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Synthetic Biogenesis of Bacterial Amyloid Nanomaterials with Tunable Inorganic-Organic Interfaces and Electrical Conductivity
Abstract
Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires. Specifically, we used cell-synthesized curli fibers as templates for nucleating and growing gold nanoparticles and showed that nanoparticle size could be modulated as a function of curli fiber gold-binding affinity. Furthermore, we demonstrated that gold nanoparticles can be pre-seeded onto curli fibers and followed by gold enhancement to form nanowires. Using these two approaches, we created artificial cellular systems that integrate inorganic-organic materials to achieve tunable electrical conductivity. We envision that cell-synthesized amyloid nanofibers will be useful for interfacing abiotic and biotic systems and constructing living biomaterials within a framework of engineered bacterial systems.
... In addition to these natural examples, there are examples of synthetic proteinaceous nanowires that have been engineered to facilitate micrometre-scale electron transport (Table 2). Synthetic nanowires use scaffolding proteins to self-assemble into nano-or micro-metre length fibres that are often modified with metal binding moieties or metalloproteins that endow these fibres with conductive properties (Altamura et al., 2017;Nguyen et al., 2014;Scheibel et al., 2003;Seker et al., 2017). Frequently, amyloid-fibril-forming proteins are used for the scaffold, such as fungal prions (Altamura et al., 2017;Scheibel et al., 2003) and bacterial curlins (Nguyen et al., 2014;Seker et al., 2017). ...
... Synthetic nanowires use scaffolding proteins to self-assemble into nano-or micro-metre length fibres that are often modified with metal binding moieties or metalloproteins that endow these fibres with conductive properties (Altamura et al., 2017;Nguyen et al., 2014;Scheibel et al., 2003;Seker et al., 2017). Frequently, amyloid-fibril-forming proteins are used for the scaffold, such as fungal prions (Altamura et al., 2017;Scheibel et al., 2003) and bacterial curlins (Nguyen et al., 2014;Seker et al., 2017). These scaffold proteins have relatively low intrinsic conductivities (Del Mercato et al., 2007;Dorval Courchesne et al., 2018;Kalyoncu et al., 2017). ...
... These scaffold proteins have relatively low intrinsic conductivities (Del Mercato et al., 2007;Dorval Courchesne et al., 2018;Kalyoncu et al., 2017). To confer metallic conductivity to these nanowire scaffolds, the monomer can be functionalized to bind exogenously supplied metal nanoparticles by fusing them to metal binding moieties such as surface-exposed cysteines (Scheibel et al., 2003), poly-histidine (Chen et al., 2014;Seker et al., 2017), or metal-binding peptides (Nguyen et al., 2014;Seker et al., 2017). Redox-hopping conductivity has been engineered into nanofibres by fusing amyloid-forming bs_bs_banner monomers to rubredoxin, a metalloprotein containing an Fe 2+/3+ centre enabling electron hopping with a conductivity of ~10 −6 S cm −1 in both hydrated electrochemical measurements and in solid-state two-electrode measurements (Altamura et al., 2017). ...
Biology leverages a range of electrical phenomena to extract and store energy, control molecular reactions and enable multicellular communication. Microbes, in particular, have evolved genetically encoded machinery enabling them to utilize the abundant redox-active molecules and minerals available on Earth, which in turn drive global-scale biogeochemical cycles. Recently, the microbial machinery enabling these redox reactions have been leveraged for interfacing cells and biomolecules with electrical circuits for biotechnological applications. Synthetic biology is allowing for the use of these machinery as components of engineered living materials with tuneable electrical properties. Herein, we review the state of such living electronic components including wires, capacitors, transistors, diodes, optoelectronic components, spin filters, sensors, logic processors, bioactuators, information storage media and methods for assembling these components into living electronic circuits.
... An important feature of amyloids in biofilms is their propensity to interact with other matrix components and modify the viscoelastic properties of the extracellular matrices, which could aid to resist environmental fluctuations 6,7 . Considering the intrinsic properties of the amyloids, researches have made use of these structures in order to construct tunable living materials or functionalized biofilms for many technical and applicative biotechnological purposes [8][9][10][11][12] . A well characterized functional amyloid system from bacterial biofilm matrix is the curli fimbriae from Escherichia coli and other enterobacteria 13,14 . ...
... Recently, novel strategies have been developed to create biofilms as artificial platforms for self-assembling functional tags 32,34,35 . One of these approaches is based on engineering extracellular matrix components, focusing on amyloids as potential targets 8,9,11 . In biofilms, bacteria secrete a wide variety of molecules that assemble to form the extracellular matrix, including polysaccharides, proteins, and nucleic acids 36 . ...
Biofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap.
... Engineering of 'truly' living materials where the living component actively facilitates material fabrication and organization is much more challenging. True ELMs have been created by engineering Escherichia coli to produce an extracellular matrix from curli fibers [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . Other types of extracellular matrices for ELM fabrication were created from secreted bacterial cellulose to embed microbial cells 26,27 or from elastin-like polypeptides to attach Caulobacter cells via their protein S-layers 28 . ...
... To prepare samples for colony PCR, a single colony of B. subtilis was mixed in 300 µL sterile water. Cells were spun down (21,130 x g) and 200 µL of the supernatant was removed. Cells were resuspended in the remaining 100 µL of sterile water followed by 20 mins of sonication in a coldwater bath sonicator (Bransonic 3510R-MTH, CT, USA). ...
Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.
... T he nascent field of engineered living materials (ELMs) aims to recapitulate desirable properties of natural living biomaterials to create useful new materials using genetically engineered organisms [1][2][3][4] . Existing ELMs exhibit a remarkable range of functions but often require intensive manual processing steps for purification, functionalization or patterning [5][6][7][8][9][10][11][12][13][14] . Others make use of the multiple functionalities of living cells by embedding them within synthetic hydrogels [15][16][17][18][19][20] . ...
... We characterized a range of other co-culture parameters, including pellicle yields, co-culture stability, cell distribution and reproducibility (Supplementary text and Supplementary Figs. [3][4][5][6][7][8]. Notably, we provide evidence that the growth enhancement conferred by yeast is based on secretion of invertase enzyme, which hydrolyses sucrose to monosaccharide sugars that are more efficiently metabolized by the bacteria (Supplementary Fig. 6). ...
Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically engineered organisms. Here, we describe an approach to fabricating functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulose-based engineered living materials with potential applications in biosensing and biocatalysis.
... 7 Bacterial biofilms, which are highly regulated, complex and dynamic systems, resistant to harsh environmental conditions with their ECM, have a great potential for the production and development of new generation materials. 8,9 In recent years there is an increasing trend towards the use of amyloids and amyloid-like fibrils as building blocks and as functional biomaterial systems [9][10][11][12][13][14] . Functional amyloid curli fiber, . ...
... 7 Bacterial biofilms, which are highly regulated, complex and dynamic systems, resistant to harsh environmental conditions with their ECM, have a great potential for the production and development of new generation materials. 8,9 In recent years there is an increasing trend towards the use of amyloids and amyloid-like fibrils as building blocks and as functional biomaterial systems [9][10][11][12][13][14] . Functional amyloid curli fiber, . ...
Self-assembling protein subunits hold great potential as biomaterials with improved functions. Among the self-assembled protein structures functional amyloids are promising unique properties such as resistance to harsh physical and chemical conditions their mechanical strength, and ease of functionalization. Curli proteins, which are functional amyloids of bacterial biofilms can be programmed as intelligent biomaterials. In order to obtain controllable curli based biomaterials for biomedical applications, and to understand role of each of the curli forming monomeric proteins (namely CsgA and CsgB from Escherichia coli) we characterized their binding kinetics to gold, hydroxyapatite, and silica surfaces. We demonstrated that CsgA, CsgB, and their equimolar mixture have different binding strengths for different surfaces. On hydroxyapatite and silica surfaces, CsgB is the crucial element that determines the final adhesiveness of the CsgA-CsgB mixture. On the gold surface, on the other hand, CsgA controls the behavior of the mixture. Those findings uncover the binding behavior of curli proteins CsgA and CsgB on different biomedically valuable surfaces to obtain a more precise control on their adhesion to a targeted surface.
... could be triggered to regulate the assembly of curli amyloid accordingly [ Fig. 3(a)]. 14,28 They also demonstrated that the composition of curli could be tuned to mimic the block copolymer using two different CsgA variants with separate inducible systems. This work offers an entry point toward creating diversified functional biofilm extracellular matrices by genetic manipulation. ...
Natural biological materials are programmed by genetic information and able to self-organize, respond to environmental stimulus, and couple with inorganic matter. Inspired by the natural system and to mimic their complex and delicate fabrication process and functions, the field of engineered living materials emerges at the interface of synthetic biology and materials science. Here, we review the recent efforts and discuss the challenges and future opportunities.
... Furthermore, the growing field of bio-inspired nanotechnology is capitalizing on the selfassembly of amyloid proteins. CsgA has been shown to have range of uses such as the passage of electric current, the extraction of rare earth metals, and many others (Nguyen et al., 2014;Seker et al., 2017;Tay et al., 2018). The work presented here will allow material scientists to use amyloid fibers to build systems of higher complexity than is currently possible. ...
Amyloids are a class of protein assembly known for their extreme stability and ordered, fibrous structure. Functional amyloids are a subclass of amyloids that can be found in all domains of cellular life. Functional amyloids fulfill key biological roles that help the organism that produce them grow and thrive in their environment. Functional amyloids can act as structural scaffolds, storage mechanisms, antibacterial agents, and major components of microbial biofilms. The most well studied functional amyloid is curli, the predominant protein component of the E. coli biofilm. The major protein subunit of curli is a fast-aggregating amyloid protein called CsgA. E. coli secretes intrinsically disordered CsgA to the outside of the cell where it can then fold into an amyloid-competent state and form fibers. Though CsgA has a high propensity to form amyloid fibers its polymerization can be controlled through different biochemical mechanisms. In this dissertation I focused on two ways that functional amyloid formation is controlled, both in vivo and in vitro. E. coli uses an amyloid inhibitor protein called CsgC to control CsgA aggregation within the cell. CsgC is a periplasmic chaperone-like protein that maintains CsgA in in an intrinsically disordered state. In collaboration with the Olofsson (Umeå University, Sweden) and Ruotolo (University of Michigan) labs, I investigated the interaction between CsgC and CsgA. We observed a 1:1 heterodimeric complex of CsgC:CsgA that suggested that CsgC interacts with monomers to keep CsgA unfolded and non-amyloidogenic. We found that after interacting transiently with CsgC, CsgA monomers aggregate much more slowly than compared to a negative control. To learn more about how CsgC interacts with CsgA, I developed an in vivo screening assay that was able to identify CsgC residues that are important for structural stability and amyloid inhibition activity. Controlling CsgA aggregation in vitro is also a useful research endeavor. Upon purification, intrinsically disordered CsgA proteins immediately begin oligomerizing and within a few hours stable, mature amyloid fibers will have formed. To aid the study of amyloid proteins, I created a CsgA variant called CsgACC that remains monomeric and unfolded while oxidized. When CsgACC is incubated under reducing conditions, an intramolecular disulfide bond is broken, and the protein adopts the amyloid conformation. Therefore, the aggregation propensity of CsgACC can be manipulated by the addition of a reducing agent. CsgACC will enable scientists to control amyloid formation in ways that were never possible before, hopefully leading to insights into how aggregation occurs. In addition, an amyloid protein that responds to a reducing agent to begin polymerization could prove to be a useful building block for constructing complex structures using functional amyloid fibers as the core structural element.
... Seker et al. [31] produced yet another self-assembled amyloids which are highly organized and ordered nanoassemblies. E. coli curli fibers that are formed through polymerization of monomeric proteins were produced in extracellular space and primarily composed of curli subunit CsgA, which helped to form higher-order nanofibers. ...
One of the principal aims of synthetic biology is to design and build novel biological systems that can perform the desired function. Natural materials such as bone, scales and shells are composed of organic and inorganic components. These possess the properties of forming self-assembly, self-healing and are highly stable. Lately, there has been an increase in the demand for sustainable production of self-assembled biomaterials for usage in a wide range of basic biology to biotechnology applications. Synthetic biology plays a key role in designing novel self-assembled biomaterials having strong strength, stability and structure. Here, we discuss the design of self-assembled biomaterials based on mainly the proteins that can be fine-tuned to alter the property of materials to be used for different applications. In this chapter, we underline the basics of self-assembled living materials and their applications in a number of areas including biomedical sciences, biotechnology and nanotechnology.
... At the same time, the polymer surface can be modified by ligands or targeted to achieve multifunctional drug delivery. In recent years, polymer nanomaterials have received more and more attention [23]. In recent studies, polymer nanoparticles in the form of chitosan and dextran are very popular. ...
More and more people are beginning to recognize the important role of intelligent rehabilitation training equipment in rehabilitation treatment and continue to carry out related researches. The use of intelligent robot technology for rehabilitation treatment has been rapidly developed, and it has achieved rapid progress on a global scale. Especially in some developed countries, this field has also received corresponding attention in some developed cities in China in recent years. Mesoporous nanomaterials have unique physical, chemical, and biological properties. Mesoporous nanomaterials can be combined with chemotherapy drugs to minimize the harm caused by chemotherapy drugs to the human body and improve the therapeutic effect. As a result, the cure rate has been improved, and it has shown deep potential in breast cancer chemotherapy. Fifty breast cancer patients were selected as the research objects and randomly divided into a control group and an experimental group, each with 25 cases. The control group was treated with conventional chemotherapeutics, and the experimental group was treated with molecular targeted therapy to compare the treatment effects of the two groups. Studies have shown that the recurrence rate and the occurrence probability of complications in the experimental group are significantly lower than those in the control group. Molecular targeted therapy for breast cancer has obvious effects, which reduces the recurrence rate of complications or diseases, and is less toxic.
... Programming these electronic nanoparticles onto biofilm surfaces can confer electronic characteristics to living biofilms (Dorval Courchesne et al., 2018;Kalyoncu et al., 2017). Moreover, the interaction between living biofilms and inorganic electroactive nanoparticles can be exploited for electronic utilization (Chen et al., 2014;Seker et al., 2017). ...
Historically, biofilms have been perceived as problematic or detrimental. However, biofilms possess favorable traits such as self-regeneration, sustainability, scalability, and tunability, which make them candidates for diverse applications. Traditional applications of biofilms, such as environmental remediation, bioleaching, microbial fuel cells, and corrosion protection, are often built on the basis of wild-type or metabolically engineered strains. In this review, we further comment on the design strategies for multiple innovative applications of living functional biofilms. With the integration of signaling pathways, engineering of metabolic pathways and modification of extracellular polymeric substances, living functional biofilms have been constructed by researchers through various strategies. Functional biofilms for diverse applications, including catalysis, electric conduction, bioremediation, and medical therapy have been demonstrated in the literature. The mechanical properties of biofilms can be tuned through genetic editing, metal ion curing and synthetic gene circuits, etc. In addition, the improvement of 3D printing to use bioinks has also achieved significant progresses in fabricating living functional biofilms with specific structures. In the future, the combination of synthetic biology and techniques from other disciplines will lead to practical large-scale applications of biofilms.
... As a case-study, we used KTK cloning to express E. coli's Curli system, 64 which produces the best-studied bacterial amyloid; a polymeric protein structurally rich in beta-65 sheets that forms a fibre with exceptional strength, stability and resistance (Evans and Chapman, 66 2014). Curli has been a focus for many ELM-associated studies (Birnbaum et al., 2020;Chen et al., 67 2014; Nguyen et al., 2014;Seker et al., 2017), and offers promise for making a novel composite with 68 BC in cells that can co-produce both materials. The Curli production system is also representative of 69 a Type VIII secretion system (T8SS) and so its expression offers a tractable solution for protein 70 secretion from BC-producing cells. ...
... As a case-study, we used KTK cloning to express E. coli's Curli system, 64 which produces the best-studied bacterial amyloid; a polymeric protein structurally rich in beta-65 sheets that forms a fibre with exceptional strength, stability and resistance (Evans and Chapman, 66 2014). Curli has been a focus for many ELM-associated studies (Birnbaum et al., 2020;Chen et al., 67 2014; Nguyen et al., 2014;Seker et al., 2017), and offers promise for making a novel composite with 68 BC in cells that can co-produce both materials. The Curli production system is also representative of 69 a Type VIII secretion system (T8SS) and so its expression offers a tractable solution for protein 70 secretion from BC-producing cells. ...
Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.
... Cell-synthesized curli nanofibers to controllably form gold nanoparticles and gold nanowires [210] Bioremediation Mercury-absorbing self-assembling curli nanofibers [199] Biofilm-Integrated Nanofiber Display (BIND) ...
Materials in nature have fascinating properties that serve as continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.
... Similarly, optical inputs can drive the on-demand production of curli fibres, allowing light-patterning of adhesive biofilms 119 . To further functionalize the biofilm, exogenous protein modules can be fused to the amyloid-forming domain of CsgA, leading to curli fibres capable of electrical conduction 120,121 , enzymatic catalysis 122,123 , bioremediation 90,124 and templating inorganic materials 125,126 . To enable more complex tasks, functionalized curli fibres can work in concert with computation and communication modules (Table 1), achieving simple Boolean decision-making 49 and intrafibre patterning 25 , or performing autonomous damage repair as 'smart' living glues 127 . ...
Synthetic biology applies genetic tools to engineer living cells and organisms analogous to the programming of machines. In materials synthetic biology, engineering principles from synthetic biology and materials science are integrated to redesign living systems as dynamic and responsive materials with emerging and programmable functionalities. In this Review, we discuss synthetic-biology tools, including genetic circuits, model organisms and design parameters, which can be applied for the construction of smart living materials. We investigate non-living and living self-organizing multifunctional materials, such as intracellular structures and engineered biofilms, and examine the design and applications of hybrid living materials, including living sensors, therapeutics and electronics, as well as energy-conversion materials and living building materials. Finally, we consider prospects and challenges of programmable living materials and identify potential future applications.
... Many approaches have been explored for producing ELMs by incorporating these living, programmable units into human-made materials (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). ELMs have also been fabricated using natural polymers harvested from microbes (15)(16)(17)(18)(19)(20)(21). But unlike the living materials found in nature, these ELMs require specialized labor and instrumentation to assemble the biomaterial into the desired architecture. ...
Bacterial cellulose (BC) has excellent material properties and can be produced cheaply and sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli . Here, we describe a simple approach for producing highly programmable BC materials through incorporation of engineered E. coli . The acetic acid bacterium Gluconacetobacter hansenii was co-cultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which were then colonized by the E. coli upon transfer to selective lysogeny broth media. We show that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, we produced capsules capable of altering their own bulk physical properties through enzyme-induced biomineralization. This novel system, based on autonomous biological fabrication, significantly expands the functionality of BC-based living materials.
... Bacterial biofilms are new tools for bionanomaterial fabrication and can be engineered for different applications under bacteria growth-controlled conditions. Seker et al. [134] engineered E. coli biofilms for use as conductive biopolymers to interface with electrodes and connect bacterial populations to electronic gadgets. Three repeats of aromatic amino acids were added to fiber-forming peptide sequences to produce delocalized π-clouds such as those observed in conductive polymers. ...
Amyloid proteins are linked to the pathogenesis of several diseases including Alzheimer’s disease, but at the same time a range of functional amyloids are physiologically important in humans. Although the disease pathogenies have been associated with protein aggregation, the mechanisms and factors that lead to protein aggregation are not completely understood. Paradoxically, unique characteristics of amyloids provide new opportunities for engineering innovative materials with biomedical applications. In this review, we discuss not only outstanding advances in biomedical applications of amyloid peptides, but also the mechanism of amyloid aggregation, factors affecting the process, and core sequences driving the aggregation. We aim with this review to provide a useful manual for those who engineer amyloids for innovative medicine solutions.
... Bacterial biofilms are new tools for bionanomaterial fabrication and can be engineered for different applications under bacteria growth-controlled conditions. Seker et al. [134] engineered E. coli biofilms for use as conductive biopolymers to interface with electrodes and connect bacterial populations to electronic gadgets. Three repeats of aromatic amino acids were added to fiber-forming peptide sequences to produce delocalized π-clouds such as those observed in conductive polymers. ...
Amyloid proteins are linked to the pathogenesis of several diseases including Alzheimer's disease, but at the same time a range of functional amyloids are physiologically important in humans. Although the disease pathogenies have been associated with protein aggregation, the mechanisms and factors that lead to protein aggregation are not completely understood. Paradoxically, unique characteristics of amyloids provide new opportunities for engineering innovative materials with biomedical applications. In this review, we discuss not only outstanding advances in biomedical applications of amyloid peptides, but also the mechanism of amyloid aggregation, factors affecting the process, and core sequences driving the aggregation. We aim with this review to provide a useful manual for those who engineer amyloids for innovative medicine solutions.
... In some biological systems the nucleating agent has been identified. Cadmium sulfide nanoparticles can nucleate on a cystathionine y-lyase enzyme (Dunleavy et al., 2016), and other known nucleation materials including enzymes, peptides and cells extracts have been reported (Merzlyak and Lee, 2006;Spoerke and Voigt, 2007;Varma, 2012;Seker et al., 2017). For arsenic sulfide nanomaterial, the nucleating agent can be provided by either E. coli or ANA-3 cells. ...
Microbes naturally build nanoscale structures, including structures assembled from inorganic materials. Here we combine the natural capabilities of microbes with engineered genetic control circuits to demonstrate the ability to control biological synthesis of chalcogenide nanomaterials in a heterologous host. We transferred reductase genes from both Shewanella sp. ANA-3 and Salmonella enterica serovar Typhimurium into an heterologous host ( Escherichia coli) and examined the mechanisms that regulate the properties of biogenic nanomaterials. Expression of arsenic reductase genes and thiosulfate reductase genes in E. coli resulted in the synthesis of arsenic sulfide nanomaterials. In addition to processing the starting materials via redox enzymes, cellular components also nucleated the formation of arsenic sulfide nanomaterials. The shape of the nanomaterial was influenced by the bacterial culture, with the synthetic E. coli strain producing nanospheres and conditioned media or cultures of wild type Shewanella sp. producing nanofibers. The diameter of these nanofibers also depended on the biological context of synthesis. These results demonstrate the potential for biogenic synthesis of nanomaterials with controlled properties by combining the natural capabilities of wild microbes with the tools from synthetic biology.
... amylase). (12,13) Additional work has focused on ELMs that function as catalytic surfaces, filtration membranes, under-water adhesives, pressure sensors, conductive films, gut adhesives, etc. (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) In spite of rapid progress in the field, it should be noted that there are few examples of ELMs that combine the structure-building capabilities of cells with their other capabilities, like self-regeneration, self-healing, or environmental responsiveness. ELM technologies that can streamline fabrication by relying more on autonomous cellular functions could help advance the field from a fundamental perspective and lead to ELMs compatible with scalable manufacturing techniques. ...
Living systems have not only the exemplary capability to fabricate materials ( e.g. wood, bone) under ambient conditions but they also consist of living cells that imbue them with properties like growth and self-regeneration. Like a seed that can grow into a sturdy living wood, we wondered: can living cells alone serve as the primary building block to fabricate stiff materials? Here we report the fabrication of stiff living materials (SLMs) produced entirely from microbial cells, without the incorporation of any structural biopolymers ( e.g. cellulose, chitin, collagen) or biominerals ( e.g. hydroxyapatite, calcium carbonate) that are known to impart stiffness to biological materials. Remarkably, SLMs are also lightweight, strong, resistant to organic solvents and can self-regenerate. This living materials technology can serve as a powerful biomanufacturing platform to design and develop sustainable structural materials, biosensors, self-regulators, self-healing and environment-responsive smart materials.
... Furthermore, the growing field of bio-inspired nanotechnology is capitalizing on the self-assembly of amyloid proteins. CsgA has been shown to have range of uses such as the passage of electric current, the extraction of rare earth metals, and many others (Nguyen et al., 2014;Seker et al., 2017;Tay et al., 2018). The work presented here will allow material scientists to use amyloid fibers to build systems of higher complexity than is currently possible. ...
Many organisms produce “functional” amyloid fibers, which are stable protein polymers that serve many roles in cellular biology. Certain Enterobacteriaceae assemble functional amyloid fibers called curli that are the main protein component of the biofilm extracellular matrix. CsgA is the major protein subunit of curli and will rapidly adopt the polymeric amyloid conformation in vitro. The rapid and irreversible nature of CsgA amyloid formation makes it challenging to study in vitro. Here, we engineered CsgA so that amyloid formation could be tuned to the redox state of the protein. A double cysteine variant of CsgA called CsgACC was created and characterized for its ability to form amyloid. When kept under oxidizing conditions, CsgACC did not adopt a β-sheet rich structure or form detectable amyloid-like aggregates. Oxidized CsgACC remained in a soluble, non-amyloid state for at least 90 days. The addition of reducing agents to CsgACC resulted in amyloid formation within hours. The amyloid fibers formed by CsgACC were indistinguishable from the fibers made by CsgA WT. When measured by thioflavin T fluorescence the amyloid formation by CsgACC in the reduced form displayed the same lag, fast, and plateau phases as CsgA WT. Amyloid formation by CsgACC could be halted by the addition of oxidizing agents. Therefore, CsgACC serves as a proof-of-concept for capitalizing on the convertible nature of disulfide bonds to control the aggregation of amyloidogenic proteins.
... These studies have shown that tunable and reversible phase behaviors can, to some degree, be encoded into de novo synthetic proteins by combining low complexity domains in particular arrangements [183]. Using this knowledge, researchers have generated synthetic peptides with predictable phase behaviors [8,182,183,185], synthetic prion domains [186], as well as redesigned bacterial amyloid proteins for application as structural biomaterials [187,188], adhesives [189], and nanowires [190]. For example, researchers fused the Escherichia coli CsgA protein, responsible for curli amyloid fiber formation, to the mussel foot protein (Mfps) in order to create a synthetic protein capable of generating a fibrillary structure of adhesive proteins that outperforms mussel foot in adhesion to underwater surfaces [189]. ...
The traditional view of protein aggregation as being strictly disease-related has been challenged by many examples of cellular aggregates that regulate beneficial biological functions. When coupled with the emerging view that many regulatory proteins undergo phase separation to form dynamic cellular compartments, it has become clear that supramolecular assembly plays wide-ranging and critical roles in cellular regulation. This presents opportunities to develop new tools to probe and illuminate this biology, and to harness the unique properties of these self-assembling systems for synthetic biology for the purposeful manipulation of biological function.
... The nascent field of engineered living materials (ELMs) aims to recapitulate the desirable properties of natural living biomaterials to create novel, useful materials using geneticallyengineered organisms [1][2][3][4] . Most ELMs to date have either synthesised novel materials from natural macromolecules and polymers harvested from microbial cells [5][6][7][8][9][10][11][12][13][14] or have made use of the multiple functionalities of living cells by embedding these within man-made hydrogels [15][16][17][18][19][20] . However, the long-term goal of ELMs research is to use engineered cells, rationally reprogrammed at the DNA level, to both make the material and incorporate novel functionalities into it at the same time -thus 'growing' functional biomaterials in situ 3 . ...
Biological systems assemble tissues and structures with advanced properties in ways that cannot be achieved by man-made materials. Living materials self-assemble under mild conditions, are autonomously patterned, can self-repair and sense and respond to their environment. Inspired by this, the field of engineered living materials (ELMs) aims to use genetically-engineered organisms to generate novel materials. Bacterial cellulose (BC) is a biological material with impressive physical properties and low cost of production that is an attractive substrate for ELMs. Inspired by how plants build materials from tissues with specialist cells we here developed a system for making novel BC-based ELMs by addition of engineered yeast programmed to add functional traits to a cellulose matrix. This is achieved via a synthetic 'symbiotic culture of bacteria and yeast' (Syn-SCOBY) approach that uses a stable co-culture of Saccharomyces cerevisiae with BC-producing Komagataeibacter rhaeticus bacetria. Our Syn-SCOBY approach allows inoculation of engineered cells into simple growth media, and under mild conditions materials self-assemble with genetically-programmable functional properties in days. We show that co-cultured yeast can be engineered to secrete enzymes into BC, generating autonomously grown catalytic materials and enabling DNA-encoded modification of BC bulk material properties. We further developed a method for incorporating S. cerevisiae within the growing cellulose matrix, creating living materials that can sense chemical and optical inputs. This enabled growth of living sensor materials that can detect and respond to environmental pollutants, as well as living films that grow images based on projected patterns. This novel and robust Syn-SCOBY system empowers the sustainable production of BC-based ELMs.
... 21,23 In their native state, amyloidogenic peptides are water-soluble and present at low concentrations in healthy organisms. 24,25 Further, these peptides were also identified as functional species in organisms, 26,27 and it was proposed that many peptides can form amyloid fibril structures, 28,29 including antimicrobial peptides, such as the peptide uperin 3.5 (U3.5, GVGDLIR-KAVSVIKNIV-NH 2 ) from the Australian toadlet Uperoleia mjobergii. 15,30−34 In this work, we systematically studied the adsorption of the amyloidogenic peptides GNNQQNY, NNFGAIL, VQIVYK, and U3.5 on unmodified and functionalized gold sensors (see Figure 1). ...
Surfaces are abundant in living systems, such as in the form of cellular membranes, and govern many biological processes. In this study, the adsorption of the amyloidogenic model peptides GNNQQNY, NNFGAIL, and VQIVYK as well as the amyloid-forming antimicrobial peptide uperin 3.5 (U3.5) were studied at low concentrations (100 μM) to different surfaces. The technique of a quartz crystal microbalance with dissipation monitoring (QCM-D) was applied as it enables the monitoring of mass binding to sensors at nanogram sensitivity. Gold-coated quartz sensors were used as unmodified gold surfaces or functionalized with self-assembled monolayers (SAMs) of alkanethiols (terminated as methyl, amino, carboxyl, and hydroxyl) resulting in different adsorption affinities of the peptides. Our objective was to evaluate the underlying role of the nature and feature of interfaces in biological systems which could concentrate peptides and impact or trigger peptide aggregation processes. In overall, the largely hydrophobic peptides adsorbed with preference to hydrophobic or countercharged surfaces. Further, the glycoprotein lubricin (LUB) was tested as an antiadhesive coating. Despite its hydrophilicity, the adsorption of peptides to LUB coated sensors was similar to the adsorption to unmodified gold surfaces, which indicates that some peptides diffused through the LUB layer to reach the underlying gold sensor surface. The LUB protein-antiadhesive is thus more effective as a biomaterial coating against larger biomolecules than small peptides under the conditions used here. This study provides directions toward a better understanding of amyloid peptide adsorption to biologically relevant interfaces, such as cellular membranes.
... Inorganic nanostructures, such as metal nanoparticles or carbon nanotubes were shown to be an effective way to improve bioelectronic charge transfer [71]. Recently genetically controlled conductive protein nanofibers have been demonstrated that will be useful for interfacing biotic and abiotic systems [72,73]. ...
Conventional microchip fabrication is energy and resource intensive. Thus, the discovery of new manufacturing approaches that reduce these expenditures would be highly beneficial to the semiconductor industry. In comparison, living systems construct complex nanometer-scale structures with high yields and low energy utilization. Combining the capabilities of living systems with synthetic DNA-/protein-based self-assembly may offer intriguing potential for revolutionizing the synthesis of complex sub-10 nm information processing architectures. The successful discovery of new biologically based paradigms would not only help extend the current semiconductor technology roadmap, but also offer additional potential growth areas in biology, medicine, agriculture and sustainability for the semiconductor industry. This article summarizes discussions surrounding key emerging technologies explored at the Workshop on Biological Pathways for Electronic Nanofabrication and Materials that was held on 16–17 November 2016 at the IBM Almaden Research Center in San Jose, CA.
Peptides and proteins are exposed to a variety of interfaces in a physiological environment, such as cell membranes, protein nanoparticles or viruses. These interfaces have a significant impact on the interaction, self-assembly, and aggregation mechanisms of biomolecular systems. Peptide self-assembly, particularly amyloid fibril formation, is associated with a wide range of functions; however, there is a link with neurodegenerative diseases such as Alzheimer's disease. This review highlights how interfaces affect peptide structure and the kinetics of aggregation leading to fibril formation. In nature, many surfaces are nanostructures such as liposomes, viruses or synthetic nanoparticles. Once exposed to a biological medium, nanostructures are coated with a corona, which then determines their activity. Both accelerating and inhibiting effects on peptide self-assembly have been observed. When amyloid peptides adsorb to a surface, they typically concentrate locally, which promotes aggregation into insoluble fibrils. Starting from a combined experimental and theoretical approach, we introduce and review models that allow for a better understanding of peptide self-assembly near hard and soft matter interfaces. We present research results from our laboratories, obtained in the last few years, and propose relationships between biological interfaces such as membranes and viruses and amyloid fibril formation. This article is protected by copyright. All rights reserved.
Extracellular electron transfer (EET) is a process via which certain microorganisms, such as bacteria, exchange electrons with extracellular materials by creating an electrical link across their membranes. EET has been studied for the reactions on solid materials such as minerals and electrodes with implication in geobiology and biotechnology. EET-capable bacteria exhibit broad phylogenetic diversity, and some are found in environments with various types of electron acceptors/donors not limited to electrodes or minerals. Oxygen has also been shown to serve as the terminal electron acceptor for EET of Pseudomonas aeruginosa and Faecalibacterium prausnitzii. However, the physiological significance of such oxygen-terminating EETs, as well as the mechanisms underlying them, remain unclear. In order to understand the physiological advantage of oxygen-terminating EET and its link with energy metabolism, in this review, we compared oxygen-terminating EET with aerobic respiration, fermentation, and electrode-terminating EET. We also summarized benefits and limitations of oxygen-terminating EET in a biofilm setting, which indicate that EET capability enables bacteria to create a niche in the anoxic zone of aerobic biofilms, thereby remodeling bacterial metabolic activities in biofilms.
Immune rejection, poor biocompatibility and cytotoxicity have seriously stalled the widespread application of biometallic materials. To overcome these problems, biometallic materials with fast and sufficient osseointegration, antibacterial properties and long-term stability have attracted the attention of researchers worldwide. Surface modification is currently used as a general strategy to develop material coatings that will overcome these challenging requirements and achieve the successful performance of implants. In this study, we proposed a substrate surface-modification strategy based on biofilm CsgA proteins that promote rapid cell attachment, proliferation, and stabilization of the cytoskeleton. CsgA-based nano-coating is easy to fabricate and has superior performance, which is expected to expand the application of medical implants.
Aggregation of unfolded or misfolded proteins into amyloid fibrils can cause various diseases in humans. However, the fibrils synthesized in vitro can be developed toward useful biomaterials under some physicochemical conditions. In this study, atomistic molecular dynamics simulations were performed to address the mechanism of beta-sheet formation of the unfolded hen egg-white lysozyme (HEWL) under a high temperature and low pH. Simulations of the protonated HEWL at pH 2 and the non-protonated HEWL at pH 7 were performed at the highly elevated temperature of 450 K to accelerate the unfolding, followed by the 333 K temperature to emulate some previous in vitro studies. The simulations showed that HEWL unfolded faster, and higher beta-strand contents were observed at pH 2. In addition, one of the simulation replicas at pH 2 showed that the beta-strand forming sequence was consistent with the ‘K-peptide’, proposed as the core region for amyloidosis in previous experimental studies. Beta-strand formation mechanisms at the earlier stage of amyloidosis were explained in terms of the radial distribution of the amino acids. The separation between groups of positively charged sidechains from the hydrophobic core corresponded to the clustering of the hydrophobic residues and beta-strand formation.
Proteins are one of the main components of the extracellular matrix in natural biological materials. They confer a unique advantage in creating engineered living materials (ELM) because they can be genetically encoded and rationally designed for constructing bioactive network structures. Advances in the design, characterization, and engineering of protein networks have been an important multidisciplinary endeavor and should be considered when designing ELM and understanding their behavior. This chapter describes the network-forming behavior of recombinant proteins, as these proteins, in principle, can be genetically programmed and synthesized directly from living cells residing in ELM. There are three major classes of protein network-forming mechanisms relevant to this topic: (1) phase separation and aggregation-induced recombinant protein networks, (2) self-assembling multi-domain artificial protein networks, and (3) chemically cross-linked recombinant protein networks. We will begin by introducing protein hydrogels and discuss their mechanism of network formation, which is a critical element in designing functionalities and mechanical properties of ELM. After introducing the network-forming mechanisms in protein hydrogels, we will discuss examples of bioactive protein networks equipped with various functionalities before concluding with future directions and remaining challenges in this field.
Global challenges require novel solutions. Microbial nanotechnology has the potential to offer an eco-friendly approach for advancing the pharmaceutical, food, environment and agriculture sectors. This review summarizes recent developments in the synthesis and applications of bionanomaterials created by intracellular and/or extracellular biosynthesis processes involving various microorganisms (e.g. bacterium, fungus, yeast, virus and microalga) and/or derived metabolites/secreted substances. Insights into the mechanisms underlying the synthesis of microbial-assisted nanomaterials, and the superiority of these ‘green’ processes and resulting microbiotic nanomaterials in various applications, are provided. Such knowledge supports the development of microfactories for the biosynthesis of diverse nanomaterials at scale under controlled conditions. This review highlights current hurdles and bottleneck issues, whilst demonstrating the need to manipulate microorganisms at both the genomic and proteomic levels, thereby tailoring the properties of the derived bionanomaterials for target applications. For many envisaged applications, toxicological and ecotoxicological studies on synthesized nanomaterials are needed, along with the establishment of suitable regulatory frameworks.
In nature, the cells are unique biofactories of various kinds of macroscale structures. These biofactories are as old as the earth. However, as technology developed and new areas of research fields developed these cellular biofactories became the center of attention. The motive was the question if we can engineer them according to the world’s needs. At that point, approaches and tools of synthetic biology came into the picture. After its development, people started to engineer biofactories and produce materials with new properties. One of those materials is classified as self-assembled soft living materials with their specific features and usage areas. To be more specific, biofilms are examples of self-assembled soft living materials due to their self-sustaining and self-assembling properties. They can be engineered starting from genetic circuits leading to creation of their building blocks and finally formation of complex biofilm systems. With the diversity in their engineering aspects, their application areas also vary. In this chapter, the design of biofilm structures from genetic circuits until the formation of complex biofilm structures and their various applications will be investigated.
Engineered living materials could have the capacity to self-repair and self-replicate, sense local and distant disturbances in their environment, and respond with functionalities for reporting, actuation or remediation. However, few engineered living materials are capable of both responsivity and use in macroscopic structures. Here we describe the development, characterization and engineering of a fungal–bacterial biocomposite grown on lignocellulosic feedstocks that can form mouldable, foldable and regenerative living structures. We have developed strategies to make human-scale biocomposite structures using mould-based and origami-inspired growth and assembly paradigms. Microbiome profiling of the biocomposite over multiple generations enabled the identification of a dominant bacterial component, Pantoea agglomerans, which was further isolated and developed into a new chassis. We introduced engineered P. agglomerans into native feedstocks to yield living blocks with new biosynthetic and sensing–reporting capabilities. Bioprospecting the native microbiota to develop engineerable chassis constitutes an important strategy to facilitate the development of living biomaterials with new properties and functionalities. Lignocellulosic waste is transformed into fungal–bacterial biocomposites that can be processed into recyclable, human-scale structural objects with biosynthetic and sensing–reporting functionalities.
Hybrid systems composed of living cells and nanomaterials have been attracting great interest in various fields of research ranging from materials science to biomedicine. In particular, the interfacing of noble metal nanoparticles and bacterial cells in a single architecture aims to generate hybrid systems that combine the unique physicochemical properties of the metals and biological attributes of the microbial cells. While the bacterial cells provide effector and scaffolding functions, the metallic component endows the hybrid system with multifunctional capabilities. This synergistic effort seeks to fabricate living materials with improved functions and new properties that surpass their individual components. Herein, we provide an overview of this research field and the strategies for obtaining hybrid systems, and we summarize recent biological applications, challenges and current prospects in this exciting new arena.
Biocompatible and nanoscale devices for biological modulation of cells and tissues possess the potential for tremendous impact on medical and industrial technologies. Typical medical devices and therapies tend to be macroscale, comprised of nonbiocompatible materials, and broadly targeted, resulting in imprecise treatments and adverse effects such as chronic immune response and tissue damage. The development of nanoenabled and biocompatible technologies—ranging from biodegradable nanoparticles for localized drug delivery to transient electronic devices for stimulation therapy to engineered biofilms with applications to nanomedicine—will continue to enable the advent of personalized medicine and precision therapies. In this review, recent research into this frontier is reviewed, first analyzing the synthesis of nanoenabled and biocompatible technologies and then presenting significant considerations regarding the development of such materials. Lastly, the latest advancements in biocompatible, nanoenabled devices are examined, followed by a discussion of the direction of future research in the field. Nanoscale devices can benefit the development of medical technologies with efficient and biocompatible solutions for precise and personalized treatments. Such a high precision of nanoenabled solution allows minimizing immune responses and tissue damage compared to traditional approaches. This review summarizes the newest developments and opportunities in the field of nanoscaled bioelectronics.
Amyloid diseases are caused due to protein homeostasis failure where incorrectly folded proteins/peptides form cross-β-sheet rich amyloid fibrillar structures. Besides proteins/peptides, small metabolite assemblies also exhibit amyloid-like features. These structures are linked to several human and animal diseases. In addition, non-toxic amyloids with diverse physiological roles are characterized as a new functional class. This finding, along with the unique properties of amyloid like stability and mechanical strength, led to a surge in the development of amyloid-based biomaterials. However, the usage of these materials by humans and animals may pose a health risk such as the development of amyloid diseases and toxicity. This is possible because amyloid-based biomaterials and their fragments may assist seeding and cross-seeding mechanisms of amyloid formation in the body. This review summarizes the potential uses of amyloids as biomaterials, the concerns regarding their usage, and a prescribed workflow to initiate a regulatory approach.
Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC‐producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli (E. coli). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in droplets of glucose‐rich media to produce robust cellulose capsules, which are then colonized by the E. coli upon transfer to selective lysogeny broth media. It is shown that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme‐induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC‐based living materials.
Living systems have not only the exemplary capability to fabricate materials (e.g., wood, bone) under ambient conditions but they also consist of living cells that imbue them with properties like growth and self‐regeneration. Like a seed that can grow into a sturdy living wood, can living cells alone serve as the primary building block to fabricate stiff materials? Here is reported the fabrication of stiff living materials (SLMs) produced entirely from microbial cells, without the incorporation of any structural biopolymers (e.g., cellulose, chitin, collagen) or biominerals (e.g., hydroxyapatite, calcium carbonate) that are known to impart stiffness to biological materials. Remarkably, SLMs are also lightweight, strong, and resistant to organic solvents and can self‐regenerate. This living materials technology can serve as a powerful biomanufacturing platform to design and develop advanced structural and cellular materials in a sustainable manner.
Glycosylation is a crucial post-translational modification for a wide range of functionalities. Adhesive protein-based biomaterials in nature rely on heavily glycosylated proteins such as spider silk and mussel adhesive proteins. Engineering protein-based biomaterials genetically enables desired functions and characteristics. Additionally, utilization of glycosylation for biomaterial engineering can expand possibilities by including saccharides to the inventory of building blocks. Here, de novo glycosylation of Bacillus subtilis amyloid-like biofilm protein TasA using Campylobacter jejuni glycosylation circuit is proposed to be a novel biomaterial engineering method for increasing adhesiveness of TasA fibrils. C. jejuni glycosylation motif is genetically incorporated to tasA gene and expressed in Escherichia coli containing C. jejuni pgl protein glycosylation pathway. Glycosylated TasA fibrils indicates enhanced adsorption on the gold surface without disruption of fibril formation. Our findings suggest that N-linked glycosylation can be a promising tool for engineering protein-based biomaterials specifically regarding adhesion.
Engineered living materials (ELMs) have drawn intense interest from both academia and industry in recent years. The essence of ELMs is the use of living cells to produce molecular building blocks, direct their hierarchical organization, and convert life into functional materials. By doing so, it confers living features on materials, including self-organization, self-maintaining, adaptability and evolvability. As the workhorse of life, proteins play an essential role in current ELM designs. To harness proteins for applications in ELMs, protein engineering is usually required to tailor their assembly, chemistry, functions, and their interactions with abiotic materials. In this review, we discuss the roles of proteins in ELMs and summarize the applications of protein engineering in developing molecular tools toward the creation of advanced ELMs with novel properties. Inspired by systems chemistry, we emphasize that future development of ELMs would benefit from a systems perspective by integrating a rich and versatile interaction network comprising multiple functional components such as genes, RNAs, proteins, and even many other abiotic components.
Intrinsically conductive protein nanowires, microbially produced from inexpensive, renewable feedstocks, are a sustainable alternative to traditional nanowire electronic materials, which require high energy inputs and hazardous conditions/chemicals for fabrication and can be highly toxic. Pilin-based nanowires can be tailored for specific functions via the design of synthetic pilin genes to tune wire conductivity or introduce novel functionalities. Other microbially produced nanowire options for electronics may include cytochrome wires, curli fibers, and the conductive fibers of cable bacteria. Proof-of-concept protein nanowire electronics that have been successfully demonstrated include biomedical sensors, neuromorphic devices, and a device that generates electricity from ambient humidity. Further development of applications will require interdisciplinary teams of engineers, biophysicists, and synthetic biologists.
Hypothesis
Self-assembling protein subunits hold great potential as biomaterials with improved functions. Among the self-assembled protein structures functional amyloids are promising unique properties such as resistance to harsh physical and chemical conditions their mechanical strength, and ease of functionalization. Curli proteins, which are functional amyloids of bacterial biofilms can be programmed as intelligent biomaterials.
Experiments
In order to obtain controllable curli based biomaterials for biomedical applications, and to understand role of each of the curli forming monomeric proteins (namely CsgA and CsgB from Escherichia coli) we characterized their binding kinetics to gold, hydroxyapatite, and silica surfaces.
Findings
We demonstrated that CsgA, CsgB, and their equimolar mixture have different binding strengths for different surfaces. On hydroxyapatite and silica surfaces, CsgB is the crucial element that determines the final adhesiveness of the CsgA-CsgB mixture. On the gold surface, on the other hand, CsgA controls the behavior of the mixture. Those findings uncover the binding behavior of curli proteins CsgA and CsgB on different biomedically valuable surfaces to obtain a more precise control on their adhesion to a targeted surface.
Identification of curli-specific dyes for biofilm communities of microorganisms is an important task. We describe here a curli fluorescent light-up probe called bromophenol blue, which bind to curli via recognizing...
Synthetic biologists are exploiting biofilms as an effective mechanism for producing various outputs. Metabolic optimization has become commonplace as a method of maximizing system output. In addition to production pathways, the biofilm itself contributes to the efficacy of production. The purpose of this review is to highlight opportunities that might be leveraged to further enhance production in preexisting biofilm production systems. These opportunities may be used with previously established production systems as a method of improving system efficiency further. This may be accomplished through the reduction in the cost of establishing and maintaining biofilms, and maintenance of the enhancement of product yield per unit of time, per unit of area, or per unit of required input.
Biosynthesis of metal nanoparticles (MNPs) has recently emerged as novel ecofriendly process for the preparation of supported MNPs. However, accumulation of MNPs suspension in environment after intended use adversely affects the ecosystem. These neo contaminants have drawn significant attention with respect to the environmental fate and consequent health issues. Synthesis of MNPs on a solid support has emerged as a prospective solution that may prevent the accumulation of waste MNPs in the ecosystem through recycling process. However, the prevalent synthesis process of supported MNPs production requires highly flammable organic solvents, huge amounts of toxic chemicals, high temperature and pressure; thus frequently raising toxicity and health concerns. To counter these adverse effects, the eco-friendly biosynthesis process can be integrated with other green chemistry principle for preparation of sustainable MNPs through regeneration and recycling of the MNPs. With the aim to reduce the MNPs toxicity, a novel bioinspired approach has been adopted recently for the synthesis of supported MNPs. In this review, we have highlighted the current development on bioinspired and biomimetic synthesis of solid supported (including bio-support) MNPs focusing on sustainable design of engineered nanoparticles (NPs). Special attention has been given to biosynthetic mechanism of supported MNPs formation and application of the bioinspired solid supported MNPs in environment oriented technologies including sensing, treatment of wastewater, catalysis, water disinfection, and anti-fouling activity.
Genetically controlled synthetic biosystems are being developed to create nanoscale materials. These biosystems are modeled on the natural ability of living cells to synthesize materials: many organisms have dedicated proteins that synthesize a wide range of hard tissues and solid materials, such as nanomagnets and biosilica. We designed an autonomous living material synthesizing system consisting of engineered cells with genetic circuits that synthesize nanomaterials. The circuits encode a nanomaterial precursor-sensing module (sensor) coupled with a materials synthesis module. The sensor detects the presence of cadmium, gold, or iron ions, and this detection triggers the synthesis of the related nanomaterial-nucleating extracellular matrix. We demonstrate that when engineered cells sense the availability of a precursor ion, they express the corresponding extracellular matrix to form the nanomaterials. This proof-of-concept study shows that endowing cells with synthetic genetic circuits enables nanomaterial synthesis, and has the potential to be extended to the synthesis of a variety of nanomaterials and biomaterials using a green approach.
Distinct spatial patterning of naturally produced materials is observed in many cellular structures and even among communities of microorganisms. Reoccurrence of spatially organized materials in all branches of life is clear proof that organization is beneficial for survival. Indeed, organisms can trick the evolutionary process by using organized materials in ways can help the organism to avoid unexpected conditions. To expand the toolbox for synthesizing patterned living materials, Boolean type “AND” and “OR” control of curli fibers expression is demonstrated using recombinases. Logic gates are designed to activate the production of curli fibers. The gates can be used to record the presence of input molecules and give output as CsgA expression. Two different curli fibers (CsgA and CsgA‐His‐tag) production are then selectively activated to explore distribution of monomers upon coexpression. To keep track of the composition of fibers, CsgA‐His‐tag proteins are labeled with nickel–nitrilotriacetic acid (Ni–NTA‐) conjugated gold nanoparticles. It is observed that an organized living material can be obtained upon inducing the coexpression of different CsgA fibers. It is foreseen that living materials with user‐defined curli composition hold great potential for the development of living materials for many biomedical applications. Synthetic‐biology‐enabled devices can be used for biomaterial assembly and patterning. Biological polymers including biofilm fibers and spider silk are formed by specific proteins that are synthetized in monomeric form and assembled into higher ordered structures. Recombinase‐integrated genetic logic gates are employed to control patterning of bacterial biofilm nanofibers for the assembly of gold nanoparticles.
Living cells can impart materials with advanced functions, such as sense‐and‐respond, chemical production, toxin remediation, energy generation and storage, self‐destruction, and self‐healing. Here, an approach is presented to use light to pattern Escherichia coli onto diverse materials by controlling the expression of curli fibers that anchor the formation of a biofilm. Different colors of light are used to express variants of the structural protein CsgA fused to different peptide tags. By projecting color images onto the material containing bacteria, this system can be used to pattern the growth of composite materials, including layers of protein and gold nanoparticles. This is used to pattern cells onto materials used for 3D printing, plastics (polystyrene), and textiles (cotton). Further, the adhered cells are demonstrated to respond to sensory information, including small molecules (IPTG and DAPG) and light from light‐emitting diodes. This work advances the capacity to engineer responsive living materials in which cells provide diverse functionality.
Cellular biocatalysts hold great promise to synthesize hard‐to‐get compounds such as complex active molecules. Whole‐cell biocatalysts can be programmed through genetic circuits to be more efficient; yet, they are suffering due to low stability. The catalytic activity of whole‐cells decays in stressful conditions such as in prolonged incubation times or at high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, we have shown that the utilization of biofilm structures can enhance the stability of whole‐cell biocatalysts. We have employed two different strategies to increase the longevity of whole cell catalysts and decrease its susceptibility to high temperature. In the first approach, we have induced the formation of biofilm structure by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of the cells in the biofilm structure. In the second approach, the ALP enzyme was fused to CsgA curli fiber subunit to utilize the protective properties of the biofilm on the enzyme biofilms. Furthermore, we introduced an "AND" logic gate between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of whole cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.
Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogs of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundations for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled based on the location of the insertion within the S-layer protein, RsaA. Moreover, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm². Finally, we show that ligation of quantum dots to the cell surface does not impair cell viability and this composite material remains intact over a period of two weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step towards building hierarchically-ordered engineered living materials with emergent properties.
Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.
Many natural biological systems-such as biofilms, shells and skeletal tissues-are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.
Coupling of polyhistidine-appended biomolecules to inorganic nanocrystals driven by metal-affinity interactions is a greatly promising strategy to form hybrid bioconjugates. It is simple to implement and can take advantage of the fact that polyhistidine-appended proteins and peptides are routinely prepared using well established molecular engineering techniques. A few groups have shown its effectiveness for coupling proteins onto Zn- or Cd-rich semiconductor quantum dots (QDs). Expanding this conjugation scheme to other metal-rich nanoparticles (NPs) such as AuNPs would be of great interest to researchers actively seeking effective means for interfacing nanostructured materials with biology. In this report, we investigated the metal-affinity driven self-assembly between AuNPs and two engineered proteins, a His7-appended maltose binding protein (MBP-His) and a fluorescent His6-terminated mCherry protein. In particular, we investigated the influence of the capping ligand affinity to the nanoparticle surface, its density, and its lateral extension on the AuNP-protein self-assembly. Affinity gel chromatography was used to test the AuNP-MPB-His7 self-assembly, while NP-to-mCherry-His6 binding was evaluated using fluorescence measurements. We also assessed the kinetics of the self-assembly between AuNPs and proteins in solution, using time-dependent changes in the energy transfer quenching of mCherry fluorescent proteins as they immobilize onto the AuNP surface. This allowed determination of the dissociation rate constant, Kd-1 ~ 1-5 nM. Furthermore, a close comparison of the protein self-assembly onto AuNPs or QDs provided additional insights into which parameters control the interactions between imidazoles and metal ions in these systems.
Diverse mechanisms have been proposed to explain biological pattern formation. Regardless of their specific molecular interactions, the majority of these mechanisms require morphogen gradients as the spatial cue, which are either predefined or generated as a part of the patterning process. However, using Escherichia coli programmed by a synthetic gene circuit, we demonstrate here the generation of robust, self-organized ring patterns of gene expression in the absence of an apparent morphogen gradient. Instead of being a spatial cue, the morphogen serves as a timing cue to trigger the formation and maintenance of the ring patterns. The timing mechanism enables the system to sense the domain size of the environment and generate patterns that scale accordingly. Our work defines a novel mechanism of pattern formation that has implications for understanding natural developmental processes.
Although the term ‘Biocomputing’ may bring to mind biological replacements of silicon processors; this type of application is far in the future. Use of bacteria-based Biocomputing for biosensors and industrial fermentation control, however, is presently attainable by using genetically-engineered bacterial cells that can process signals in a logical operation via one or a few pathways. Here, we refer to these systems as ‘Cellular Computing Circuits’ and focus on their possible future implementations. We also briefly discuss concepts from Synthetic Biology and enzyme-based Biocomputing because they will be important during future development. Our lab has already transformed an idea from enzyme-based Biocomputing into a bacteria-based Boolean logic gate with a digital output signal of direct electric current and we suggest future applications in this perspective. We predict useful functions for Cellular Computing Circuits in the near future.
Microorganisms produce and secrete secondary metabolites to assist in their survival. We report that the gold resident bacterium Delftia acidovorans produces a secondary metabolite that protects from soluble gold through the generation of solid gold forms. This finding is the first demonstration that a secreted metabolite can protect against toxic gold and cause gold biomineralization.
Escherichia coli and a few other members of the Enterobacteriales can produce functional amyloids known as curli. These extracellular fibrils are involved in biofilm formation and studies have shown that they may act as virulence factors during infections. It is not known whether curli fibrils are restricted to the Enterobacteriales or if they are phylogenetically widespread. The growing number of genome-sequenced bacteria spanning many phylogenetic groups allows a reliable bioinformatic investigation of the phylogenetic diversity of the curli system. Here we show that the curli system is phylogenetically much more widespread than initially assumed, spanning at least four phyla. Curli fibrils may consequently be encountered frequently in environmental as well as pathogenic biofilms, which was supported by identification of curli genes in public metagenomes from a diverse range of habitats. Identification and comparison of curli subunit (CsgA/B) homologs show that these proteins allow a high degree of freedom in their primary protein structure, although a modular structure of tightly spaced repeat regions containing conserved glutamine, asparagine and glycine residues has to be preserved. In addition, a high degree of variability within the operon structure of curli subunits between bacterial taxa suggests that the curli fibrils might have evolved to fulfill specific functions. Variations in the genetic organization of curli genes are also seen among different bacterial genera. This suggests that some genera may utilize alternative regulatory pathways for curli expression. Comparison of phylogenetic trees of Csg proteins and the 16S rRNA genes of the corresponding bacteria showed remarkably similar overall topography, suggesting that horizontal gene transfer is a minor player in the spreading of the curli system.
Bacterial pathogens secrete chemically diverse iron chelators called siderophores, which may exert additional distinctive functions in vivo. Among these, uropathogenic Escherichia coli often coexpress the virulence-associated siderophore yersiniabactin (Ybt) with catecholate siderophores. Here we used a new MS screening approach to reveal that Ybt is also a physiologically favorable Cu(II) ligand. Direct MS detection of the resulting Cu(II)-Ybt complex in mice and humans with E. coli urinary tract infections demonstrates copper binding to be a physiologically relevant in vivo interaction during infection. Ybt expression corresponded to higher copper resistance among human urinary tract isolates, suggesting a protective role for this interaction. Chemical and genetic characterization showed that Ybt helps bacteria resist copper toxicity by sequestering host-derived Cu(II) and preventing its catechol-mediated reduction to Cu(I). Together, these studies reveal a new virulence-associated function for Ybt that is distinct from iron binding.
Curli are extracellular proteinaceous functional amyloid aggregates produced by Escherichia coli, Salmonella spp., and other enteric bacteria. Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit. In vitro, purified CsgA and CsgB exhibit intrinsically disordered properties, and both are capable of forming amyloid fibers similar in morphology to those formed in vivo. However, in vivo, CsgA alone cannot form curli fibers, and CsgB is required for filament growth. Thus, we studied the aggregation of CsgA and CsgB both alone and together in vitro to investigate the different roles of CsgA and CsgB in curli formation. We found that though CsgA and CsgB individually are able to self-associate to form aggregates/fibrils, they do so using different mechanisms and with different kinetic behavior. CsgB rapidly forms structured oligomers, whereas CsgA aggregation is slower and appears to proceed through large amorphous aggregates before forming filaments. Substoichiometric concentrations of CsgB induce a change in the mechanism of CsgA aggregation from that of forming amorphous aggregates to that of structured intermediates similar to those of CsgB alone. Oligomeric CsgB accelerated the aggregation of CsgA, in contrast to monomeric CsgB, which had no effect. The structured β-strand oligomers formed by CsgB serve as nucleators for CsgA aggregation. These results provide insights into the formation of curli in vivo, especially the nucleator function of CsgB.
Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.
Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult
environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of
one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied.
C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT
of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all
these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations.
Only corA1 expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of
each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability,
CorA1 as the main secondary magnesium uptake system, and CorA2 and CorA3 as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent
metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with
efflux systems that “worry later” about surplus cations.
Nanotechnology is having a major impact on medicine and the treatment of disease, notably in imaging and targeted drug delivery. It may, however, be possible to go even further and design 'pseudo-cell' nanofactories that work with molecules already in the body to fight disease.
In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation.
FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic
protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The
crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral
geometry composed of His44, Met90, His97, and His127, and CuB, a second degenerate octahedral geometry with the addition of Glu46. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I)
binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport
by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease
and an iron-scavenging and potentially iron-reducing periplasmic protein.
Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.
Gold nanoparticles are useful in biomedical applications due to their distinct optical properties and high chemical stability. Reports of the biogenic formation of gold colloids from gold complexes has also led to an increased level of interest in the biomineralization of gold. However, the mechanism responsible for biomolecule-directed gold nanoparticle formation remains unclear due to the lack of structural information about biological systems and the fast kinetics of biomimetic chemical systems in solution. Here we show that intact single crystals of lysozyme can be used to study the time-dependent, protein-directed growth of gold nanoparticles. The protein crystals slow down the growth of the gold nanoparticles, allowing detailed kinetic studies to be carried out, and permit a three-dimensional structural characterization that would be difficult to achieve in solution. Furthermore, we show that additional chemical species can be used to fine-tune the growth rate of the gold nanoparticles.
Viral Battery
In developing materials for batteries, there is a trade-off between charge capacity, conductivity, and chemical stability. Nanostructured materials improve the conductivity for some resistive materials, but fabricating stable materials at nanometer-length scales is difficult. Harnessing their knowledge of viruses as toolkits for materials fabrication, Lee et al. (p. 1051; published online 2 April) modified two genes in the filamentous bacteriophage M13 to produce a virus with an affinity for nucleating amorphous iron phosphate along its length and for attaching carbon nanotubes at one of the ends. In nanostructured form, the amorphous iron phosphate produced a useful cathode material, while the carbon nanotubes formed a percolating network that significantly enhanced conductivity.
Binding kinetics of platinum-, silica-, and gold-binding peptides were investigated using a modified surface plasmon resonance spectroscopy (SPR). Platinum binding septa-peptides, quartz-binding dodecapeptides, and gold-binding 14-aa peptides were originally selected using phage or cell surface display libraries using the mineral or pure forms of these materials. All of the peptides were synthesized singly to investigate their binding kinetics and to assess quantitatively the specific affinity of each to its material of selection. The peptides were also postselection engineered to contain multiple copies of the same original sequences to quantify the effects of repeating units. SPR spectroscopy, normally using gold surfaces, was modified to contain a thin film (a few nm thick) of the material of interest (silica or platinum) on gold to allow the quantitative study of the adsorption kinetics of specific solid-binding peptides. The SPR experiments, carried out at different concentrations, on all three materials substrates, resulted in Langmuir behavior that allowed the determination of the kinetic parameters, including adsorption, desorption, and equilibrium binding constants for each of the solids as well as free energy of adsorption. Furthermore, we also tested multiple repeats of the peptide sequences, specifically three repeats, to see if there is a general trend of increased binding with increased number of binding domains. There was no general trend in the binding strength of the peptides with the increase of the repeat units from one to three, possibly because of the conformational changes between the single and multiple repeat polypeptides. In all cases, however, the binding was strong enough to suggest that these inorganic binding peptides could potentially be used as specific molecular linkers to bind molecular entities to specific solid substrates due to their surface recognition characteristics.
Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.
The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations
in the ompRgene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes
biofilm formation by binding the csgDpromoter region and stimulating its transcription. ThecsgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBAoperon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase
in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor
RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR;cpxR-mediated repression of csgB andcsgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.
Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases. Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coli called curli were amyloid. The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli. In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively. Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery.
Proteins, through their unique and specific interactions with other macromolecules and inorganics, control structures and functions of all biological hard and soft tissues in organisms. Molecular biomimetics is an emerging field in which hybrid technologies are developed by using the tools of molecular biology and nanotechnology. Taking lessons from biology, polypeptides can now be genetically engineered to specifically bind to selected inorganic compounds for applications in nano- and biotechnology. This review discusses combinatorial biological protocols, that is, bacterial cell surface and phage-display technologies, in the selection of short sequences that have affinity to (noble) metals, semiconducting oxides and other technological compounds. These genetically engineered proteins for inorganics (GEPIs) can be used in the assembly of functional nanostructures. Based on the three fundamental principles of molecular recognition, self-assembly and DNA manipulation, we highlight successful uses of GEPI in nanotechnology.
Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell. Here we present a post-transcriptional regulation system in Escherichia coli that uses RNA to both silence and activate gene expression. We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5'-untranslated region of the mRNA. The stem-loop structure interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.
Siderophores are extracellular iron-binding compounds that mediate iron transport into many cells. We present evidence of
analogous molecules for copper transport from methane-oxidizing bacteria, represented here by a small fluorescent chromopeptide
(C45N12O14H62Cu, 1216 daltons) produced by Methylosinus trichosporium OB3b. The crystal structure of this compound, methanobactin, was resolved to 1.15 angstroms. It is composed of a tetrapeptide,
a tripeptide, and several unusual moieties, including two 4-thionyl-5-hydroxy-imidazole chromophores that coordinate the copper,
a pyrrolidine that confers a bend in the overall chain, and an amino-terminal isopropylester group. The copper coordination environment includes a dual nitrogen- and sulfur-donating system derived
from the thionyl imidazolate moieties. Structural elucidation of this molecule has broad implications in terms of organo-copper
chemistry, biological methane oxidation, and global carbon cycling.
The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF). Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur. Absence of FieF led to decreased growth of E. coli cells in complex growth medium but only if fur was additionally deleted. The presence of EDTA was partially able to relieve this growth deficiency. Expression of fieF in trans rendered the double deletion strain more tolerant to iron. Furthermore, E. coli cells exhibited reduced accumulation of (55)Fe when FieF was expressed in trans. FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles. Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching. This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.
The selection and assembly of materials are central issues in the development of smaller, more flexible batteries. Cobalt
oxide has shown excellent electrochemical cycling properties and is thus under consideration as an electrode for advanced
lithium batteries. We used viruses to synthesize and assemble nanowires of cobalt oxide at room temperature. By incorporating
gold-binding peptides into the filament coat, we formed hybrid gold–cobalt oxide wires that improved battery capacity. Combining
virus-templated synthesis at the peptide level and methods for controlling two-dimensional assembly of viruses on polyelectrolyte
multilayers provides a systematic platform for integrating these nanomaterials to form thin, flexible lithium ion batteries.
We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).
Viruses have been used as scaffolds for the peptide-directed synthesis of magnetic and semiconducting materials, and have been further exploited in the formation of nanowires and liquid crystals. Reviewed in this manuscript is the work of Douglas, Mann, Fraden, Belcher, DeYoreo and others who have either exploited native viral structures to grow or assemble materials, or have genetically modified existing viral structures to specifically affect the growth and mineralization of inorganic materials. Rod-shaped viruses, including M13 bacteriophage and tobacco mosaic viruses, have been used in the synthesis of nanowires of metals, semiconductors and magnetic materials. The cowpea chlorotic mottle and the cowpea mosaic viruses have been used as nucleation cages for the mineralization of materials such as iron oxide and polyoxometalates. The exterior of such cages has been chemically modified with conjugating linkers as well as with polymeric materials and fluorophores. Further, viral-inorganic complexes have been incorporated into liquid crystal systems as well as self-supporting viral thin films and viral fibers.
Recent advances in the design and construction of synthetic multicelled systems in E. coli and S. cerevisiae suggest that it may be possible to implement sophisticated distributed algorithms with these relatively simple organisms. However, existing design frameworks for synthetic biology do not account for the unique morphologies of growing microcolonies, the interaction of gene circuits with the spatial diffusion of molecular signals, or the relationship between multicelled systems and parallel algorithms. Here, we introduce a framework for the specification and simulation of multicelled behaviors that combines a simple simulation of microcolony growth and molecular signaling with a new specification language called gro. The framework allows the researcher to explore the collective behaviors induced by high level descriptions of individual cell behaviors. We describe example specifications of previously published systems and introduce two novel specifications: microcolony edge detection and programmed microcolony morphogenesis. Finally, we illustrate through example how specifications written in gro can be refined to include increasing levels of detail about their bimolecular implementations.
PbS colloidal quantum dot heterojunction solar cells have shown significant improvements in performance, mostly based on devices that use high-temperature annealed transition metal oxides to create rectifying junctions with quantum dot thin films. Here, we demonstrate a solar cell based on the heterojunction formed between PbS colloidal quantum dot layers and CdS thin films that are deposited via a solution process at 80 °C. The resultant device, employing a 1,2-ethanedithiol ligand exchange scheme, exhibits an average power conversion efficiency of 3.5%. Through a combination of thickness-dependent current density-voltage characteristics, optical modeling, and capacitance measurements, the combined diffusion length and depletion width in the PbS quantum dot layer is found to be approximately 170 nm.
Electromicrobiology deals with the interactions between microorganisms and electronic devices and with the novel electrical properties of microorganisms. A diversity of microorganisms can donate electrons to, or accept electrons from, electrodes without the addition of artificial electron shuttles. However, the mechanisms for microbe-electrode electron exchange have been seriously studied in only a few microorganisms. Shewanella oneidensis interacts with electrodes primarily via flavins that function as soluble electron shuttles. Geobacter sulfurreducens makes direct electrical contacts with electrodes via outer-surface, c-type cytochromes. G. sulfurreducens is also capable of long-range electron transport along pili, known as microbial nanowires, that have metallic-like conductivity similar to that previously described in synthetic conducting polymers. Pili networks confer conductivity to G. sulfurreducens biofilms, which function as a conducting polymer, with supercapacitor and transistor functionalities. Conductive microorganisms and/or their nanowires have a number of potential practical applications, but additional basic research will be necessary for rational optimization.