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bioRxiv2024

Ultrahigh yields of giant vesicles obtained through mesophase evolution and breakup

Self-assembly odry amphiphilic lipid films on surfaces upon hydration is a crucial step in the formation of cell-like giant unilamellar vesicles (GUVs). GUVs are useful as model biophysical systems, as chassis for synthetic biology, and in biomedical applications. Here via combined quantitative measurements of the molar yield and distributions of sizes and high-resolution imaging of the evolution of lipid films on surfaces, we report the discovery of a previously unknown pathway for the assembly of GUVs which can lead to ultrahigh yields of > 50 %. This yield is about 60 % higher than any GUV yield reported to date. The "shear-induced fragmentation" pathway occurs in membranes containing 3 mol % of the poly(ethylene glycol) modified lipid PEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), when a lipid-dense foam-like mesophase forms upon hydration. The membranes in the mesophase fragment and close to form GUVs upon application of fluid shear. Experiments with varying mol % of PEG2000-DSPE and with lipids with partial molecular similarity to PEG2000-DSPE show that ultrahigh yields are only achievable under conditions where the lipid-dense mesophase forms. The increased yield of GUVs compared to mixtures without PEG2000-DSPE was general to other flat supporting surfaces such as stainless steel sheets and to various lipid mixtures. Since FDA-approved liposomal and lipid nanoparticle formulations use PEG2000-DSPE, these results provide a useful route to obtaining ultrahigh yields of GUVs that are suitable for biomedical applications.

biophysics↗

bioRxiv2024

Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3

Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been hindered by challenges involved in their expression and extraction in a homogeneous, functionally-active form. The inherent enzymatic activity of receptor tyrosine kinases (RTKs) presents additional challenges. Oncogenic fusions of RTKs with heterologous partners represent a particularly difficult-to-express protein subtype due to their high flexibility, aggregation propensity and the lack of a known method for extraction within the native lipid environment. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 hours. The resultant protein was reconstituted using SMA copolymers with a specific yield of 300 {micro}g/mL of lysate. Functionally, the protein demonstrated significant kinase domain phosphorylation (t<0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins.

biochemistry↗

bioRxiv2024

TurboID-mediated surface protein biotinylation to inhibit the growth of Staphylococcus aureus

Staphylococcus aureus (S. aureus) infection is major cause of nosocomial infections. Antibiotic treatment for S. aureus remains the primary solution for managing S. aureus infections, which, however, increases the risk of antibiotic resistance. To broaden the resolutions on S. aureus infection, here we report TurboID-mediated protein proximity technologies to inhibit the growth of S. aureus. To achieve this goal, we utilized synthetic biology techniques to create a fusion protein named N-AgrD-TurboID (Agr-ID). The N-AgrD domain includes auto-inducer peptide (AIP) which combined to the surface AgrC protein on S. aureus. As such, TurboID then catalyzed the production of biotinoyl-5-AMP anhydride, triggering the biotinylation of surface proteins on S. aureus 25923 which were visualized by using fluorescence microscopy after incubating with Alexa Fluor 647-conjugated streptavidin. The biotinylation of surface protein on S. aureus 25923, S. aureus 43300, and S. aureus 6538 (MRSA) also resulted in growth inhibition and impaired colonization. Moreover, the biotinylation on surface protein further inhibited virulence protein production in S. aureus 25923, as indicated by reduced apoptosis of HEK 293T cells after treatment with S. aureus 25923 lysates. Overall, our work reveals that the biotinylation of surface proteins can inhibit the growth and toxicity of S. aureus 25923, S. aureus 43300, and S. aureus 6538 (MRSA), indicating therapeutic potential in clinical treatment.

microbiology↗

bioRxiv2024

Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

High-throughput DNA transformation techniques are invaluable when creating high-diversity mutant libraries, and the success rate of any protein engineering endeavors is directly dependent on both the size and diversity of the mutant library that is to be screened. It is also widely accepted that in both bacteria and yeast there is an inverse correlation between the DNA transformation efficiency and the likelihood of transforming multiple DNA molecules into each cell. However, most successful high-throughput mutant screening efforts require high quality libraries, i.e., libraries comprised of cells with a clear phenotype-to-genotype relationship (one genotype/cell). Thus, DNA transformation methods with a high multiplicity of transformation are highly undesirable and detrimental to most mutant screening assays. Here we describe a simple, robust, and highly efficient yeast plasmid DNA transformation methodology, using a dual heat-shock and electroporation approach (HEEL) that generates more than 2 x 107 plasmid-transformed yeast cells per reaction, while simultaneously increasing the fraction of mono-transformed cells from 20% to more than 70% of the transformed population. By also using an automated yeast genotyping workflow coupled with a dual-barcoding approach, consisting of a SNP and high-diversity barcode (10N), we can consistently identify and enumerate unique plasmid genotypes within a heterogeneous population merely through Sanger sequencing. We demonstrate here that the size and quality of a transformed library no longer need to be inversely correlated when transforming large mutant DNA libraries in yeast using highly efficient DNA electroporation methods. SignificanceWith the recent expansion of artificial intelligence in the field of synthetic biology there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput, but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.

microbiology↗

bioRxiv2024

Discovery of the missing cytochrome P450 monooxygenase cyclases that conclude glyceollin biosynthesis in soybean

Glyceollins are isoflavonoid-derived metabolites produced by soybean that hold great promise in improving human and animal health due to their antimicrobial, and other medicinal properties. They play important roles in agriculture by defending soybean against one of its most destructive pathogens, Phytophthora sojae. Longstanding research efforts have focused on improving accessibility to glyceollins, yet chemical synthesis remains uneconomical. The fact that some of the key genes involved in the final step of glyceollin biosynthesis have not been identified, engineering the accumulation of these important compounds in microbes is not yet possible. Although the activity of a P450 cyclase was inferred to catalyze the final committed step in glyceollin biosynthesis forty years ago, the enzyme in question has never been conclusively identified. This study reports, for the first time, the identification of three cytochrome P450 monooxygenase cyclases that catalyze the final steps of glyceollin biosynthesis. Utilizing P. sojae-soybean transcriptome data, along with genome mining tools and co-expression network analysis, we have identified 16 candidate glyceollin synthases (GmGS). Heterologous expression of these candidate genes in yeast, coupled with in vitro enzyme assays, enabled us to discover three enzymes capable of producing two glyceollin isomers. GmGS11A and GmGS11B catalyzed the conversion of glyceollidin to glyceollin I, whereas GmGS13A converted glyceocarpin to glyceollin III. The functionality of these candidates was further confirmed in planta through gene silencing and overexpression in soybean hairy roots. This groundbreaking study not only contributes to the understanding of glyceollin biosynthesis, but also demonstrates a new synthetic biology strategy that could potentially be scaled up to produce valuable molecules for crop and disease management.

biochemistry↗

bioRxiv2024

Cybernetic control of a natural microbial co-culture

A key obstacle in the widespread application of microbial co-cultures in bioprocesses is their compositional instability, as faster-growing species outcompete and dominate the culture. While several synthetic biology approaches have demonstrated control over co-culture composition, there has been an increased interest in computer-based cybernetic control approaches that can offload burdensome genetic control circuitry to computers and enable dynamic control and real-time noise rejection. This work extends that approach, demonstrating a cybernetic control method that is not reliant on any genetic engineering, instead interfacing cells with computers by exploiting their natural characteristics to measure and actuate the composition. We apply this to a Pseudomonas putida (P. putida) and Escherichia coli (E. coli) co-culture grown in Chi.Bio bioreactors, first showing how composition estimates calculated from different bioreactor measurements can be combined with a system model using an extended Kalman filter to generate accurate estimates of a noisy system. We also demonstrate that because the species have different optimal temperature niches, adjusting the temperature of the culture can drive the composition in either direction. By using a proportional-integral control algorithm to calculate the temperature that would bring the measured composition towards the desired composition, we are able to track dynamic references and stabilised the co-culture for 7 days ([~]250 generations), with the experiment ending before the cells could adapt out of the control. This cybernetic framework is broadly applicable, with different microbes unique features and specific growth niches enabling robust control over diverse co-cultures.

systems biology↗

bioRxiv2024

Droplet-AI-empowered high-throughput screening of cell-free gene expression

Cell-free gene expression (CFE) systems enable transcription and translation using crude cellular extracts, offering a versatile platform for synthetic biology by eliminating the need to maintain living cells. This allows direct manipulation of molecular components and the focused synthesis of specific products. However, the optimization of CFE systems is constrained by cumbersome composition, high costs, and limited yields due to numerous additional components required to maintain biocatalytic efficiency. While optimizing such complicated systems is daunting for existing high-throughput screening means, we introduce DropAI, a droplet-based, AI-driven screening strategy designed to optimize CFE systems with high throughput and economic efficiency. DropAI employs microfluidics to generate picoliter reactors and utilizes a fluorescent color-based coding-decoding system to address and screen a vast array of additive combinations. The in-droplet screening is complemented by in silico optimization, where experimental results train a machine-learning model to estimate the contribution of the components and predict high-yield combinations, which are then validated in vitro. Applying DropAI to an Escherichia coli-based CFE system, we simplified a set of 12 additives to only 3 essential components. Through further optimization, we achieved a 2.1-fold cost reduction and a 1.9-fold increase in yield for the expression of superfolder green fluorescent protein (sfGFP). This optimized formulation was further validated across 12 different proteins. Notably, the established E. coli model is successfully adapted to a Bacillus subtilis-based system through transfer learning, leading to doubled yield through prediction. DropAI thus offers a generalizable and scalable method for optimizing CFE systems, enhancing their potential for biochemical engineering and biomanufacturing applications.

bioengineering↗

bioRxiv2024

Pressorum Sensing: Growth-induced Compression Activates cAMP Signaling in Pseudomonas aeruginosa

Bacteria employ various strategies to coordinate population-level behaviors, with quorum sensing being a well-established mechanism. Here, we report a novel population-level regulatory mechanism in Pseudomonas aeruginosa, which we term pressorum sensing. This mechanism allows bacteria to modulate their collective behavior in response to growth-induced mechanical compression in confined spaces. Using a highly sensitive cAMP biosensor in combination with microfluidics, we demonstrate that when compressive forces reach approximately 30 nN, P. aeruginosa cells rapidly increases intracellular cAMP levels via the Pil-Chp chemosensory system. This response leads to up-regulation of the Type III Secretion System, a key virulence factor. Unlike quorum sensing, which relies on diffusible chemical signals, pressorum sensing utilizes mechanical cues to gauge population density and spatial confinement. In bacterial colonies, this mechanism generates striking spatial patterns of cAMP signaling, including traveling rings that coincide with step-like structures in colony morphology. Our findings reveal a previously unknown link between mechanical compression and bacterial virulence, providing new insights into how P. aeruginosa coordinates population-level responses in confined environments. This work also expands our knowledge of mechanogenetics and opens up new possibilities in synthetic biology and bioengineering applications.

microbiology↗

bioRxiv2024

Bioenergetic stress potentiates antimicrobial resistance and persistence

Antimicrobial resistance (AMR) is a global health crisis and there is an urgent need to better understand AMR mechanisms. Antibiotic treatment alters several aspects of bacterial physiology, including increased ATP utilization, carbon metabolism, and reactive oxygen species (ROS) formation. However, how the "bioenergetic stress" induced by increased ATP utilization affects treatment outcomes is unknown. Here we utilized a synthetic biology approach to study the direct effects of bioenergetic stress on antibiotic efficacy. We engineered a genetic system that constitutively hydrolyzes ATP or NADH in Escherichia coli. We found that bioenergetic stress potentiates AMR evolution via enhanced ROS production, mutagenic break repair, and transcription-coupled repair. We also find that bioenergetic stress potentiates antimicrobial persistence via potentiated stringent response activation. We propose a unifying model that antibiotic-induced antimicrobial resistance and persistence is caused by antibiotic-induced. This has important implications for preventing or curbing the spread of AMR infections.

microbiology↗

bioRxiv2024

CRISPR/dCas-mediated counter-silencing: Reprogramming dCas proteins into antagonists of xenogeneic silencers

Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, AT-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel CRISPR/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in Corynebacterium glutamicum or Lsr2 in Streptomyces venezuelae. Systematic in vivo reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding - independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in S. venezuelae when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in C. glutamicum strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference (CRISPRi) effects based on the target position and strand. Genome-wide transcriptome profiling in sgRNA/dCas9 co-expressing C. glutamicum wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology. IMPORTANCELsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, AT-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel CRISPR/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in Corynebacterium glutamicum and Streptomyces venezuelae. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise activation of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.

microbiology↗

bioRxiv2024

Exchange, promiscuity, and orthogonality in de novo designed coiled-coil peptide assemblies

De novo protein design is delivering new peptide and protein structures at a rapid pace. Many of these synthetic polypeptides form well-defined and hyperthermal-stable structures. Generally, however, less is known about the dynamic properties of the de novo designed structures. Here, we explore one aspect of dynamics in a series of de novo coiled-coil peptide assemblies: namely, peptide exchange within and between different oligomers from dimers through to heptamers. First, we develop a fluorescence-based reporter assay for peptide exchange that is straightforward to implement, and, thus, would be useful to others examining similar systems. We apply this assay to explore both homotypic exchange within single species, and heterotypic exchange between coiled coils of different coiled-coil oligomer states. For the former, we provide detailed study for the dimeric coiled coil CC-Di finding a half-life for exchange of 4.2 {+/-} 0.3 minutes when the concentration of CC-Di is 200 {micro}M. Interestingly, more broadly when assessing exchange across all of the oligomeric states, we find that some of the designs are faithful and only undergo homotypic strand exchange, whereas others are promiscuous and exchange to form unexpected hetero-oligomers. Finally, we develop two design strategies to improve the orthogonality of the different oligomers: (i) using alternate positioning of salt bridge interactions; and (ii) incorporating of non-canonical repeats into the designed sequences. In so doing, we reconcile the promiscuity and deliver a set of faithful homo-oligomeric de novo coiled-coil peptides. Our findings have implications for the application of these and other coiled coils as modules in chemical and synthetic biology.

systems biology↗

bioRxiv2024

Unlocking expanded flagellin perception through rational receptor engineering

The surface-localized receptor kinase FLS2 detects the flg22 epitope from bacterial flagella. FLS2 is conserved across land plants, but bacterial pathogens exhibit polymorphic flg22 epitopes. Most FLS2 homologs possess narrow perception ranges, but four with expanded perception ranges have been identified. Using diversity analyses, AlphaFold-Multimer modeling, and amino acid properties, key residues enabling expanded recognition were mapped to FLS2s concave surface, interacting with the co-receptor and polymorphic flg22 residues. Synthetic biology enabled engineering of expanded recognition from QvFLS2 (Quercus variabilis) and FLS2XL (Vitis riparia) into homologs with canonical perception. Evolutionary analyses across three plant orders showed residues under positive selection aligning with those binding the co-receptor and flg22s C-terminus, suggesting more alleles with expanded perception exist. Our experimental data enabled the identification of specific receptor amino acid properties and AlphaFold3 metrics that facilitate predicting FLS2-flg22 recognition. This study provides a framework for rational receptor engineering to enhance pathogen restriction.

plant biology↗

bioRxiv2024

Profiling the physiological impact of aberrant folded-state protein filamentation in cells

The formation of large polymeric structures such as cytoskeletal and enzyme filaments is crucial for normal cellular function. However, such filaments can also form due to mutations that create self-interactions at the surface of symmetric proteins. Often, the proteins forming these structures maintain a folded state and thereby differ from aggregates and amyloids that involve misfolding. We refer to this type of assemblies as agglomerates to mark this difference. While cells have quality control mechanisms to identify, buffer, and eliminate misfolded proteins, it is unclear whether similar mechanisms exist for agglomerates, or whether agglomerates are toxic to cells. Here, we profiled the physiological impact of mutation-induced folded-state protein filamentation in yeast cells. First, we devised a simple strategy to distinguish fluorescently labeled proteins forming agglomerates versus aggregates. We then profiled exogenous protein agglomerates in terms of their recognition by known quality control mechanisms, their effects on specific cellular processes and overall fitness on S. cerevisiae cultures. We found that agglomerates do not colocalize with the proteostasis machinery and do not result in measurable fitness defects. Proteomics profiling of cells expressing the wild type protein, agglomerating or misfolded variants revealed a consistent picture, with only minor, agglomerate-size-dependent changes observed and linked to the cell-wall and plasma-membrane proteins. Overall, our findings indicate that agglomerates form mostly benign structures in cells when compared to aggregates, and thereby offer a promising route for synthetic biology applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/612878v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@7136a4org.highwire.dtl.DTLVardef@ba62dborg.highwire.dtl.DTLVardef@b02bbaorg.highwire.dtl.DTLVardef@6cbf9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

in vitro analysis of a competitive inhibition model for T7 RNA polymerase biosensors

T7 RNA polymerase (T7 RNAP) biosensors, in which T7 RNAP transcribes some reporter gene or signal in response to external stimuli, have wide applications in synthetic biology and metabolic engineering. We adapted a biochemical reaction network model and used an in vitro transcription assay to determine network parameters for different T7 RNAP constructs. Under conditions where template DNA is limiting, the EC50 values of native and engineered T7 RNAPs ranged from 33 nM (29 -37 95% c.i.) to 570 nM (258 -714 95% c.i.) (wild-type T7 RNAP). The measured EC50 values were largely insensitive to free magnesium, pH, or other buffer conditions. Many biosensor configurations use a split RNAP construct, where the C-terminal (CT7) and N-terminal T7 (NT7) are fused to proximity induced dimerization modules. We used proteolysis and ion exchange chromatography to prepare a CT7 (80 kDa) product. The impact of free CT7 on T7 RNAP transcriptional activity was well described by a competitive inhibition model, with an inhibitory constant KI = 24 nM (22-26 95% c.i.) of the sensor. These model parameters will be useful for forward modeling and design of T7 RNAP-based genetic circuits. HighlightsO_LIEC50 values for natural and engineered T7 RNAPs measured in vitro C_LIO_LIC-terminal T7 RNAP is a competitive inhibitor of full-length T7 RNAP C_LIO_LIThe inhibition constant of C-terminal T7 RNAP is lower than the EC50 of T7 RNAP C_LI

bioengineering↗

bioRxiv2024

Robust and highly efficient transformation method for a minimal Mycoplasma cell

Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining of the genome of Mycoplasma mycoides. When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, CFU, and transformation efficiency, we found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early-exponential growth phase. Notably, we attained the transformation efficiency up to 4.4 x 10-2 transformants per cell per g plasmid DNA. We developed a method to obtain several hundred to several thousand transformants with less than 0.2 mL of cultured cells and 10 ng of plasmid DNA. Moreover, we established a transformation method using frozen stock of transformation-ready cells. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCEMycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand cellular process of life and for bioengineering because of their simple metabolism, small genomes and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtainable only 10 nanograms of plasmid DNA, which is around one-thousandth amount required for traditional mycoplasma transformations. Moreover, we established a convenient method using frozen stocks of transformation-ready cells. These improved methods play a crucial role in further studies using minimal cells.

microbiology↗

bioRxiv2024

Creased ciliary flocks shape unfolding dynamics via information bottlenecks in an aneural animal

Multicellular organisms utilize thin sheet folding to achieve functional three-dimensional forms. During embryonic development, stereotypical epithelial folds emerge from active cellular and molecular processes including cell shape change and differential cell growth. Active thin sheet folding promises to be a powerful design technique in the fields of active solids, soft robotics, and synthetic biology. However, the general principles of active thin sheet folding remain poorly understood. Here we discover a non-canonical cilia-driven thin sheet folding behavior exhibited by basal animal Trichoplax adhaerens. Through volumetric imaging, we found that, despite having no nervous system, T. adhaerens has the remarkable ability to resolve complex body folding states in a non-stereotypical fashion using a carpet of collectively flocking cilia. Cilia-resolved imaging revealed that folds create crease defects in the animals ciliary carpet, which act as information bottlenecks to break collective behavior of cilia. In turn, these bottlenecks enable the disjointed locomotion required for fold removal. These findings point to a two-way coupling mechanism, wherein ciliary activity shapes the animals folding state and vice versa. Our work demonstrates the broad configuration space of non-stereotypical active folding and highlights the power of distributed activity to drive folding and unfolding of a thin multicellular sheet. We anticipate our study to be a starting point for the establishment of a new class of distributed active origami wherein fold lines themselves are dynamic and motile, with implications in engineering of self-folding materials. Additionally, our work reveals a new facet of the Placozoan behavioral repertoire, which extends our understanding of mechanical intelligence in the absence of a nervous system.

biophysics↗

bioRxiv2024

Regulating Human T Lymphocytes Through Magnetogenetic Tools

The field of synthetic biology has expanded the possibilities for controlling cellular functions, particularly in the development of mammalian cells for therapeutic applications. This study explored the application of magnetogenetic tools to regulate T cell activity, a crucial aspect of developing advanced immunotherapies. Magnetogenetic tools use magnetic fields to remotely control engineered ion channels and protein domains, providing non-invasive, deep-tissue stimulation that overcomes the limitations of traditional methods. We investigated the effects of three magnetogenetic tools - engineered TRPV1 (TRP1-Fer) and TRPV4 (TRP4-Fer) channels, and Electromagnetic Perceptive Gene (EPG) - in Jurkat cells. First, calcium concentration measurements confirmed the activity of these tools within the cells. Using qPCR and proteomics analysis, we then analyzed their impact on T cell activation, calcium signaling, mitochondrial function, membrane integrity, and gene expression under both stimulated (with antigens) and non-stimulated conditions. Our results revealed significant upregulation of activation and calcium-handling proteins in stimulated cells, indicating enhanced activation and cytoskeletal dynamics compared to controls. However, in non-stimulated cells, the magnetogenetic tools unexpectedly led to deactivation of T cells. This investigation showed that while magnetic induction alone deactivated the cells, antigen stimulation in conjunction with magnetic induction amplified cell activation. This study highlights the potential of magnetogenetics to precisely modulate T cell functions, presenting promising avenues for more effective and controlled immunotherapies. However, the findings also underscore the need for careful optimization to mitigate potential adverse effects on cellular integrity and function.

bioengineering↗

bioRxiv2024

Cell-free immuno-profiling on a genetically programmed biochip

Emerging cell-free synthetic biology approaches provide biosafe, cheap, and versatile genetic tools to advance therapeutic research and development. Combined with micro-fabrication technology, we developed a platform to quantitatively reconstitute interactions of cell-free synthesized antigens with antibodies and human receptors in miniaturized compartments on a silicon chip. Photolithographic surface patterning of protein traps and on chip expression from high density gene brushes generated a continuous surface density gradient of fluorescently labeled antigens. Antibodies binding to the antigen gradient generate a full binding curve in each single compartment for affinity determination. We used the SARS-CoV-2 antigens as a model to profile the specificity and affinity of monoclonal antibodies to > 30 viral epitopes synthesized simultaneously on one chip in a genotype-phenotype linked compartments. We further profiled polyclonal antibodies in minute volumes of human sera, revealing patient-specific epitope profiles that are difficult to detect by conventional approaches. Cell-free co-synthesis of the human ACE2 receptor with the viral Receptor-Binding-Domain yielded relative binding affinities to different SARS-CoV-2 variants. This rapid, quantitative, and on-chip genetically programmed approach allows to study complex protein-protein interactions independent of protein purification steps for human immuno-profiling with a fast response time for combating emerging pathogens.

bioengineering↗