Synthetic Biology Engineering Research Articles

Synthetic Biology in T-cell Engineering Research

Synthetic biology has emerged as a transformative discipline, enabling precise genetic and functional reprogramming of cellular systems. In T-cell engineering, it offers groundbreaking potential to revolutionize immunotherapy by endowing T cells with enhanced specificity, adaptability, and resilience against complex diseases such as cancer and autoimmune disorders. By integrating advanced genome-editing tools like CRISPR-Cas9 with modular synthetic constructs, researchers can design T cells with bespoke functionalities, such as tunable antigen recognition, controlled cytokine release, and resistance to immunosuppressive tumor microenvironments. This approach not only overcomes the limitations of conventional T-cell therapies but also facilitates the development of novel therapeutic paradigms, including "smart" cellular systems capable of sensing and responding to dynamic biological cues. Furthermore, synthetic circuits allow for the incorporation of logic-gated mechanisms to minimize off-target effects and enhance therapeutic precision. Despite these advancements, challenges remain in optimizing safety, scalability, and regulatory compliance. This research aims to explore the intersection of synthetic biology and T-cell engineering, highlighting cutting-edge methodologies, therapeutic applications, and emerging trends. By addressing current limitations and envisioning future possibilities, this work seeks to contribute to the growing body of knowledge driving synthetic biology toward clinical and industrial breakthroughs in cellular immunotherapy.

Genetically Engineered Liposwitch-Based Nanomaterials.

Fusion of intrinsically disordered and globular proteins is a powerful strategy to create functional nanomaterials. However, the immutable nature of genetic encoding restricts the dynamic adaptability of nanostructures postexpression. To address this, we envisioned using a myristoyl switch, a protein that combines allostery and post-translational modifications─two strategies that modify protein properties without altering their sequence─to regulate intrinsically disordered protein (IDP)-driven nanoassembly. A typical myristoyl switch, allosterically activated by a stimulus, reveals a sequestered lipid for membrane association. We hypothesize that this conditional exposure of lipids can regulate the assembly of fusion proteins, a concept we term "liposwitching". We tested this by fusing recoverin, a calcium-dependent myristoyl switch, with elastin-like polypeptide, a thermoresponsive model IDP. Biophysical analyses confirmed recoverin's myristoyl-switch functionality, while dynamic light scattering and cryo-transmission electron microscopy showed distinct calcium- and lipidation-dependent phase separation and assembly. This study highlights liposwitching as a viable strategy for controlling DP-driven nanoassembly, enabling applications in synthetic biology and cellular engineering.

Current Technologies in Snake Venom Analysis and Applications

This comprehensive review explores the cutting-edge advancements in snake venom research, focusing on the integration of proteomics, genomics, transcriptomics, and bioinformatics. Highlighting the transformative impact of these technologies, the review delves into the genetic and ecological factors driving venom evolution, the complex molecular composition of venoms, and the regulatory mechanisms underlying toxin production. The application of synthetic biology and multi-omics approaches, collectively known as venomics, has revolutionized the field, providing deeper insights into venom function and its therapeutic potential. Despite significant progress, challenges such as the functional characterization of toxins and the development of cost-effective antivenoms remain. This review also discusses the future directions of venom research, emphasizing the need for interdisciplinary collaborations and new technologies (mRNAs, cryo-electron microscopy for structural determinations of toxin complexes, synthetic biology, and other technologies) to fully harness the biomedical potential of venoms and toxins from snakes and other animals.

A Review of Antimicrobial Peptides: Structure, Mechanism of Action, and Molecular Optimization Strategies

Antimicrobial peptides (AMPs) are bioactive macromolecules that exhibit antibacterial, antiviral, and immunomodulatory functions. They come from a wide range of sources and are found in all forms of life, from bacteria to plants, vertebrates, and invertebrates, and play an important role in controlling the spread of pathogens, promoting wound healing and treating tumors. Consequently, AMPs have emerged as promising alternatives to next-generation antibiotics. With advancements in systems biology and synthetic biology technologies, it has become possible to synthesize AMPs artificially. We can better understand their functional activities for further modification and development by investigating the mechanism of action underlying their antimicrobial properties. This review focuses on the structural aspects of AMPs while highlighting their significance for biological activity. Furthermore, it elucidates the membrane targeting mechanism and intracellular targets of these peptides while summarizing molecular modification approaches aimed at enhancing their antibacterial efficacy. Finally, this article outlines future challenges in the functional development of AMPs along with proposed strategies to overcome them.

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model

Background and Objectives: The rapid spread of Newcastle disease (ND), driven by extensive commercial exchange in the poultry industry, necessitates urgent preventive measures. Although effective vaccines against the Newcastle disease virus (NDV) have been used since 1940, recent outbreaks and the limitations of current vaccines highlight the need for improved solutions. Advances in synthetic biology, reverse vaccinology, molecular biology, and recombinant DNA technology over the past 20 years have led to the development of recombinant vaccines, which offer enhanced protection and broader immuno- genic coverage against NDV. This study aimed to express the immunogenic domains of Hemagglutinin Neuraminidase (HN) and Fusion (F) glycoproteins, linked to the heat-labile enterotoxin B subunit (LTB) bio-adjuvant, to develop an effective and reliable recombinant vaccine for NDV. Materials and Methods: In this study, the L(HN)2F protein, composed of the LTB bio-adjuvant and the immunogenic regions of the doubled Hemagglutinin Neuraminidase (HN-HN) and Fusion (F) epitope, was expressed in Escherichia coli. Subcutaneous injection was used to evaluate the humoral immune response in mice and the result was compared with B1 vaccine. Results: The induction of strong humoral immune responses proved the strong immunoreactivity of the recombinant protein. Conclusion: The IgG elicited by the recombinant proteins was comparable to that of the commercial B1 vaccine against NDV, indicating its potential as a viable candidate for further development and evaluation as a recombinant vaccine against NDV.

Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.

Efficient Simulation of Viral Transduction and Propagation for Biomanufacturing.

The design of biomanufacturing platforms based on viral transduction and/or propagation poses significant challenges at the intersection between synthetic biology and process engineering. This paper introduces vitraPro, a software toolkit composed of a multiscale model and an efficient numeric technique that can be leveraged for determining genetic and process designs that optimize transduction-based biomanufacturing platforms and viral amplification processes. Viral infection and propagation for up to two viruses simultaneously can be simulated through the model, considering viruses in either the lytic or lysogenic stage, during batch, perfusion, or continuous operation. The model estimates the distribution of the viral genome(s) copy number in the cell population, which is an indicator of transduction efficiency and viral genome stability. The infection age distribution of the infected cells is also calculated, indicating how many cells are in an infection stage compatible with recombinant product expression or viral amplification. The model can also consider the presence of defective interfering particles in the system, which can severely compromise the productivity of biomanufacturing processes. Model benchmarking and validation are demonstrated for case studies of the baculovirus expression vector system and influenza A propagation in suspension cultures.

Enhancing fatty acid and omega-3 production in Schizochytrium sp. using developed safe-harboring expression system

BackgroundSchizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production.ResultsTransgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain.ConclusionThis study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.

Advances in Metabolic Engineering for the Accumulation of Astaxanthin Biosynthesis.

Astaxanthin, a lipophilic carotenoid renowned for itsstrong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application inmedicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longercompared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged asa promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use ofthe natural host Xanthophyllomyces dendrorhous, andthe heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed.

Biosafety risks, control, and biocontainment of engineered microalgae

Microalgae, with the ability to harness solar energy to fix CO2 and convert it into organic compounds, have emerged as promising green cell factories. With the rapid development of cutting-edge biotechnologies, the research and application of photosynthetic microalgae have been expanding, leading to comprehensive and in-depth engineering of microalgae. The synthetic biology and genome editing technologies have enabled the applications of microalgae in medicine, agriculture, food, energy, and the environment. However, the survival and spreading of engineered microalgae in the natural environment pose potential safety risks to ecosystems and human health. To curb the risks caused by the spreading of engineered microalgae in the environment, biosafety policies should be formulated for engineered microalgae and the prevention and control technologies should be developed. Toward this goal, researchers have developed biocontainment systems, including positive strategies such as the design of toxic protein-based kill switches and passive strategies such as knocking out essential genes to construct the strains with nutritional deficiencies, thereby spatially containing engineered microalgae. This article summarizes the application of cutting-edge biotechnologies in the engineering of microalgae, the biosafety risks and management regulations associated with the escape of engineered microalgae, and the progress in novel biocontainment technologies established for engineered microalgae. Finally, this article gives insights into the future development direction of microalgae biocontainment.

In vitro transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade.

In vitro metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing, and metabolic engineering. Yet, the analysis of the activity of such in vitro networks is very often restricted by time- and cost-intensive methods. To overcome these limitations, we sought to develop an in vitro transcription (IVT)-based biosensing workflow that is compatible with the complex conditions of in vitro metabolism, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a 27-component in vitro metabolic system that converts CO2 into glycolate. As proof of concept, we constructed a novel glycolate sensor module that is based on the transcriptional repressor GlcR from Paracoccus denitrificans and established an IVT biosensing workflow that allows us to quantify glycolate from CETCH samples in the micromolar to millimolar range. We investigate the influence of 13 (shared) cofactors between the two in vitro systems to show that Mg2+, adenosine triphosphate , and other phosphorylated metabolites are critical for robust signal output. Our optimized IVT biosensor correlates well with liquid chromatography-mass spectrometry-based glycolate quantification of CETCH samples, with one or multiple components varying (linear correlation 0.94-0.98), but notably at ∼10-fold lowered cost and ∼10 times faster turnover time. Our results demonstrate the potential and challenges of IVT-based systems to quantify and prototype the activity of complex reaction cascades and in vitro metabolic networks.

Recent Advances in the Biosynthesis of Mid- and Long-Chain Dicarboxylic Acids Using Terminally Oxidizing Unconventional Yeasts.

As high-performance monomers for the manufacture of polyamide materials, mid- and long-chain dicarboxylic acids (DCAi, i ≥ 6) have received extensive attention from researchers. Biosynthesis is gradually replacing chemical synthesis due to its outstanding advantages in the industrial production of mid- and long-chain dicarboxylic acids, which is mostly achieved by using the strong terminal oxidation ability of nonmodel microorganisms such as Candida tropicalis to oxidize hydrophobic substrates such as alkanes. Here, we first summarize the metabolic pathways of oxidative alkane conversion into dicarboxylic acid by terminally oxidizing unconventional yeasts and the corresponding metabolic engineering strategies. Then, we summarize the research progress on new dicarboxylic acid production processes. Finally, the future development directions in the biosynthesis of mid- and long-chain dicarboxylic acids are prospected from synthetic biology and bioprocess engineering, which can also provide a reference for the synthesis of other biobased chemicals and biomaterials.

Intracellular Construction of Organelle-like Compartments Facilitates Metabolic Flux in Escherichia coli.

The formation of well-designed synthetic compartments or membraneless organelles for applications in synthetic biology and cellular engineering has aroused enormous interest. However, establishing stable and robust intracellular compartments in bacteria remains a challenge. Here, we use the structured DIX domains derived from Wnt signaling pathway components, more specifically, Dvl2 and Axin1, as building blocks to generate intracellular synthetic compartments in Escherichia coli. Moreover, the aggregation behaviors and physical properties of the DIX-based compartments can be tailored by genetically embedding a specific dimeric domain into the DIX domains. Then, a pair of interacting motifs, consisting of the aforementioned dimeric domain and its corresponding binding ligand, was incorporated to modify the client recruitment pattern of the synthetic compartments. As a proof of concept, the human milk oligosaccharide lacto-N-tetraose (LNT) biosynthesis pathway was selected as a model metabolic pathway. The fermentation results demonstrated that the co-compartmentalization of sequential pathway enzymes into intracellular compartments created by DIX domain, or by the DIX domain in conjunction with interacting motifs, prominently enhanced the metabolic flux and increased LNT production. These synthetic protein compartments may provide a feasible and effective tool to develop versatile organelle-like compartments in bacteria for applications in cellular engineering and synthetic biology.

Integrated Review of Transcriptomic and Proteomic Studies to Understand Molecular Mechanisms of Rice's Response to Environmental Stresses.

Rice (Oryza sativa L.) is grown nearly worldwide and is a staple food for more than half of the world's population. With the rise in extreme weather and climate events, there is an urgent need to decode the complex mechanisms of rice's response to environmental stress and to breed high-yield, high-quality and stress-resistant varieties. Over the past few decades, significant advancements in molecular biology have led to the widespread use of several omics methodologies to study all aspects of plant growth, development and environmental adaptation. Transcriptomics and proteomics have become the most popular techniques used to investigate plants' stress-responsive mechanisms despite the complexity of the underlying molecular landscapes. This review offers a comprehensive and current summary of how transcriptomics and proteomics together reveal the molecular details of rice's response to environmental stresses. It also provides a catalog of the current applications of omics in comprehending this imperative crop in relation to stress tolerance improvement and breeding. The evaluation of recent advances in CRISPR/Cas-based genome editing and the application of synthetic biology technologies highlights the possibility of expediting the development of rice cultivars that are resistant to stress and suited to various agroecological environments.

Degradable living plastics programmed by engineered spores.

Plastics are widely used materials that pose an ecological challenge because their wastes are difficult to degrade. Embedding enzymes and biomachinery within polymers could enable the biodegradation and disposal of plastics. However, enzymes rarely function under conditions suitable for polymer processing. Here, we report degradable living plastics by harnessing synthetic biology and polymer engineering. We engineered Bacillus subtilis spores harboring the gene circuit for the xylose-inducible secretory expression of Burkholderia cepacia lipase (BC-lipase). The spores that were resilient to stresses during material processing were mixed with poly(caprolactone) to produce living plastics in various formats. Spore incorporation did not compromise the physical properties of the materials. Spore recovery was triggered by eroding the plastic surface, after which the BC-lipase released by the germinated cells caused near-complete depolymerization of the polymer matrix. This study showcases a method for fabricating green plastics that can function when the spores are latent and decay when the spores are activated and sheds light on the development of materials for sustainability.

CILF: CRISPR/Cas9 based integration of large DNA fragments in Saccharomyces cerevisiae.

Genome integration technology has markedly expedited the construction of cell factories. However, its application is currently limited by the inefficient integration of large DNA fragments. Here, we report a CRISPR/Cas9 based integration of large DNA fragments (CILF) method to efficiently integrate large DNA fragments in Saccharomyces cerevisiae. In this approach, a fusion protein, Cas9-Brex27-FadR, was employed for the targeted delivery of donor plasmid to double-strand breaks (DSBs), while simultaneously recruiting Rad51 to enhance the efficiency of homologous recombination (HR). Our findings demonstrate that this method can achieve an integration efficiency of 98% for 10 kb DNA fragments and nearly 80% for 40 kb DNA fragments at a single site, using donor plasmids with 1000 bp homology arms (HAs) and 12 FadR binding sites (BSs). The CILF technique significantly enriches the synthetic biology toolbox of S. cerevisiae, offering significant potential to propel advancements in both synthetic biology and metabolic engineering.

Systematic metabolic engineering enables highly efficient production of vitamin A in Saccharomyces cerevisiae

Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food, cosmetics, pharmaceutical, and nutraceutical industries. Synthetic biology and metabolic engineering enable microbes, especially the model organism Saccharomyces cerevisiae (generally recognised as safe) to possess great potential for the production of vitamin A. Herein, we first generated a vitamin A-producing strain by mining β-carotene 15,15′-mono(di)oxygenase from different sources and identified two isoenzymes Mbblh and Ssbco with comparable catalytic properties but different catalytic mechanisms. Combinational expression of isoenzymes increased the flux from β-carotene to vitamin A metabolism. To modulate the vitamin A components, retinol dehydrogenase 12 from Homo sapiens was introduced to achieve more than 90 % retinol purity using shake flask fermentation. Overexpressing POS5Δ17 enhanced the reduced nicotinamide adenine dinucleotide phosphate pool, and the titer of vitamin A was elevated by almost 46 %. Multi-copy integration of the key rate-limiting step gene Mbblh further improved the synthesis of vitamin A. Consequently, the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level. Eventually, the highest reported titer of 5.21 g/L vitamin A in S. cerevisiae was achieved in a 1-L bioreactor. This study unlocked the potential of S. cerevisiae for synthesising vitamin A in a sustainable and economical way, laying the foundation for the commercial-scale production of bio-based vitamin A.

Review of the Proteomics and Metabolic Properties of Corynebacterium glutamicum.

Corynebacterium glutamicum (C. glutamicum) has become industrially important in producing glutamic acid and lysine since its discovery and has been the subject of proteomics and central carbon metabolism studies. The proteome changes depending on environmental conditions, nutrient availability, and stressors. Post-translational modification (PTMs), such as phosphorylation, methylation, and glycosylation, alter the function and activity of proteins, allowing them to respond quickly to environmental changes. Proteomics techniques, such as mass spectrometry and two-dimensional gel electrophoresis, have enabled the study of proteomes, identification of proteins, and quantification of the expression levels. Understanding proteomes and central carbon metabolism in microorganisms provides insight into their physiology, ecology, and biotechnological applications, such as biofuels, pharmaceuticals, and industrial enzyme production. Several attempts have been made to create efficient production strains to increase productivity in several research fields, such as genomics and proteomics. In addition to amino acids, C. glutamicum is used to produce vitamins, nucleotides, organic acids, and alcohols, expanding its industrial applications. Considerable information has been accumulated, but recent research has focused on proteomes and central carbon metabolism. The development of genetic engineering technologies, such as CRISPR-Cas9, has improved production efficiency by allowing precise manipulation of the metabolic pathways of C. glutamicum. In addition, methods for designing new metabolic pathways and developing customized strains using synthetic biology technology are gradually expanding. This review is expected to enhance the understanding of C. glutamicum and its industrial potential and help researchers identify research topics and design studies.

High Throughput Screening of Electroactive Bacteria Using a Cross-Bar Architecture in a 3D Design Array

Promoting extracellular electron transfer (EET) in bacteria has many widespread applications including wastewater treatment and environmental remediation. With the development of synthetic biology technologies that can alter microbial electron transfer routes and enhance their electrogenic capacity, there is a need for high throughput systems to identify the responsible genes and the consequent metabolic pathways in an expeditious way. Conventional platforms utilizing single electrodes, or electrochemical cells, and colorimetric detection suffer from low sensitivity and low throughput. Moreover, they require bulky equipment for readout and fluid handling. Moving towards a nuanced miniaturized electrochemical detection, we propose individually addressable microwells used to monitor electron flux from EET-capable bacteria with the ability to screen a large number of electroactive bacteria for various applications. Materials and method : The device is simple and fabricated with cost-effective electrodes. Carbon felt is used as the working electrode to improve bacterial entrapment and assist in capturing maximum electrons donated by the electroactive bacteria. Silver-silver chloride ink (Ag/AgCl) as the reference electrode will help maintain the potential of with respect to the working electrode. Conductive carbon ink as the counter electrode will promote current collection. The proposed device has the elements of a traditional three electrode system arranged in two different planes resulting in a 3D configuration of electrodes. The reference and counter electrode are on the bottom plane with acrylic wells housed right above each electrode pair, while the working electrode is used in the cross-bar architecture from the top plane as shown in Figure 1(a). The fabrication steps are illustrated in Figure 1(b). This system can be scaled to have 10000 microwells on a single platform with individual addressability of each micro-well array. With this layout, connections from the innermost wells can be drawn effortlessly without any signal damping from the surrounding wells. As a proof of concept, we have fabricated a 9 by 9 array (20cm by 20 cm) as shown in Figure 1(a). Five strains of Shewanella oneidensis MR-1, the wild type organism and four modified strains overexpressing variants of MtrA (unmodified MtrA, IV-205, IV-261 and an empty vector), were screened for their current producing capacity [1]. Results:A bacterial load of OD600= 0.065 was sufficient to give a reliable current signal. For each strain, the working electrode was biased at 0.205 V with respect to the reference electrode (Ag/AgCl), and the resulting chronoamperometry signal was recorded as shown in Figure 1(c). The values obtained helped identify the current-producing capacity of each mutant. Among them, the highest peak current was given by mtr A+ (20 μA), and IV – 261 gave a low peak current (2 μA) within 3 minutes, validating the difference in the genetic make-up and EET capabilities. Technical and biological replicates were also conducted for all the 5 strains. The average standard deviation for the technical and biological replicates (n=3, OD600=0.065) was 0.09 μA and 0.56 μA respectively. A similar current trend for the same set of strains was obtained by Ian et al., [1] using a traditional bioelectrochemical system, thus validating our platform’s functionality and integrity. This system allows for parallel screening of wild type and mutant variations of multiple electroactive bacteria as demonstrated. The high throughput feature enabled by this device can also find application to characterize mutants generated by directed evolution workflows. Additionally, mechanical and electrical multiplexing can further improve the electronic instrumentation and connections between the wells for parallel readout and will also minimize human intervention during the current measurements. Chronoamperometry measurements is an important technique to characterize EET. Thus, a low-cost system like ours will help do that in a shorter time frame with minimal bacterial inoculum and for a larger number of electroactive bacterial species. In conclusion, we aim to show that a 3D carbon felt platform is miniaturized, and can be scaled to understand and characterize EET from electroactive bacteria in a high throughput manner. References Ian J. Campbell, Joshua T. Atkinson, Matthew D. Carpenter, Dru Myerscough, Lin Su, Caroline M. Ajo-Franklin, and Jonathan J. Silberg Biochemistry 2022 61 (13), 1337-1350 Figure 1

Thermo-responsive aqueous two-phase system for two-level compartmentalization

Hierarchical compartmentalization responding to changes in intracellular and extracellular environments is ubiquitous in living eukaryotic cells but remains a formidable task in synthetic systems. Here we report a two-level compartmentalization approach based on a thermo-responsive aqueous two-phase system (TR-ATPS) comprising poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX). Liquid membraneless compartments enriched in PNIPAM are phase-separated from the continuous DEX solution via liquid-liquid phase separation at 25 °C and shrink dramatically with small second-level compartments generated at the interface, resembling the structure of colloidosome, by increasing the temperature to 35 °C. The TR-ATPS can store biomolecules, program the spatial distribution of enzymes, and accelerate the overall biochemical reaction efficiency by nearly 7-fold. The TR-ATPS inspires on-demand, stimulus-triggered spatiotemporal enrichment of biomolecules via two-level compartmentalization, creating opportunities in synthetic biology and biochemical engineering.