Living Synthelectronics: A New Era for Bioelectronics Powered by Synthetic Biology.

Bioelectronics, which converges biology and electronics, has attracted great attention due to their vital applications in human-machine interfaces. While traditional bioelectronic devices utilize nonliving organic and/or inorganic materials to achieve flexibility and stretchability, a biological mismatch is often encountered because human tissues are characterized not only by softness and stretchability but also by biodynamic and adaptive properties. Recently, a notable paradigm shift has emerged in bioelectronics, where living cells, and even viruses, modified via gene editing within synthetic biology, are used as core components in a new hybrid electronics paradigm. These devices are defined as "living synthelectronics," and they offer enhanced potential for interfacing with human tissues at informational and substance exchange levels. In this Perspective, the recent advances in living synthelectronics are summarized. First, opportunities brought to electronics by synthetic biology are briefly introduced. Then, strategic approaches to designing and making electronic devices using living cells/viruses as the building blocks, sensing components, or power sources are reviewed. Finally, the challenges faced by living synthelectronics are raised. It is believed that this paradigm shift will significantly contribute to the real integration of bioelectronics with human tissues.

Engineering functional materials through bacteria-assisted living grafting.

Functionalizing materials with biomacromolecules such as enzymes has broad applications in biotechnology and biomedicine. Here, we introduce a grafting method mediated by living cells to functionalize materials. We use polymeric scaffolds to trap engineered bacteria and micron-sized particles with chemical groups serving as active sites for grafting. The bacteria synthesize the desired protein for grafting and autonomously lyse to release it. The released functional moieties are locally grafted onto the active sites, generating the materials engineered by living grafting (MELGs). MELGs are resilient to perturbations because of both the bonding and the regeneration of functional domains synthesized by living cells. The programmability of the bacteria enables us to fabricate MELGs that can respond to external input, decompose a pollutant, reconstitute synthetic pathways for natural product synthesis, and purify mismatched DNA. Our work establishes a bacteria-assisted grafting strategy to functionalize materials with a broad range of biological activities in an integrated, flexible, and modular manner. A record of this paper's transparent peer review process is included in the supplemental information.

Self-lysis microbial consortia for predictable multi-proteins assembly.

The scope of bioengineering is expanding from the design of single strain to the microbial communities, allowing for the division-of-labor in synthesizing the multi-protein systems. Predicting the composition of the final product during the biomanufacturing process, however, can be difficult. Consortia-based manufacturing has the potential to boost production efficiency, but this benefit primarily holds in the upstream. The current format of downstream process heavily relies on the centralized facility, and is not economical and flexible to address the demands in small-scale. Here, we present a concise and manageable platform to enable the multi-protein system assembly. We engineer a self-lysis microbial consortium, where each strain lyses autonomously at high densities and produces a single protein component. The product fraction can be precisely tuned by varying the inoculation ratio. Utilizing this platform, we assemble a classical 34-component PURE (protein synthesis using recombinant elements) system. We have further optimized the downstream process of the biomanufacturing by incorporating the porous structure of polymeric materials. The encapsulated autolysis consortium can produce and release the proteins while maintaining the cell factories enclosed in the materials by exporting the multi-protein system for collection. Our research provides a novel approach to the flexible and controllable production of multi-protein systems, opening up new possibilities for pathway assembly and portable biomanufacturing.

The regulation of liquid-liquid phase separated condensates containing nucleic acids.

Liquid-liquid phase separation (LLPS) has been recognized as a universal biological phenomenon. It plays an important role in life activities. LLPS is induced by weak interactions between intrinsically disordered regions or low complex domains. Nucleic acids are widely present in cells, and shown to be closely related to LLPS. Their structure and electronegativity provide the excellent platforms for the formation of phase-separated condensates. In this review, we summarize the interconnected regulation between nucleic acids and LLPS demonstrated in in vivo and in vitro studies. Beside homogeneous and single-phase condensates, complicated and multicompartment LLPS induced by nucleic acids is discussed as well. Recent advances about nucleic-acid-induced LLPS as a new pathogenic mechanism and drug design direction are highlighted, especially virus-mediated disease treatment and prevention.

[Engineering microbial consortia through synthetic biology approach].

There are a large number of natural microbial communities in nature. Different populations inside the consortia expand the performance boundary of a single microbial population through communication and division of labor, reducing the overall metabolic burden and increasing the environmental adaptability. Based on engineering principles, synthetic biology designs or modifies basic functional components, gene circuits, and chassis cells to purposefully reprogram the operational processes of the living cells, achieving rich and controllable biological functions. Introducing this engineering design principle to obtain structurally well-defined synthetic microbial communities can provide ideas for theoretical studies and shed light on versatile applications. This review discussed recent progresses on synthetic microbial consortia with regard to design principles, construction methods and applications, and prospected future perspectives.

Engineering Photosynthetic Microbial Consortia for Carbon-Negative Biosynthesis.

Microbial consortia consisting of phototrophs and heterotrophs have raised extensive attention due to their potential in sustainable biotechnology. The challenge remains in the selection of appropriate partners since most heterotrophic microorganisms cannot naturally use the intermediate carbohydrates secreted by autotrophic partners. In a recent study, the Ni Lab has developed a highly compatible autotrophic-heterotrophic symbiotic system comprising Synechococcus elongatus and Vibrio natriegens. V. natriegens (the sucrose utilization module) shows a high degree of nutritional complementarity and culturing compatibility with the engineered S. elongatus (the CO2 sequestration module). The combination of both species channels CO2 into various valuable chemicals, enabling carbon-negative biosynthesis.

Phosphorylation and specific DNA improved the incorporation ability of p53 into functional condensates.

The transcription factor p53 acted as a critical tumor suppressor by activating the expression of various target genes to regulate diverse cellular responses. The phosphorylation of p53 influenced the binding of p53 to promotor-specific DNA and the choice of cell fate. In this study, we found that full-length wild-type p53 and pol II CTD could form heterotypic phase separation condensates in vitro. The heterotypic condensates of p53 and pol II CTD were mediated by electrostatic and hydrophobic interactions between pol II CTD and multiple domains of p53. The mobility of heterotypic p53 and pol II CTD droplets was significantly higher than that of p53 droplet. The phosphorylation promoted p53 to be recruited into pol II CTD droplets and transcription condensates. The specific DNA could further enhance the incorporation ability of p53 into functional condensates. Therefore, we proposed that the p53 droplet might be in a mediate state, the mutations resulting in p53 mutants with gain-of-function impelled the aggregate of p53, while the phosphorylation promoted p53 to be recruited into functional condensates as a client molecule to exert its function. This study might provide insights into the regulation mechanism that the phosphorylation and nuclei acid affected the phase behavior of p53.

On-demand biomanufacturing through synthetic biology approach.

Biopharmaceuticals including protein therapeutics, engineered protein-based vaccines and monoclonal antibodies, are currently the mainstay products of the biotechnology industry. However, the need for specialized equipment and refrigeration during production and distribution poses challenges for the delivery of these technologies to the field and low-resource area. With the development of synthetic biology, multiple studies rewire the cell-free system or living cells to impact the portable, on-site and on-demand manufacturing of biomolecules. Here, we review these efforts and suggest future directions.

Engineered Living Materials For Sustainability.

Recent advances in synthetic biology and materials science have given rise to a new form of materials, namely engineered living materials (ELMs), which are composed of living matter or cell communities embedded in self-regenerating matrices of their own or artificial scaffolds. Like natural materials such as bone, wood, and skin, ELMs, which possess the functional capabilities of living organisms, can grow, self-organize, and self-repair when needed. They also spontaneously perform programmed biological functions upon sensing external cues. Currently, ELMs show promise for green energy production, bioremediation, disease treatment, and fabricating advanced smart materials. This review first introduces the dynamic features of natural living systems and their potential for developing novel materials. We then summarize the recent research progress on living materials and emerging design strategies from both synthetic biology and materials science perspectives. Finally, we discuss the positive impacts of living materials on promoting sustainability and key future research directions.

Engineering living materials by synthetic biology.

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.

Engineering consortia by polymeric microbial swarmbots.

Synthetic microbial consortia represent a new frontier for synthetic biology given that they can solve more complex problems than monocultures. However, most attempts to co-cultivate these artificial communities fail because of the winner-takes-all in nutrients competition. In soil, multiple species can coexist with a spatial organization. Inspired by nature, here we show that an engineered spatial segregation method can assemble stable consortia with both flexibility and precision. We create microbial swarmbot consortia (MSBC) by encapsulating subpopulations with polymeric microcapsules. The crosslinked structure of microcapsules fences microbes, but allows the transport of small molecules and proteins. MSBC method enables the assembly of various synthetic communities and the precise control over the subpopulations. These capabilities can readily modulate the division of labor and communication. Our work integrates the synthetic biology and material science to offer insights into consortia assembly and serve as foundation to diverse applications from biomanufacturing to engineered photosynthesis.

Loops constructing the substrate-binding site controlled the catalytic efficiency of Thermus thermophilus SG0.5JP17-16 laccase.

Laccase (EC1.10.3.2) belongs to the family of multicopper oxidases. It oxidazes various substrates and uses the dioxygen as an electron acceptor, the only by-product is the water, thus, laccase was considered as "green catalysts" in the biotechnology field. In this work, the function of amino acid residues D357 and K430 in the loops 5 and 7 of Thermus thermophilus SG0.5JP17-16 laccase (lacTT) was studied. The residues D357 and K430 were mutated into methionine by site-directed mutagenesis. Kinetic assays revealed that the catalytic efficiency of D357M and K430M mutants was 1.9, 1.8 times higher than that of the wild type enzyme, respectively. Double mutant D357MK428M was also examined, the catalytic efficiency of D357MK428M decreased by 24% as compared with that of the wild type lacTT. We also examined the decolorization ability of D357M, K430M and D357MK428M for four industrial dyes, and found that D357M, K430M and D357MK428M mutants took less time than lacTT when the same degree of decolorization was achieved for three azo dyes, Congo Red, Reactive Black WNN (RBWNN), Reactive Black B (RBB) within 3 h. For the anthraquinone dye Remazol Brilliant Blue R, the decolorization ability of D357M, K430M was improved significantly. Within 24 h, as compared with that of the wild type enzyme, the decolorization efficiency of D357M and K430M mutants was enhanced by 21.3% and 21.6%, respectively, while the decolorization efficiency of D357MK428M mutant decreased by 15.2%.

Ser392 phosphorylation modulated a switch between p53 and transcriptional condensates.

Human p53 is a transcription factor regulating the transcription of a variety of target genes. Under various stresses, its tumor suppressor function was activated by the phosphorylation of p53. In this study, we found that full-length wild-type p53 could form phase-separated condensates with the aggregation tendency in vitro and in vivo. The LLPS of p53 was regulated by multiple functional domains. Specific DNA could promote the formation of p53 condensates. Fluorescence recovery data after photobleaching revealed that the Ser392 phosphorylation enhanced the fluidity of p53 condensates. Fluorescence analysis suggested that Ser392 phosphorylation increased the p53 concentration in condensates involved in transcription initiation and the stability of p53-mediated transcriptional condensates. The experiments in cells showed that p53 was evenly dispersed in the nucleus, it formed the dynamic condensates under the UV radiation-induced DNA damage, and the Ser392 nonphosphorylatable mutant S392A p53 formed condensates with significantly reduced number and size. These findings revealed that p53 phosphorylation modified its LLPS behavior, and suggested a mechanism that phosphorylation regulated condensate preference.

Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology.

Inteins are protein segments that are capable of enabling the ligation of flanking extein into a new protein, a process known as protein splicing. Since its discovery, inteins have become powerful biotechnological tools for applications such as protein engineering. In the last 10 years, the development in synthetic biology has further endowed inteins with enhanced functions and diverse utilizations. Here we review these efforts and discuss the future directions.

Programmable living assembly of materials by bacterial adhesion.

The field of engineered living materials aims to construct functional materials with desirable properties of natural living systems. A recent study demonstrated the programmed self-assembly of bacterial populations by engineered adhesion. Here we use this strategy to engineer self-healing living materials with versatile functions. Bacteria displaying outer membrane-anchored nanobody-antigen pairs are cultured separately and, when mixed, adhere to each other to enable processing into functional materials, which we term living assembled material by bacterial adhesion (LAMBA). LAMBA is programmable and can be functionalized with extracellular moieties up to 545 amino acids. Notably, the adhesion between nanobody-antigen pairs in LAMBA leads to fast recovery under stretching or bending. By exploiting this feature, we fabricated wearable LAMBA sensors that can detect bioelectrical or biomechanical signals. Our work establishes a scalable approach to produce genetically editable and self-healable living functional materials that can be applied in biomanufacturing, bioremediation and soft bioelectronics assembly.

Living fabrication of functional semi-interpenetrating polymeric materials.

Cell-mediated living fabrication has great promise for generating materials with versatile, programmable functions. Here, we demonstrate the engineering of living materials consisting of semi-interpenetrating polymer networks (sIPN). The fabrication process is driven by the engineered bacteria encapsulated in a polymeric microcapsule, which serves as the initial scaffold. The bacteria grow and undergo programmed lysis in a density-dependent manner, releasing protein monomers decorated with reactive tags. Those protein monomers polymerize with each other to form the second polymeric component that is interlaced with the initial crosslinked polymeric scaffold. The formation of sIPN serves the dual purposes of enhancing the mechanical property of the living materials and anchoring effector proteins for diverse applications. The material is resilient to perturbations because of the continual assembly of the protein mesh from the monomers released by the engineered bacteria. We demonstrate the adoption of the platform to protect gut microbiota in animals from antibiotic-mediated perturbations. Our work lays the foundation for programming functional living materials for diverse applications.

The catalytic properties of Thermus thermophilus SG0.5JP17-16 laccase were regulated by the conformational dynamics of pocket loop 6.

BACKGROUND: Laccase is one member of the blue multicopper oxidase family. It can catalyze the oxidation of various substrates. The Thermus thermophilus SG0.5JP17-16 laccase (lacTT) is thermostable, pH-stable, and high tolerance to halides, and can decolorize the synthetic dyes. In lacTT, the function of the loop 6 constructing the substrate-binding pocket wasn't clear. METHODS: The residues Asp394 and Asp396 located in loop 6, and were used to probe how the loop 6 influenced catalytic properties of the laccase. Site-directed mutagenesis was performed for two amino acids. Kinetic assay was utilized to characterize the catalytic efficiency of mutants. Mutants with different catalytic activities were used to decolorize the synthetic dyes to clarify the relationship between the catalytic efficiency and dye decolorization. Redox potential, structural and spectral analyses were performed to explain the differences in laccase activity between wild type and mutant enzymes. RESULTS: D394M, D394E and D394R mutants with the lower laccase activity displayed a decreased decolorization efficiency, while D396A, D396M and D396E mutant enzymes with higher catalytic efficiency decolorized the synthetic dye more efficiently than the wild type enzyme. CONCLUSIONS: The pocket loop 6 might experience a conformational dynamics. The D394 residue controlled this conformation change by amino acid interaction networks containing the D396 residue at the entrance of substrate channel. GENERAL SIGNIFICANCES: These studies may provide clues to improve the activity of the laccase for the better use in industrial applications, and/or contribute to further understanding the mechanism of laccase oxidation on the substrate.

Versatile biomanufacturing through stimulus-responsive cell-material feedback.

Small-scale production of biologics has great potential for enhancing the accessibility of biomanufacturing. By exploiting cell-material feedback, we have designed a concise platform to achieve versatile production, analysis and purification of diverse proteins and protein complexes. The core of our technology is a microbial swarmbot, which consists of a stimulus-sensitive polymeric microcapsule encapsulating engineered bacteria. By sensing the confinement, the bacteria undergo programmed partial lysis at a high local density. Conversely, the encapsulating material shrinks responding to the changing chemical environment caused by cell growth, squeezing out the protein products released by bacterial lysis. This platform is then integrated with downstream modules to enable quantification of enzymatic kinetics, purification of diverse proteins, quantitative control of protein interactions and assembly of functional protein complexes and multienzyme metabolic pathways. Our work demonstrates the use of the cell-material feedback to engineer a modular and flexible platform with sophisticated yet well-defined programmed functions.

Emerging strategies for engineering microbial communities.

From biosynthesis to bioremediation, microbes have been engineered to address a variety of biotechnological applications. A promising direction in these endeavors is harnessing the power of designer microbial consortia that consist of multiple populations with well-defined interactions. Consortia can accomplish tasks that are difficult or potentially impossible to achieve using monocultures. Despite their potential, the rules underlying microbial community maintenance and function (i.e. the task the consortium is engineered to carry out) are not well defined, though rapid progress is being made. This limited understanding is in part due to the greater challenges associated with increased complexity when dealing with multi-population interactions. Here, we review key features and design strategies that emerge from the analysis of both natural and engineered microbial communities. These strategies can provide new insights into natural consortia and expand the toolbox available to engineers working to develop novel synthetic consortia.

Robust, linear correlations between growth rates and ß-lactam-mediated lysis rates.

It is widely acknowledged that faster-growing bacteria are killed faster by ß-lactam antibiotics. This notion serves as the foundation for the concept of bacterial persistence: dormant bacterial cells that do not grow are phenotypically tolerant against ß-lactam treatment. Such correlation has often been invoked in the mathematical modeling of bacterial responses to antibiotics. Due to the lack of thorough quantification, however, it is unclear whether and to what extent the bacterial growth rate can predict the lysis rate upon ß-lactam treatment under diverse conditions. Enabled by experimental automation, here we measured >1,000 growth/killing curves for eight combinations of antibiotics and bacterial species and strains, including clinical isolates of bacterial pathogens. We found that the lysis rate of a bacterial population linearly depends on the instantaneous growth rate of the population, regardless of how the latter is modulated. We further demonstrate that this predictive power at the population level can be explained by accounting for bacterial responses to the antibiotic treatment by single cells. This linear dependence of the lysis rate on the growth rate represents a dynamic signature associated with each bacterium-antibiotic pair and serves as the quantitative foundation for designing combination antibiotic therapy and predicting the population-structure change in a population with mixed phenotypes.