity line profile for silver of the Ag NP extracted from the EDS spectrum. Distance refers to the length from the start of the line scan through the particle shown in (A). (C) EDS spectrum of a particle with an electron-dense core showing the characteristic Ag Lα and Ag Lβ transitions. (D) Control EDS spectrum of an empty capsid. Biogenic Ag NPs have enhanced antimicrobial activities. Five bacterial species, including four pathogenic strains, were analyzed via disk diffusion assays on MH plates. Inhibitory activities were compared with that of AgNO3, commercial citrate-capped Ag NPs and silver-free EncTmAG4 capsids. Protein-coated and shell-free Ag NPs were tested. Both types of biogenic Ag NPs synthesized in this study were found to exhibit greater antimicrobial activity than silver nitrate or commercial Ag NPs in all cases with the largest difference found for P. aeruginosa (Figure 6, Supplemental Figure S6). Our results show comparable trends to previous studies,


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where Ag NPs were synthesized using biological or chemical methods.33 Both the size (20 ± 3 nm) and the capping (citrate) of the commercial Ag NPs are likely to have an influence on their antimicrobial activity. It has been reported that Ag NPs smaller than 20 nm have elevated antimicrobial activity due to increased cell penetration and Ag+-leaching rates which could explain the observed increased toxicity of our Ag NPs.25 In addition, citrate-capping has been shown to negatively influence ion release rates.34 Accordingly, our Ag NPs without protein shell generally showed increased antimicrobial activity compared with protein-coated particles. This can be rationalized both by an increased tendency to release silver ions due to a lack of coating and the smaller size of the shell-free particles which might be able to interact more easily with bacterial cells. In addition, the SDS-based method used to remove the protein shell might have resulted in the partial capping of our Ag NPs with SDS, which has been previously shown to increase antimicrobial activity.35 These results also show that the protein shell does not prevent Ag NP antimicrobial action. Encapsulin cages possess 5 Å pores at the points of five- and threefold symmetry which would allow Ag+ to exit the compartment. Silver-free EncTmAG4 particles did not show antimicrobial activity at the concentrations used.

Figure 6. Antimicrobial activity of EncTmAG4-synthesized Ag NPs. The strains tested are: Escherichia coli CDC B170, Salmonella typhimurium CDC 6516-60, Shigella flexneri CDC 3591-52, Pseudomonas aeruginosa 1C and Bacillus subtilis 168. A total of 5 µg of silver was


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added in the form of protein-coated and uncoated Ag NPs based on the UV-Vis absorption of uncoated Ag NPs. For AgNO3, 7.9 µg (corresponding to 5 µg of silver) was added. As protein controls, 5 µg of protein capsids were added. All samples were prepared in 20 µL distilled water, added to a 6 mm disk and then placed on MH agar. Conclusion In sum, we have established encapsulins as a platform for the synthesis of biogenic functional nanomaterials. Genetic engineering of the natural T. maritima compartment EncTm yielded a compartment with new properties that could be employed in the size-constrained synthesis of metallic nanoparticles. Our synthesized Ag NPs display a uniform size and shape distribution with long-term stability under ambient conditions. Moreover, biogenic protein-coated and shellfree Ag NPs exhibited antimicrobial activity that was superior to comparable amounts of commercial citrate-coated Ag NPs and AgNO3. Taken together, the inherent properties of encapsulins make them a new system for applications in biomedicine, materials science and catalysis. Methods Reagents, instruments and general methods All reagents were purchased from Sigma-Aldrich unless otherwise stated. The EncTmAG4 construct was ordered as a gBlock Gene Fragment (Integrated DNA Technologies, IDT). Codon usage was optimized for E. coli using the Codon Optimization Tool (IDT). Gibson Assembly® Master Mix was purchased from New England BioLabs (NEB). DNA sequencing was performed by GENEWIZ. NEB Turbo Competent E. coli (NEB) were used for all cloning procedures while BL21 (DE3) Competent E. coli (NEB) were used for protein production. pETDuet1-derived plasmids were used as vectors. Expression experiments were carried out in LB medium


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supplemented with ampicillin (100 µg/mL). Size exclusion chromatography was carried out using an ÄKTA Explorer 10 (GE Healthcare Life Sciences) equipped with a HiPrep 16/60 Sephacryl S-500 HR column (GE Healthcare Life Sciences). Protein samples were concentrated using Amicon Ultra Filters (Millipore). For SDS-PAGE, Novex Tris-Glycine Gels (ThermoFisher Scientific) were used. DNA and protein concentrations were determined with a Nanodrop ND-1000 instrument (PEQLab) and UV-Vis spectra were recorded with the same spectrophotometer. ICP-MS analysis was carried out at the Trace Metals Lab at HSPH using a Elan DRC-II Inductively Coupled Plasma-Mass Spectrometer (Perkin Elmer). 200 Mesh Gold Grids (FCF-200-Au, EMS) were used for all EM experiments. Standard TEM experiments were carried out at the HMS Electron Microscopy Facility using a Tecnai G2 Spirit BioTWIN instrument. HR-TEM and EDS-STEM was done at the Harvard Center for Nanoscale Systems. For HR-TEM a FEI Tecnai Cryo-Bio 200KV FEG TEM instrument was used while a JEOL 2010 TEM/STEM equipped with an EDS detector was used for EDS-STEM analysis. DLS analysis was carried out with a Malvern zen3600 particle sizer (Malvern Instruments). For disk diffusion assays Mueller Hinton Agar and Antibiotic Sensitivity Discs (Fisher Scientific) with or without antibiotics were used. Homology modelling To generate the homology model of EncTmAG4 and to evaluate model quality via DOPE (Discrete Optimized Protein Energy)36 score, Modeller 9.15 was used.37 Outputs were generated using Anaconda 2.2.0 and Python 2.7. Cloning techniques For the construction of expression constructs, Gibson Assembly was used. The gBlock Gene Fragment encoding EncTmAG4 and containing overlaps for direct assembly was combined with


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NdeI and PacI digested pETDuet1 yielding the expression construct pETDuet1_EncTmAG4. Chemically competent E. coli Turbo cells were then transformed with pETDuet1_EncTmAG4 and subsequently confirmed by sequencing. Silver sensitivity tests Over-night cultures of EncTm and EncTmAG4 were grown in 5 mL LB medium and used to inoculate (1:50) new 5 mL LB cultures the next day. Cultures were grown at 37°C and 200 rpm to an OD600 of 0.6. Protein production was induced with IPTG (final concentration: 0.1 mM). After 2 h, samples were back diluted to an OD600 of 0.2 using IPTG-containing LB medium. Different amounts of freshly made AgNO3 in dH2O were added and the cultures incubated at 37°C for 18 h in the dark. Different dilution were then plated on LB agar plates and incubated over night at 37°C to assess cell viability. Experiments were carried out in triplicate. Expression and purification of protein compartments The resulting expression construct was used to transform E. coli BL21 (DE3) cells. Protein expression was carried out by inoculating 50 mL LB medium (1:50) from an over-night culture, growing it at 37°C and 200 rpm to an OD600 of 0.5 and then inducing protein production using IPTG (final concentration: 0.1 mM). Cultures were grown at 30°C for 18 h and then harvested via centrifugation (4000 rpm, 15 min, 4°C). Cells were suspended in 5 mL 100 mM Hepes buffer (pH 7.4) and lysed using a 550 Sonic Dismembrator (FisherScientific). Power level 3 was used with a pulse time of 10 sec. The time between pulses was 10 sec and total pulse time was 3 min. Subsequently, 10 µg/mL DNase I was added to the lysate followed by incubation on ice for 15 min. After cell debris was removed by centrifugation (8000 rpm, 15 min, 4°C), 0.1 g NaCl and 0.5 g of PEG-8000 was added, followed by incubation at 4°C for 1 h. Precipitated protein was collected via centrifugation (8000 rpm, 15 min, 4°C), resuspended in 3 mL Hepes buffer and


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filtered (0.2 µm). The sample was then subjected to size exclusion chromatography using Hepes buffer and a flow rate of 1 mL/min. Fractions were evaluated using SDS-PAGE and compartment containing fractions were pooled and if necessary concentrated to 10 µM using Amicon filters. Samples were immediately used for subsequent analyses/assays. ICP-MS analysis For the analysis of silver content of whole cells, 1 mL (OD600 = 1.8) of induced over-night culture was pelleted and then washed four times with dH2O. The pellets were then prepared for ICP-MS analysis using nitric acid resulting in a final concentration of 2% (w/v). For the analysis of assay mixtures, the sample was pelleted (20000 rpm, 30 min, 25°C) washed for times with dH2O and prepared for ICP-MS using nitric acid. Silver nanoparticle synthesis, kinetics and long-term stability tests To prevent the precipitation of insoluble silver salts as well as photoreduction of silver salts to elemental silver, the buffer system used for the purification of EncTmAG4 and the subsequent assays consisted solely of a 100 mM Hepes buffer at pH 7.4 and assays were carried out in the dark. Freshly purified compartments (10 µM) were incubated with 20 mM freshly prepared AgNO3 (in dH2O) at 37°C in the dark. Samples were taken at different time points and subjected to UV-Vis spectroscopy. For long-term stability test, samples were taken after 24 h and the synthesized Ag NPs were pelleted via centrifugation (20000 rpm, 30 min, 25°C). AgNO3containing supernatant was removed and the pellets washed three times with dH2O. Washed Ag NPs were finally suspended in dH2O or Mueller-Hinton medium and incubated in the dark at 25°C. EM analysis


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Samples for negative-stain TEM analysis were diluted to 0.5 µM using dH2O and subsequently adsorbed onto formvar/carbon coated grids. To increase the hydrophilicity of grids a 100x glow discharge unit (EMS) was used directly prior to applying the sample (10 sec, 10 mA). After adsorption (1 min) excess liquid was blotted off using Whatman #1 filterpaper, washed with dH2O and then floated on a 10 µL drop of staining solution (0.75% uranyl formate in dH2O) for 30 sec. After removal of excess liquid the samples are ready for TEM analysis. HR-TEM and EDS-STEM analysis was done at the Center for Nanoscale Systems. For TEM analysis of fixed cells, induced over-night cultures were fixed by mixing the cell suspension with fixative (1:1, 1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% picric acid in 0.1 M sodium cacodylate buffer, pH 7.4). The sample was then incubated at 25°C for 1 h, centrifuge for 3 min at 3000 rpm and again incubated as a pellet for 1 h at 25°C. Cells were subsequently washed three times in cacodylate buffer, 4 times with malelate buffer pH 5.15 and then stained with 1% uranyl acetate for 30 min. After washing, the sample was dehydrated (15 min 70% ethanol, 15 min 90% ethanol, 2 x 15 min 100% ethanol) and incubated with propyleneoxide for 1 h. For infiltration, Epon resin was mixed with propylenoxide (1:1) and the sample incubated for 2 h at 25°C before moving it to an embedding mold fileed with freshly mixed Epon. The sample was allowed to sink and then moved to an oven for polymerization (24 h, 60°C). Ultrathin sections were then cut at -120°C using a cryo-diamond knife (Reichert cryo-ultramicrotome) and transferred to formvar/carbon coated grids. DLS analysis Analysis of EncTmAG4 before Ag NP synthesis was done by diluting samples to 0.1 µM using Hepes buffer. To analyze Ag NP cores, still protein-coated Ag NPs were precipitated via centrifugation (20000 rpm, 30 min, 25°C), then dissolved in 1% SDS solution. Proteinase K was


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added to a final concentration of 1 mg/mL and the reaction mixture incubated at 37°C for 2 h. After centrifugation (20000 rpm, 30 min, 25°C) the pellet was suspended in Hepes buffer and subjected to DLS analysis. Disk diffusion assays Disk diffusion assays to test the antimicrobial activity of Ag NPs was carried out in accordance with Kirby and Bauer.38 Over-night cultures of Escherichia coli CDC B170, Salmonella typhimurium CDC 6516-60, Shigella flexneri CDC 3591-52, Pseudomonas aeruginosa 1C and Bacillus subtilis NCIB 3610 were grown in LB medium. A lawn of each bacterium was streaked out on Mueller-Hinton agar plates. After 10 min incubation at 25°C disks (diameter: 6 mm) containing 20 µL of the respective sample were placed on the agar plate and incubated at 37°C for 24 h. Experiments were carried out in triplicate.

ASSOCIATED CONTENT Supporting Information Additional TEM data, additional disk diffusion data. This material is available free of charge via the Internet at http://pubs.acs.org.. AUTHOR INFORMATION Corresponding Author *[email protected]


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Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We thank the Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network (NSF award number: ECS-0335765) for help with HR-TEM and EDSSTEM analysis. We thank Nick Lupoli and the Trace Metals Laboratory at HSPH for ICP-MS analyses. This work was supported by a Leopoldina Research Fellowship (LPDS 2014-05) from the German National Academy of Sciences Leopoldina (T.W.G), a DARPA Living Foundries: 1000 Molecules grant (award number: HR0011-14-C-0072; P.A.S) and an Office of Naval Research grant (award number: N00014-11-1-0725; P.A.S.). REFERENCES 1. Breitling, R.; Takano, E. Synthetic biology advances for pharmaceutical production. Curr. Opin. Biotechnol. 2015, 35, 46-51. 2. Haellman, V.; Fussenegger, M. Synthetic Biology-Toward Therapeutic Solutions. J. Mol. Biol. 2015. 3. Edmundson, M. C.; Capeness, M.; Horsfall, L. Exploring the potential of metallic nanoparticles within synthetic biology. New Biotechnol. 2014, 31, 572-578. 4. Li, F.; Wang, Q. Fabrication of nanoarchitectures templated by virus-based nanoparticles: strategies and applications. Small 2014, 10, 230-245. 5. Park, T. J.; Lee, S. Y.; Heo, N. S.; Seo, T. S. In vivo synthesis of diverse metal nanoparticles by recombinant Escherichia coli. Angew. Chem. Int. Ed. Engl. 2010, 49, 70197024. 6. Jutz, G.; van Rijn, P.; Santos Miranda, B.; Boker, A. Ferritin: a versatile building block for bionanotechnology. Chem. Rev. 2015, 115, 1653-1701. 7. Meldrum, F. C.; Wade, V. J.; Nimmo, D. L.; Heywood, B. R.; Mann, S. Synthesis of inorganic nanophase materials in supramolecular protein cages. Nature 1991, 349, 684-687. 8. Douglas, T.; Young, M. Host-guest encapsulation of materials by assembled virus protein cages. Nature 1998, 393, 152-155. 9. Kramer, R. M.; Li, C.; Carter, D. C.; Stone, M. O.; Naik, R. R. Engineered protein cages for nanomaterial synthesis. J. Am. Chem. Soc. 2004, 126, 13282-13286. 10. Theil, E. C. Ferritin: the protein nanocage and iron biomineral in health and in disease. Inorg. Chem. 2013, 52, 12223-12233.


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Figure 1. Engineering of the T. maritima protein compartment. (A) Schematic depiction of capsid engineering. The ferritin-like protein (Flp, orange) was removed from the wild-type encapsulin system and the AG4 peptide (red) was fused to the N-terminus of EncTm yielding EncTmAG4 which would then allow the size-constrained synthesis of silver nanoparticles in its interior when exposed to silver ions. 28x9mm (300 x 300 DPI)


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Figure 2. Characterization of EncTmAG4 in vivo. (A) Relative cell viability of EncTm- and EncTmAG4expressing E. coli in the presence of different concentrations of AgNO3. Cell viability was based on colony forming units (CFUs). (B) Left: ICP-MS analysis of washed cells after overnight exposure to 0, 50 and 100 µM AgNO3. Right: TEM micrographs of fixed cells, .expressing EncTm or EncTmAG4 after overnight exposure to 50 µM AgNO3. Intracellular electron-dense material can only be seen in EncTmAG4 cells. Size-exclusion chromatogram of EncTmAG4. The inset shows the SDS-PAGE gel of the indicated fractions (red asterisk), a negative stain TEM micrograph and the size distribution of purified EncTmAG4 (determined by DLS). 85x86mm (300 x 300 DPI)



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Figure 3. In vitro characterization of the EncTmAG4 compartment. (A) Size-exclusion chromatogram of EncTmAG4. The inset shows the SDS-PAGE gel of the indicated fractions (red asterisk), a negative stain TEM micrograph and the size distribution of purified EncTmAG4 (determined by DLS). (B) UV-Vis spectrum showing the characteristic Ag NP surface plasmon resonance absorption band at 420 nm when EncTmAG4 is incubated with AgNO3 for 24 h. EncTmAG4 and AgNO3 in Hepes buffer as well as buffer alone (blank) were used as controls. (C) Time-course of silver nanoparticle formation. Assays were carried out in triplicate. (D) Stability of synthesized Ag NPs. UV-Vis spectra were recorded for samples stored in distilled water (left) or MH liquid medium (right) at 25°C in the dark for 1 and 30 days. Both protein-coated and shell-free Ag NPs were used. 100x62mm (300 x 300 DPI)


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Figure 4. TEM imaging, ICP-MS and size-distribution of EncTmAG4-synthesized Ag NPs. (A) TEM micrographs of Ag NPs without (left) and with (right) uranyl formate (UF) stain. In the UF-stained sample the protein shell surrounding the electron-dense core can be seen while only Ag cores are visible in the unstained sample. (B) Negative stain TEM micrograph showing an empty, partially filled and completely filled capsid. The inset shows a HR-TEM image of a Ag NP with a characteristic line spacing (d) of 0.25 nm. (C) ICP-MS analysis of assays containing EncTmAG4, EncTm and no capsids. After 24 h synthesized Ag NPs were collected via centrifugation, washed with distilled water and subjected to ICP-MS analysis. (D) Size distribution of Ag NPs after capsid removal as determined by DLS. 71x44mm (300 x 300 DPI)



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Figure 5. Elemental composition of EncTmAG4-encapsulated Ag NPs. (A) Dark-field STEM (200 keV) image of a single Ag NP. (B) EDS intensity line profile for silver of the Ag NP extracted from the EDS spectrum. Distance refers to the length from the start of the line scan through the particle shown in (A). (C) EDS spectrum of a particle with an electron-dense core showing the characteristic Ag Lα and Ag Lβ transitions. (D) Control EDS spectrum of an empty capsid. 66x57mm (300 x 300 DPI)


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Figure 6. Antimicrobial activity of EncTmAG4-synthesized Ag NPs. The strains tested are: Escherichia coli CDC B170, Salmonella typhimurium CDC 6516-60, Shigella flexneri CDC 3591-52, Pseudomonas aeruginosa 1C and Bacillus subtilis 168. A total of 5 µg of silver was added in the form of protein-coated and uncoated Ag NPs based on the UV-Vis absorption of uncoated Ag NPs. For AgNO3, 7.9 µg (corresponding to 5 µg of silver) was added. As protein controls, 5 µg of protein capsids were added. All samples were prepared in 20 µL distilled water, added to a 6 mm disk and then placed on MH agar. 43x24mm (300 x 300 DPI)


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