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December 19, 1:21 AM
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Protein secretion in mammalian cells is the active transport of proteins from the cytoplasm to the extracellular space. It plays a fundamental role in mammalian physiology and signaling, as well as biotherapeutics production and cell and gene therapies. The efficacy of protein secretion, however, is impacted by features of the secreted protein itself, and the host-cell machinery that supports each step of the secretion process. High-throughput techniques such as microfluidics, cell display, and cell encapsulation assays for the study and engineering of secreted proteins are transforming biomedical knowledge and our ability to modulate protein secretion. In addition, computational advances, including signal peptide modeling, whole-protein machine learning models, and genome-scale simulations, are opening new pathways for rational design of protein secretion. Here, we highlight recent developments in secretion engineering that are leading to the convergence of high-throughput experimentation and machine learning methods and can help address current challenges in bioproduction and support future efforts in cell and gene therapy while enabling new modalities.
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December 19, 1:15 AM
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Extracting fungal hyphae with their naturally associated microbiota from soil samples presents a significant challenge due to their small size, typically in the micrometer range, and the formation of dynamic fungal networks. We combined elements of previous protocols and automated the wet-sieving steps of the methodology to efficiently extract fungal hyphae from various soil types, including natural loamy soils. This approach reduces manual handling, minimizes operator-dependent variability, and shortens processing time by up to 2.5-fold. Unlike earlier methods that require sand or glass bead supplementation, which can introduce artificial conditions and limit large-scale field applications, our Sieving and Sucrose Centrifugation (SSC) method avoids these drawbacks. The SSC technique enables both quantification of hyphal length density (HLD) and, importantly, preserves surface-associated microbes for downstream analyses. Among the tested methods, SSC yielded the highest hyphal length density. Using a combination of microscopy, molecular techniques, and next-generation sequencing (NGS), we demonstrate that this method allows targeted study of bacteria tightly attached to fungal hyphae. Furthermore, the SSC approach effectively enriched fungal hyphae from a highly diverse soil community, establishing a dependable tool for advancing research on fungal hyphae as microbial hotspots in soil ecosystems.
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December 19, 12:22 AM
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Two-component signaling pathways allow bacteria to sense and respond to environmental changes, yet the sensory mechanisms of many remain poorly understood. In the pathogen Vibrio cholerae, the DbfRS TCS controls the biofilm lifecycle, a critical process for environmental persistence and host colonization. Here, we identified DbfQ, a small periplasmic protein encoded adjacent to dbfRS, as a direct modulator of pathway activity. DbfQ directly binds the sensory domain of the histidine kinase DbfS, shifting it toward phosphatase activity and promoting biofilm dispersal. In contrast, outer membrane perturbations, caused by mutations in lipopolysaccharide biosynthesis genes or membrane-damaging antimicrobials, activate phosphorylation of the response regulator DbfR. Transcriptomic analyses reveal that DbfR phosphorylation leads to broad transcriptional changes spanning genes involved in biofilm formation, central metabolism, and cellular stress responses. Constitutive DbfR phosphorylation imposes severe fitness costs in an infection model, highlighting this pathway as a potential target for anti-infective therapeutics. We find that dbfQRS-like genetic modules are widely present across bacterial phyla, underscoring their broad relevance in bacterial physiology. Collectively, these findings establish DbfQ as a new class of periplasmic regulator that influences TCS and bacterial adaptation. A TCS, DbfRS, regulates biofilm formation in Vibrio cholerae. Here, Nguyen et al. identify a small periplasmic protein that controls the activity of the system’s receptor, and show that DbfRS responds to membrane stress and regulates additional processes such as metabolism and cell envelope biosynthesis.
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December 19, 12:03 AM
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Antibiotic resistance stands as a formidable global challenge to public health. Herein, we present a bacterial nanoinducer (bacNID) designed for targeted protein degradation in treating bacterial infections. Specifically, bacNID is engineered by grafting targeting peptides of MurD and SspB onto gold nanoparticles (GNPs). MurD plays a pivotal role in peptidoglycan production for cell wall synthesis, while SspB recruits SsrA-tagged proteins for degradation by ClpXP protease. The effectiveness of bacNIDs in targeted MurD degradation via ClpXP is demonstrated across both Gram-positive and Gram-negative bacterial strains. Importantly, prolonged exposure to bacNIDs does not result in the acquisition of resistance in either Staphylococcus aureus (S. aureus) or Salmonella typhimurium (S. typhimurium), even after 25 successive treatment passages. This stands in stark contrast to the rapid emergence of robust resistance observed with norfloxacin, evidenced by a 243-fold reduction in antibacterial activity against S. aureus after just 15 passages, and a 7410-fold decrease in activity against S. typhimurium over 22 passages. Moreover, the antimicrobial potential of bacNIDs is evaluated in vivo using S. aureus-infected nonhealing skin and corneal wounds. In summary, this study unveils a potent nanotechnology-driven strategy for targeted bacterial protein degradation with promising implications for in vivo antimicrobial applications. In this work, authors present a nanoinducer (bacNID), which enables targeted bacterial protein degradation via ClpXP, leading to the effective treatment of bacterial infection (Staphylococcus aureus and Salmonella typhimurium) without inducing resistance.
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December 18, 11:35 PM
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Dairy waste, such as whey resulting from cheese production, is produced in massive volumes worldwide and is regarded as environmentally difficult to dispose of due to its high organic content. Harnessing the potential of this waste material to support the synthesis of valuable products such as hydrogen fuel or reduced graphene oxide, which may be utilized for conductive thin films and energy storage can reduce waste and add revenue streams for dairy farmers. Here, we demonstrate a circular bioeconomy using alginate-encapsulated co-cultures of Shewanella oneidensis together with lactic-acid-producing bacteria Klebsiella pneumoniae. These co-cultures can directly metabolize unprocessed cheese-making waste as an electron source instead of costly, environmentally high-impact lactic acid. Alginate-encapsulated co-cultures fed unprocessed dairy waste showed a 2-to-3-fold higher graphene oxide reduction rate compared to S. oneidensis monocultures with no supplemental electron source. Encapsulated co-cultures were able to be recycled for more than 30 days with no measurable decrease in graphene oxide reduction efficiency, showing compatibility with future industrial scaling. Photocatalytic hydrogen generation with cadmium selenide quantum dots as the catalyst resulted in 6-fold increases in hydrogen produced by co-cultures using milk as an electron source precursor for the system in comparison to S. oneidensis monocultures without any added electron sources. Thus, dairy waste may be processed to drive the synthesis of valuable products utilizing microbial electron transfer processes, converting a significant fluvial environmental pollutant into a valuable renewable energy resource that could provide a robust alternative revenue stream for dairy farmers in a volatile industry.
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December 18, 10:04 PM
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The production of lactic acid, a crucial platform chemical by microbial fermentation, is currently hindered by its low production efficiency. Herein, this study aims to enhance the synthetic efficiency of lactic acid in widely used Komagataella phaffii by reconfiguring the synthetic pathway and a metabolite damage-repair system. First, through the introduction of lactic acid dehydrogenase (BsLDH) from Bacillus subtilis and the transporter LutP from Bacillus coagulans, the titer of lactic acid was increased from 1.5 g/L in the original strain to 55.4 g/L using glucose. In particular, the titer was improved to 87.2 g/L by introducing metabolite damage-repair genes for NAD(P)HX detoxification and phosphate-based inhibitor elimination. In addition, the carbon source transport and metabolism pathway were strengthened, resulting in titers of 18.7 and 7.7 g/L from glycerol and methanol. Finally, the strains were scaled up in a 5 L bioreactor, achieving lactic acid titer values of 153.0, 133.2, and 37.4 g/L from glucose, glycerol, and methanol, respectively. This study significantly improved the yield of lactic acid production from low-cost carbon sources by microbial fermentation, demonstrating the potential of engineered K. phaffii for industrial production.
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December 18, 6:37 PM
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The detection of small molecules (e.g., pesticides, pollutants, antibiotics, drug residues) is critical for human health and environmental surveys as well as for many industrial applications. Fluorogenic RNA-based biosensors (FRBs) are aptamer-based tools specifically reporting on the presence of such targets by converting a specific binding event into fluorescence emission, thus making them of particular interest for on-site sensing applications. However, new sensor aptamers suited for FRB development are tedious to develop. In this study, we show that Capture-SELEX used in tandem with our microfluidic-assisted screening technology (μIVC) accelerates de novo FRB development by reprogramming the specificity of an existing sensor aptamer, followed by fluorescence-based functional selection. Furthermore, we show that these new sensor aptamers can be repurposed into functional biotechnological tools, such as gene-regulating aptazymes, thus demonstrating the versatility of our pipeline for the development of sensor aptamers adapted for various applications.
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December 18, 6:25 PM
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Synthetic biology has emerged as a rapidly advancing field, with its promising applications in human health attracting increasing attention. Engineered bacteria are designed according to synthetic biology principles to perform specific biological functions. In recent years, the application of engineered bacteria focuses on engineered live biotherapeutic products with the aim of improving health in vivo and there is increasing evidence supporting that these products possess huge application potential covering diagnosis, treatment and prevention. Compared with conventional host strains of engineered bacteria, Clostridium butyricum as a probiotic, has significant advantages including the ability to produce butyrate and the resistance to low pH, high temperature as well as the bile salts, which is an ideal host to be designed as a engineered bacterium. Despite these advantages, the application potential of engineered Clostridium butyricum has been long underestimated, primarily due to the challenges associated with genetic manipulation. However, with the advances of emerging genetic tools, it restrikes researchers’ attention. In this review, we first emphasized its advantages. Then we focused on the current research on its applications in the health field, highlighting its application potential, challenges and future directions in this promising field.
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December 18, 6:12 PM
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Bacterial chemotaxis is an important behavior to study to understand spatial segregation of species in mixed communities and the assembly of host microbiomes. This is particularly relevant in the rhizosphere, where chemoattraction toward root exudates is an important determinant of plant colonization. However, current methods to screen chemoeffectors are limited in their throughput, creating a barrier to generating comprehensive data sets describing chemotactic profiles for species of interest. Here, we describe a novel 3D-printed capillary tube holder approach, which facilitates up to 384 simultaneous capillary tube chemotaxis assays. We optimized and benchmarked our assay using Escherichia coli K12 and Bacillus subtilis 3610 with known chemoattractants: serine and aspartate. We then tested the threshold concentration of these chemoattractants using our assay and found that we could detect chemoattraction toward concentrations spanning multiple orders of magnitude. In this paper, we describe our high-throughput chemotaxis assay in detail and provide the necessary files for 3D printing the capillary tube holder.
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December 18, 4:52 PM
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Building a universal biochemical constructor, an autonomously self-replicating biochemical system, is a major challenge in synthetic biology. The PURE cell-free system is an ideal starting point for exploring self-regeneration, and its 36 non-ribosomal proteins constitute the primary macromolecular components that must be regenerated. Here, we demonstrate that the PURE system can be reconstituted from proteins synthesized by PURE itself. We first show that each of the 36 non-ribosomal proteins can be individually synthesized in PURE. We then purify the PURE synthesized proteins as pooled subsets and reconstitute a fully functional PURE system by combining the subsets. Finally, we show that all 36 non-ribosomal PURE proteins can be synthesized simultaneously in a single PURE reaction and, after purification, can reconstitute a functional PURE system. Together, these results establish that the non-ribosomal protein components of the PURE system can be self-regenerated, representing a critical step toward the realization of a universal biochemical constructor.
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December 18, 1:27 AM
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The growing demand for bio-based and biodegradable plastics has intensified interest in producing polyhydroxyalkanoates (PHAs). Short- and medium-chain-length (SCL-MCL)-PHA copolymers are particularly attractive because of their enhanced flexibility and desirable thermal properties. However, their high-level production from inexpensive carbon sources, such as glucose derived from lignocellulosic biomass, remains challenging, largely due to limited precursor supply and inefficient polymerization. Here, we report high-level de novo production of SCL-MCL-PHAs from glucose in metabolically engineered E. coli. We employed a modular metabolic engineering strategy comprising three modules: 1) construction of the 3-hydroxybutyryl-CoA monomer pathway; 2) enhancement of fatty acid biosynthesis to strengthen MCL-fatty acyl-CoA supply; and 3) screening of PHA synthases with broad substrate specificity. PHA synthase (PhaC) variants from Pseudomonas sp. MBEL 6–19 were identified to efficiently polymerize both SCL and MCL monomers. Fed-batch cultures of the engineered strains achieved two distinct outcomes: one strain produced 82.88 g L−1 PHA with 5 mol% MCL fraction, while another accumulated 17.35 g L−1 PHA with 19.52 mol% MCL fraction. Notably, these values fall within the 5–20 mol% MCL fraction range allowing good polymer applications, underscoring the industrial relevance of our results. This modular approach provides a versatile framework for tunable, sustainable production of SCL-MCL-PHAs.
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December 18, 1:13 AM
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Recently, engineered bacterial cells have been shown to behave as optically-active photonic devices comparable to industrially fabricated microlenses. Bacterial cells can be encapsulated within a layer of polysilicate through surface display of the sea sponge enzyme silicatein, which mineralizes a polysilicate coating. The addition of this polysilicate layer significantly enhances the ability of these cells to guide, scatter, and focus light. However, this previous technique was limited to creating rod-shaped microlenses, which are not ideal for all applications. Here we expand upon this technology by engineering the shapes of silicatein-displaying bacterial cells. Through the overexpression of the genes bolA and sulA or through the use of the drug A22, we are able to alter Escherichia coli cells from their characteristic rod-like shape to either spherical or filamentous forms. Round cells encapsulated in polysilicate were shown to scatter light more intensely and symmetrically than rod-shaped cells, while encapsulated filamentous cells were shown to guide light similarly to an optical fiber. This control over the size and shape of optically-active cells is a major advancement towards developing bio-engineered photonic devices such as nanophotonic waveguides, spherical microlens arrays, and advanced biosensors.
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December 18, 1:01 AM
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Establishing synthetic microbial consortia in competitive environments is often compromised by stochastic colonization bottlenecks, where founder effects lead to the unpredictable dominance of a single strain. Here, we overcome this challenge by engineering a differentiation abacus, a scalable, single-layer recombinase architecture that enables a single progenitor cell to differentiate into up to twelve distinct subpopulations. By arranging competitive excision sites in a linear array, we demonstrate that differentiation ratios can be programmed through rationally tuning recombination-site kinetics and inter-site spacing. This architecture allows the generation of strictly mutually exclusive phenotypes with tunable composition, scaling from simple two-state systems to complex multi-state ensembles without the need for multilayered regulation. Finally, we validate the utility of the system in a mouse tumor model, showing that in situ differentiation establishes robust, homogeneous consortia that overcome the colonization variability associated with pre-assembled mixtures. This work provides a versatile and scalable framework for reliably controlling consortia composition for bioproduction, synthetic ecology, and engineered living therapies.
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December 19, 1:19 AM
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Under severe nutrient-limiting conditions, Bacillus subtilis is able to form highly resilient endospores for survival. However, to avoid this irreversible process, it employs an adaptive strategy termed cannibalism, a form of programmed cell death, to outcompete siblings and delay sporulation. One of the three cannibalism toxins, the epipeptide EPE, is encoded by the epeXEPAB operon. The pre-pro-peptide EpeX undergoes post-translational modification and processing to be secreted as the mature EPE toxin. While EPE production is tightly regulated at multiple levels, this study focuses on the post-transcriptional control by the small regulatory RNA FsrA, which is transcriptionally regulated by the global iron response regulator Fur. Electrophoretic mobility shift assays and RNA structure probing revealed two binding sites of FsrA within the intergenic region between epeX and epeE flanking the annotated epeX terminator structure and potentially interfering with RNA stability and epeXEP expression. Reporter assays revealed decreased levels of EPE-dependent stress response in the absence of FsrA, indicative of a positive FsrA effect on gene expression under iron-limited conditions; in contrast to the normally inhibitory activity of FsrA. Together, our findings suggest that under iron starvation, FsrA promotes RNA processing and enables epeE translation, ultimately enhancing EPE production.
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December 19, 12:30 AM
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Base editors enable precise genome modification but are constrained by bystander edits that limit their applicability. Existing strategies to enhance precision often compromise efficiency and remain highly sequence dependent. Here we present a parallel engineering approach that optimizes both guide RNAs and the deaminase enzyme to minimize bystander editing without sacrificing activity. We designed a library of 3′-extended guide RNAs and identified context-dependent variants that improved specificity. Using a precision-driven phage-assisted evolution system and protein language models, we evolved adenine base editor variants two- to threefold more precise than adenine base editor ABE8e while maintaining high efficiency across a library of thousands of human pathogenic contexts in vitro. Our findings establish a scalable framework for precision engineering of base editors, addressing a major challenge in genome editing. Base editors are made more precise through deaminase and gRNA engineering.
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December 19, 12:12 AM
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Riboswitches are structured allosteric RNA molecules that change conformation upon metabolite binding, triggering a regulatory response. Here we focus on the de novo design of riboswitch-like aptamers, the core part of the riboswitch undergoing structural changes. We use Restricted Boltzmann machines (RBM) to learn generative models from homologous sequence data. We first verify, on four different riboswitch families, that RBM-generated sequences correctly capture the conservation, covariation and diversity of natural aptamers. The RBM model is then used to design new SAM-I riboswitch aptamers. To experimentally validate the properties of the structural switch in designed molecules, we resort to chemical probing (SHAPE and DMS), and develop a tailored analysis pipeline adequate for high-throughput tests of diverse sequences. We probe a total of 476 RBM-designed and 201 natural sequences. Designed molecules with high RBM scores, with 20% to 40% divergence from any natural sequence, display ≈ 30% success rate of responding to SAM with a structural switch similar to their natural counterparts. We show how the capability of the designed molecules to switch conformation is connected to fine energetic features of their structural components. Riboswitches are allosteric RNA molecules that change conformation upon ligand binding to regulate downstream genes. Here, the authors use Restricted Boltzmann machines trained on natural sequences to design new riboswitch aptamer domains, and validate their functionality via chemical probing.
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December 18, 11:48 PM
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With some exceptions, wine fermentation has increasingly relied on the use of Saccharomyces cerevisiae starters since the last decades of the past century. However, there is growing interest on the understanding of spontaneous wine fermentation, as well as on the use of complementary non-Saccharomyces wine starters. This in turn raises the question of the importance of interspecific interactions in winemaking and the underlying mechanisms. An important question about these interspecies recognition mechanisms is whether or not it is mediated by physical contact between yeast cells. To address this topic, different laboratories have developed diverse devices to cultivate at least two yeast species in the same growth medium without cell-to-cell contact between them. In this work, we compared four of the most popular systems and found that one of them (twin-bottles exchange through a flat membrane) showed very limited metabolite exchange. Among the other systems (Transwell, dialysis tube, or active exchange through hollow fiber cross-flow filtration devices), each one showed specific characteristics that made them more or less suitable, depending on the objectives of each experiment. The option showing the best versatility and efficiency was the use of active exchange. Our results highlight the importance of carefully characterizing the compartmentalization system when drawing conclusions about the impact of cell-to-cell contact in fermentation experiments.
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December 18, 11:30 PM
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Anaerobic ammonium oxidizing bacteria in the class “Candidatus Brocadiia” in the Planctomycetota are the only known group of bacteria capable of producing energy by coupling the oxidation of ammonium to the reduction of nitrite within a unique bacterial organelle called the anammoxosome. Due to the lack of homologs in other species, it is hypothesized that the key enzyme in this process, the hydrazine synthase complex, originated by de novo birth. We performed extensive searches for proteins that exhibited similarity in sequence and structure to the hydrazine synthase subunits and identified distantly related homologs in anaerobic bacteria from the phyla Planctomycetota and Desulfobacterota. However, key residues of importance for the enzymatic function were not conserved, rejecting the hypothesis that the identified genes represent previously unrecognized anammox bacteria. Phylogenetic analyses indicate that the anammox pathway has been assembled from genes acquired by horizontal gene transfer from a variety of anaerobic bacteria. The ancestral states of enzymes in the hydroxylamine oxidoreductase family were inferred, and transitions between reductive and oxidative forms of the enzymes were mapped onto the phylogenetic tree. Finally, it is shown that the signal sequences of key enzymes in the anammox pathway are able to transport a reporter gene into the periplasm of Escherichia coli cells. In conclusion, our findings suggest that the hydrazine synthase complex has evolved from already existing heme-binding periplasmic proteins and that the anammoxosome has an endogenous origin.
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December 18, 9:51 PM
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The impressive capabilities of natural pattern recognition systems have inspired their synthetic recreation for many chemical and biological applications. However, developing artificial receptors for pattern recognition is currently constrained by a laborious trial-and-error process within a limited selection space of synthetically generated molecules/materials. Here, we propose pattern recognition aptamers (PRAs)─a set of single-stranded nucleic acid ligands with quasi-specificity for multiple targets─that can be evolved through systematic exponential enrichment from a nucleic acid library for high-precision target identification. Our approach allows for the reliable generation of customized artificial receptors over a limited number of selection rounds. Using bacteria as model analytes, we developed 9 PRAs targeting 15 common bacteria through 3 rounds of evolutionary screening, achieving an identification accuracy of 98.5% in blinded unknown bacterial identification. This approach provides a generalized pipeline for creating customized pattern recognition arrays, supporting their potential to meet rapidly increasing application demands.
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December 18, 6:31 PM
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The convergence of synthetic biology and biosensor technology is driving a scientific and technological revolution in biotechnology. Characterized by its interdisciplinary nature, synthetic biology applies engineering principles to design and construct novel biological components and systems, significantly advancing biosensor development. As a key interface between the digital and biomolecular worlds within synthetic biology, cell-free biosensors (CFBs) leverage core advantages─including rapid prototyping, high controllability, excellent biosafety, and tolerance to potentially toxic substances─to demonstrate increasingly significant strategic value and broad application potential in numerous frontier fields. These fields encompass the precise design of biological circuits, intelligent optimization of metabolic pathways, and efficient in vitro diagnostics. CFBs overcomes limitations inherent in traditional cell-based sensors, offering a powerful platform for instant, on-demand biomolecular detection. This review aims to explore the innovative potential unlocked by the integration of synthetic biology and biosensors and to systematically summarize the latest research progress and technological breakthroughs in the CFB field.
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December 18, 6:19 PM
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Vibrio parahaemolyticus is a significant marine pathogen causing gastroenteritis and wound infections in humans. Its pathogenicity is mediated by key virulence factors, including the type VI secretion system (T6SS). This review comprehensively synthesizes current knowledge on the functional divergence and regulatory networks of T6SSs in V. parahaemolyticus, with emphasis on T6SS1 and T6SS2. T6SS1, enriched in clinical isolates, is activated under high-salt and warm conditions and primarily facilitates antibacterial competition and adhesion to human epithelial cells in strains such as RIMD2210633. T6SS2, nearly ubiquitous across strains, operates optimally under low-salt/low-temperature conditions and regulates adhesion, biofilm formation, motility, macrophage autophagy induction, and virulence as demonstrated in strains including RIMD2210633 and SH112. Both systems deploy diverse effectors (e.g., Tme1, PoNe, and RhsP) targeting membrane integrity, DNA, or peptidoglycan. Their expression is intricately controlled by environmental cues (e.g., salinity, temperature, and metal ions), stress responses (e.g., antibiotics, ethanol, and curcumin), quorum sensing regulators (e.g., AphA and OpaR), and transcriptional factors (e.g., H-NS, TfoY, and CalR). Strain-specific functional variations highlight the complexity of T6SS biology. Understanding these mechanisms offers insights for developing anti-virulence strategies against V. parahaemolyticus infections.
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December 18, 5:05 PM
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Phages are the most abundant biological entities on Earth and play central roles in bacterial evolution and the emergence of new pathogens. Many phages encode proteins that specifically inhibit host RNA polymerase activity, thereby sabotaging and, in some cases, hijacking the host transcription machinery to serve their needs. Identification and characterization of these transcription inhibitors not only provide insights into the logic of transcription regulation but also inspire the design of antibiotics targeting bacterial transcription. Traditional methods for identifying new phage proteins that inhibit bacterial transcription are labor-intensive and require access to live phages. To overcome these limitations, we developed a highly efficient pipeline for AlphaFold 3-guided discovery of phage proteins that inhibit bacterial transcription. Using this pipeline, three phage proteins were identified and characterized. Structural and biochemical analyses demonstrated that these phage proteins bind to distinct sites on RNA polymerase and inhibit transcription via unprecedented mechanisms. This study showcases the power of AlphaFold 3 in discovering novel binders of large protein complexes, and the pipeline developed here could be readily adapted to screen modulators of other large targets, such as the ribosome, proteasome, and CRISPR-Cas systems.
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December 18, 1:15 PM
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Bacterial nanocellulose (BC) from Komagataeibacter spp. is an ideal scaffold for biological Engineered Living Materials (ELMs). Engineering Komagataeibacter to simultaneously synthesize and functionalize BC could transform current workflows, but protein secretion in this organism remains poorly understood. Here, we demonstrate recombinant protein secretion in K. rhaeticus iGEM by leveraging its genome-encoded Sec-translocase. Using secretome analysis to identify native Sec signal peptides (SPs), secretion efficiencies of mScarlet and β-lactamase variant libraries were benchmarked under pellicle-forming and non-pellicle conditions. Inducible expression eliminated metabolic burden and enabled secretion without cell lysis, as confirmed by fluorescence and scanning electron microscopy. Comparative analyses revealed strong condition-dependent variations in secretion performance, with native SPs exhibiting higher efficiency under pellicle-forming conditions. Finally, BC functionalization using secreted β-lactamase was demonstrated, with no detrimental effects on the BC material. This work establishes the first comparative framework for SP-mediated secretion in Komagataeibacter and provides a foundation for next-generation BC-based bioELMs.
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December 18, 1:24 AM
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Pathogens that are harmless in one environment can cause serious disease in another. Among host-associated bacteria, transition between hosts can have serious consequences for animal and human health. However, much remains unknown about how adaptation shapes bacterial distribution in the wild. Here, investigating the ecological genomics of Escherichia coli from diverse hosts and environments, we address the idea that bacteria disperse freely, and challenge the “everything is everywhere” paradigm. Using comparative genomics and parallelised high throughout pangenome-wide association studies (900 experiments) we investigate lineage distribution and identify adaptive genomic signatures associated with host species, physiology and ecology. Our findings provide insights into bacterial niche adaptation, emphasize the impact of agriculture on microbial evolution, and inform One Health frameworks by linking genomics, host ecology, and the emergence of antimicrobial resistance.
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December 18, 1:09 AM
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Cell-free biosensor systems offer a promising platform for portable diagnostics. However, most employ fluorescent reporter proteins that require complex instrumentation and can be affected by photo-bleaching and auto-fluorescence, limiting translatability. Electrochemical reporters do not suffer from these drawbacks. Here, we evaluate horseradish peroxidase (HRP) as a redox enzyme reporter for cell-free biosensor systems. HRP was synthesized in an E. coli cell-free transcription-translation system supplemented with hemin, calcium acetate, and commercial disulfide bond enhancers. The electrochemical detection of its activity was established by chronoamperometry, with H2O2 as a substrate and tetramethylbenzidine as a redox mediator. Cell-free expressed HRP produced a strong steady state current compared to a catalytically inactive mutant and a no-template control. Kinetic analysis showed a Km for the cell-free expressed HRP close to that of the native enzyme. To explore the potential of HRP as an electrochemical reporter, we placed it under the control of a tetracycline-responsive regulatory promoter and demonstrated a 3-fold current increase in the presence of anhydrotetracycline. These results support HRP as an electrochemical reporter for cell-free biosensors, offering a practical alternative to optical reporters for future use in handheld analytical devices.
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