涩里番

Muhammad Algiffari, Hana Kiyama, Makoto Miyata

Graduate School of Science, Osaka Metropolitan University, Japan.

Abstract

The 涩里番-syn3B provides a unique platform for studying the physical principles for minimal life. However, its spherical shape and small diameter limit analysis by optical microscopy. Here, we present approaches to manipulate syn3B morphology through (i) elongation, (ii) division, and (iii) fusion, shown as follows. (i) Elongated cells were obtained by cultivation in alkaline conditions (pH 7.5 and 8.0) and early-phase harvesting. Expression of a Mycoplasma genitalium cytoskeletal protein MG218 also induced cell elongation. (ii) Cell division was induced by treatment with low concentration, 0.01% of detergents, Tween 60 and Tween 80. (iii) Cell fusion on glass slide was induced by polyethylene glycol (PEG), showing various cell morphologies. The highest fusion efficiency was achieved with 50% PEG 8000, at 37 掳C, and with a complex membrane lipid composition. These methods for controlling syn3B morphology may open new perspectives for physical analyses of syn3B cells.

Ali Ahsan, Hana Kiyama, Makoto Miyata

Graduate School of Science, Osaka Metropolitan University, Japan

Abstract

The helical bacterium Spiroplasma eriocheiris swims through viscous media via processive helicity switching driven by an internal cytoskeletal ribbon composed of MreB isoforms and the major 60-kDa Fibril protein. Although Fibril accounts for nearly half of the ribbon mass, its precise contribution apart from actin-like MreBs remains unclear. Previously, syn3 cell worked as a chassis to study Spiroplasma motility related genes. Here, we expressed a Fibril protein in syn3, alone or combined with swimming-associated MreB proteins (SMreB4 and SMreB5). Phase-contrast and fluorescence microscopy demonstrated that Fibril independently forms filamentous structures, induces cell elongation, and promotes left-handed helical geometry. Co-expression of Fibril with SMreB45 significantly enhanced net cell translocation, kink-traveling distance, and swimming efficiency. Furthermore, osmotic downshift assays revealed that Fibril confers structural robustness and mechanical stability, enabling reversible cell-length recovery under osmotic perturbation. These findings indicate that while MreBs drive the active helicity switch, Fibril acts as a structural stabilizer that enhances cell rigidity, processivity, and swimming efficiency.

Andrew K. Maytin鹿虏, Ron Acda虏鲁, Zaida Luthey-Schulten鹿虏鲁

鹿Department of Physics, University of Illinois Urbana-Champaign, Urbana, IL, USA 虏Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA 鲁Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA

Abstract

The genetically minimal bacterium 涩里番-Syn3A most commonly undergoes symmetric division into daughter cells of approximately spherical morphology, but also occasionally exhibits alternative morphologies, including cells with inward buds and elongated, filament-like cells. Here, we combine continuum membrane theory with dynamically triangulated surface (FreeDTS) and particle-based (MesoMem) simulations to investigate the physical mechanisms underlying these morphologies. We connect these approaches directly to three-dimensional fluorescence imaging and cryo-electron tomography of 涩里番-Syn3A. In particular, we investigate whether cell-to-cell variation in membrane properties, such as intrinsic curvature, can explain differences in cell morphology, using values for hydraulic permeability and membrane rigidity informed by MD (Martini 3) simulations. By varying membrane properties potentially associated with differences in membrane protein expression, we reproduce complete cell cycles as well as filamentous and inward-budding trajectories. Comparisons across 涩里番-Syn3A and 涩里番-Syn3.0 genomic contexts allow us to investigate when membrane mechanics alone can drive division and when additional mechanical contributions from the FtsZ Z-ring are required. Finally, we present preliminary work incorporating formation of the FtsZ Z-ring and its mechanical effects on membrane remodeling during cell division.

Enguang Fu^1,2, Troy Brier虏, Zaida Luthey-Schulten^1,2.

鹿NSF Science and Technology Center for Quantitative Cell Biology, Beckman Institute, 虏Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Abstract

涩里番-syn1.0 (Syn1.0) and 涩里番-syn3A (Syn3A), genetically synthetic and genome-reduced versions of the naturally occurring bacterium Mycoplasma mycoides, are landmark platforms for defining the gene set required for life, yet how their genomes are expressed at the RNA level remains uncharacterized. We combined full-length PacBio and native long-read RNA sequencing with short-read quantification and complementary proteomics to map transcription, RNA processing, and protein abundance in both cells. In Syn1.0, full-length sequencing resolved 459 operons encompassing 911 genes, revealing pervasive RNA processing with a strong 3' bias. Analysis of the division and cell-wall cluster showed how transcriptional context explained the restoration of genes required for normal cell division in Syn3A. Most antisense and intergenic transcription in Syn1.0 reflected low-level transcriptional noise arising from inherited mis-annotation, read-through, and synthetic sequences. Much of that transcription was lost in Syn3A after genome minimization. Reducing the genome unexpectedly altered the expression of several retained genes by deleting promoters, most prominently reducing expression of the nucleoid protein HupA and central-carbon enzymes. Meanwhile, one third of the coding mRNA pool was allocated to a single 21-gene ribosomal-protein operon, while several other ribosomal proteins had reduced transcript abundance, which possibly led to imbalanced ribosome assembly. Expression of RNA polymerase and central-carbon metabolism declined at both mRNA and protein levels whereas RNase Y degradosome abundance increased. These shifts in the synthetic evolution of Syn3A suggest plausible mechanisms for its reduced chromosome contacts and slower growth. The analysis and visualization that are shared via Jupyter Notebook provide the RNA-level foundation for whole-cell modeling of the minimal cell.

The above analysis motivated the following refinements to the 4DWCM of Syn3A. A new transcription scheme tracks the positions of RNA polymerase along the genome, combining stochastic initiation and termination events with elongation to simulate co-transcription in polycistronic operons. This allows stoichiometric protein synthesis in single cells, a requirement for efficient complex assembly and balanced cellular activities. Overall gene expression kinetics are constrained by the experimentally measured mRNA abundances reported above. These developments provide a framework for assessing how cooperativity between co-transcribed genes shapes protein output in a minimal cell and enhance the predictability of the 4DWCM.

Authors: Roksana Riddle, Satya Kulkarni, Jayla Zink, Kenneth Hunyh, Christopher Contag

Michigan State University

Abstract

Over 2 billion years of evolution, ancestral eukaryotic cells acquired intracellular parasitic microbes that eventually became organelles such as mitochondria. To model this phenomenon for therapeutic applications, we have been developing intracellular organelle-like entities, called quasiorganelles, that can produce mammalian proteins and guide host cells towards a desired phenotype. Syn3B is a synthetic minimal genome bacterium, derived from Mycoplasma mycoides capri. Syn3B does not parasitize mammalian cells and appears to be poorly immunogenic in animals. For these reasons, it is an ideal quasiorganelle chassis for in vivo cellular reprogramming and applications in regenerative medicine. Since intravenously injected microbes largely localize to the liver, we chose to target liver disease, a pathological process where hepatocytes die and are replaced by fibrotic scar tissue. To enable uptake of syn3B into mammalian cells, we engineered syn3B to express internalin B (InIB) and invasin (inv), derived from Listeria monocytogenes and Yersinia pseudotuberculosis, respectively. These bind to host receptors (c-MET, beta1-integrin) and enable internalization by a zipper mechanism. Using these strains, we developed a synthetic transcriptional circuit to visualize the dynamics of host-syn3B interactions. In this system, syn3B produced a synthetic transcription factor (tTA) that localized to a synthetic promoter element (TRE3G) within the host cell to activate transcription of a downstream reporter gene (luciferase-tdTomato). Bioluminescence and fluorescence imaging revealed population- and cellular-level dynamics of transcriptional activation by syn3B in a mouse Kupffer cell line. Further, we measured the transcriptional response to syn3B-tTA in a transgenic mouse model, and optimized the dose of syn3B for future studies in liver regeneration. Future studies will evaluate these transcriptional dynamics in other reporter cell lines and engineer syn3B to express therapeutic proteins, such as Wilm鈥檚 Tumor Factor 1 (WT-1), for liver regeneration.

Ali Navid

Lawrence Livermore National Laboratory

Abstract

Minimal genomes are not necessarily unique solutions. Computational studies show that the order in which genes are lost can determine which alternative pathways remain and which genes become indispensable. Environmental conditions further shape this process by changing nutrient requirements, energetic constraints, and selection pressures. Using metabolic and whole-cell models, researchers can simulate repeated genome-reduction trajectories and identify both conserved cellular functions and historically contingent outcomes. These models reveal why individually nonessential genes may become collectively essential, and why distinct reduced genomes can support growth in the same environment. The results provide a framework for designing streamlined microbial chassis while emphasizing the need for iterative computational and experimental validation.

Institute for Computer Science & Department of Biology, Heinrich Heine University D眉sseldorf, Germany

Abstract

Independent bacterial populations evolving under identical selection pressures can follow similar or divergent evolutionary trajectories. We aim to determine how repeatable these trajectories are and whether divergent adaptations represent local fitness optima or alternative paths toward the same optimum. To address this question in a simplified system, we use 涩里番-syn3B, which is able to rapidly adapt during laboratory evolution, despite its reduced genome and limited regulatory capacity.

We conducted a two-month adaptive laboratory evolution experiment under five selection pressures consisting of pH, temperature, and metabolic perturbation, with 48 independently evolving populations per condition. Changes in fitness were monitored throughout the experiment, and samples were collected at regular intervals. We are now focusing on generating and analysing the corresponding -omics datasets to compare mutations, protein abundances, and metabolite levels across replicate populations, selection pressures, and time points. This will reveal parallel and divergent evolutionary trajectories. Ultimately, the experimental data will be compared with growth and resource-allocation predictions from a genome-scale kinetic balanced-growth model.

Wichittra Arai鹿, Masaki Mizutani虏, *Shigeyuki Kakizawa鹿藪鲁

鹿 MOLBIS, National Institute of Advanced Industrial Science and Technology (AIST), Japan

虏 Faculty of Science, Gakushuin University, Japan

鲁 IAB, Keio University, Japan

Abstract

We previously developed serum-free and chemically defined media for the minimal cell 涩里番-syn3B. To further improve the serum-free medium, we investigated the types and concentrations of lipids and lipid carriers used as serum substitutes.

First, we found that each component had an optimal concentration range, with excessively high concentrations inhibiting cell growth.

Next, we found that supplementation with five lipids (cholesterol, palmitic acid, oleic acid, sphingomyelin, and phosphatidylcholine) resulted in faster growth of 涩里番-syn3B and more efficient recovery after storage at 鈭�80潞C, compared with supplementation with only three lipids (cholesterol, palmitic acid, and oleic acid).

We also found that sphingomyelin and phosphatidylcholine were essential for the growth of "swimming syn3B" cells expressing Spiroplasma MreB proteins. Since these two lipids may affect membrane fluidity, our findings suggest that membrane fluidity may be particularly important for cells with non-spherical morphologies.

Wonsik Choi & Jeongin Choi

Abstract

It breaks a research question into explicit concepts, dependencies and rules that AI can work with reliably, and organizes established equations and theories within that structure. Human intuition supplies the direction, while AI helps turn it into models, code and testable designs. This gives the human researcher and AI a shared tool for understanding the same system.

Using this approach, I was able to develop a sequence-level computational design for a walking protein, WRRA-Motor H2, despite not being trained in biotechnology. The presentation includes this case: an inherited motor core with a designed coordination gate and explicit experimental controls. It illustrates how an intuitive idea can become a concrete candidate for biological testing.

For minimal-cell research, we apply the same approach to make the machinery, resources and dependencies behind each function easier to trace, including what a cell must regenerate for its daughters.

Jitra-Marie Jittasevi (G茅raldine Gourgues鹿, Dominick Matteau虏, Jade Jaubert鲁, Yonathan Arfi鹿, S茅bastien Rodrigue虏, Carole Lartigue鹿)

鹿 INRAE, UMR BFP, F-33882, Villenave d鈥橭rnon, France

虏 D茅partement de biologie, Universit茅 de Sherbrooke, Sherbrooke, QC, Canada

鲁 Univ. Bordeaux, Bordeaux Proteome, Bordeaux, France

Abstract

Background

Large-scale engineering of Mollicutes genomes was enabled by genome transplantation (GT), during which a donor genome is isolated and transferred into the cytoplasm of a different species, yielding a cell whose genetic and phenotypic identity becomes that of the donor after the selection process. GT in the recipient cell Mycoplasma capricolum (Mcap) is limited to a handful of donor genomes, and its efficiency is inversely correlated to the phylogenetic distance separating the donor and recipient species. We hypothesised that transcription of the donor genome by the recipient cell machinery is an essential compatibility factor during GT.

Material and methods

We designed two complementary strategies involving the RNA polymerases (RNAP) of Mcap (recipient) and Mesoplasma florum (Mflorum, donor). First, we replaced all the RNAP coding sequences in the Mcap genome with that of Mflorum鈥檚 using CRISPR-Cas9 and a yeast host. In parallel, we expressed recombinant RNAP subunits from Mflorum in Mcap using transposons.

Results

We obtained viable Mcap clones expressing only the Mflorum RNAP, as well as clones expressing several hybrid subunit combinations, but no viable cells in which the Mcap catalytic subunits (RpoB, RpoC) were replaced with Mflorum鈥檚. Meanwhile, expression of the RNAPs of both species simultaneously in Mcap led to truncating mutations appearing in the coding sequences of the Mflorum RpoB and RpoC, suggesting that their expression was detrimental to Mcap.

Conclusions

Co-expressing the transcription machineries of Mcap and Mflorum generates non-functional protein hybrids, which could decrease the probability of selecting a viable donor genome and compromise transplantation.

Authors

L茅a Wagner鹿, G茅raldine Gourgues鹿, Marie-Pierre Dubrana鹿, Laure Beven鹿, Jean-Paul Douliez鹿, Pascal Sirand-Pugnet鹿, Yonathan Arfi鹿, Carole Lartigue鹿

鹿 Univ. Bordeaux, INRAE, UMR BFP, F-33140 Villenave d鈥橭rnon, France

Abstract

Whole-genome transplantation (WGT) was a key enabling technology for the construction of the first synthetic bacterial cells, including Mycoplasma 涩里番-syn1.0 and 涩里番-syn3.0. WGT consists of transferring an entire bacterial chromosome (the donor genome) into a compatible living recipient cell, ultimately generating viable cells whose identity and functions are governed by the transplanted chromosome. Although WGT has been successfully applied to several mycoplasma species, often using Mycoplasma capricolum subsp. capricolum as the recipient cell, the molecular mechanisms underlying this process remain poorly understood.

In particular, little is known about the 鈥渂oot-up鈥� phase during which the donor genome starts to be expressed within the recipient cytoplasm and progressively establishes the molecular machinery required to sustain its own expression. How transcription of the donor genome is initiated by the recipient machinery, how this transition is regulated, and whether successful boot-up emerges from largely stochastic molecular interactions or follows a defined sequence of events remain open questions.

The ExEGeSyS project aims to dissect this process by reconstructing key features of WGT in vitro using a mycoplasma lysate-based cell-free expression system. A cell-free system derived from 涩里番-syn3A was established in 2023, paving the way toward such a system for a WGT recipient cell. We therefore aim to (i) develop a lysate-based cell-free expression system from M. capricolum subsp. capricolum cells, (ii) investigate the expression of complete genomes from different WGT donor species in this recipient-derived environment, and (iii) map the temporal expression of donor genes during genome boot-up. Ultimately, this approach should provide a controlled experimental framework to uncover the molecular principles governing the transition from a transplanted genome to a self-sustaining bacterial cell.

Key words: Whole genome transplantation, lysate-based cell-free systems, Mycoplasma 涩里番-syn3A, Mycoplasma capricolum subsp. capricolum.

Mert Bozoflu

Molecular Dynamics Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands

Abstract

The 涩里番-syn3A membrane has an unusual lipid composition dominated by cholesterol, sphingomyelin, and cardiolipin. Here, we present insights from Martini coarse-grained molecular dynamics simulations that examine the role of this lipid composition from complementary perspectives. Simulations of protein-free 涩里番-syn3A membrane models show that cholesterol strongly influences the structural, dynamic, and mechanical properties of the membrane, as well as its permeability, while the native-like composition maintains an ordered but fluid state despite its high cholesterol content. In separate cellular-scale simulations, we investigate how lipid composition shapes membrane organization in the presence of the 涩里番-syn3A membrane proteome. The native lipid mixture shows pronounced organization around membrane proteins, with cardiolipin and DOPG enriched at protein interfaces and sphingomyelin depleted. Cardiolipin enrichment at these interfaces is also associated with substantially reduced protein-mediated phospholipid translocation. Together, these studies provide complementary views of how lipid composition shapes the properties and organization of the 涩里番-syn3A membrane.

Deborah Bambil, Patricia Verdugo Pascoal, Rayane Nunes Lima, Luisa Mayumi Arake de Tacca, Marco Antonio de Oliveira, Elibio Rech

National Institute of Science and Technology in Engineering Biological Systems/Embrapa, Bras铆lia, Distrito Federal, 70770-917, Brazil

Abstract

Quantum computing offers revolutionary avenues for the analysis and engineering of biological information, particularly as genomic datasets and synthetic DNA constructs grow in size and complexity. However, practical quantum approaches for large-scale genome manipulation remain largely unexplored, constrained by Noisy Intermediate-Scale Quantum (NISQ) era hardware limitations and Out-of-Memory (OOM) bottlenecks in classical processing. Here, we present a hybrid computational architecture that virtualizes quantum circuits using the Qiskit framework and the AerSimulator backend, executing high-performance Quantum Random-Access Memory (QuRAM) operations on classical infrastructure. This modular framework integrates two modules: QuSearch, designed for high-dimensional spatial screening and sequence alignment, and QuAssembly, designed for automated multi-target genomic insertion and splicing. To overcome OOM errors and physical qubit limitations when processing whole genomes, we implement a dynamic Quantum Chunking strategy that maps localized genomic windows onto addressable virtual quantum states, isolating spatial complexity and enabling the processing of sequences of any length. In the QuSearch module, discrete DNA nucleotides are mapped onto a Hilbert space using strictly orthogonal quantum encoding logic. A specialized biological oracle maps the target sequence to activation bases and employs an uncomputation technique to restore the original computational states. QuAssembly automates the scanning and splicing of multiple targets comprising standardized genetic units Promoter, Ribosome Binding Site (RBS), Enzyme, and Terminator using a reverse-insertion strategy that preserves coordinate mapping and reference genome orientation integrity during multi-target splicing. As a proof of concept, we uploaded the minimized synthetic genome 涩里番- syn3.0 (~531 kb). The algorithms successfully identified target sequences, accurately assembled their left and right boundaries, and validated the anchor regions (scars).

Authors: Jeremy Gagnon, Dominick Matteau, Frederic Grenier, S茅bastien Rodrigue*

Affiliations:

Universit茅 de Sherbrooke, D茅partement de Biologie, 2500 Boulevard Universit茅, Sherbrooke, Qu茅bec J1K 2R1, Canada

Abstract

Genome transplantation is a key technology for synthetic genomics, enabling entire genomes to be transferred into recipient cells. Despite its importance, genome transplantation remains confined to a small number of wall-less Mollicute species and is often characterized by low efficiencies, limiting the development and testing of synthetic genomes. Because genome transplantation relies on the same polyethylene glycol mediated DNA delivery process used for plasmid transformation, improving DNA uptake is an important step toward more efficient transplantation systems. Here, we systematically optimized polyethylene glycol-mediated transformation in Mesoplasma florum, a fast-growing, non-pathogenic Mollicute with a reduced genome that represents an attractive chassis for synthetic genomics. We evaluated 20 parameters spanning cell physiology, DNA preparation, membrane conditioning, and recovery conditions. DNA topology emerged as the strongest determinant of transformation efficiency, with highly compacted DNA preparations producing up to three orders of magnitude more transformants than conventional plasmid preparations. Growth phase and polyethylene glycol concentration also strongly influenced transformation outcomes. To identify potential recipients for future genome transplantation experiments, we further screened ten strains belonging to the Mesoplasma lineage. Transformation efficiencies varied widely among strains, and Mesoplasma entomophilum W17 emerged as a particularly promising candidate. Moreover, W17 supported replication of plasmids carrying the M. florum oriC, suggesting compatibility between the replication systems of the two species. Together, these results establish an improved transformation workflow for M. florum, identify DNA topology as a major determinant of DNA uptake, and reveal a new candidate recipient for future genome transplantation studies. These advances provide practical tools and biological insights for synthetic genome engineering in Mollicutes.

^1*Emmanuel Gabriel Njoku, ^1*Isabel Buitron and 鹿Bogumil Karas

鹿Western University, London, Canada.

^*Presenting Authors

Abstract

Advances in the design, delivery, and integration of synthetic genomes and biosynthetic pathways will enable the creation of organisms with tailored traits to address challenges in agriculture, medicine, and biofuel production. Acholeplasma laidlawii is an attractive chassis for synthetic biology because of its relatively small genome (~ 1.5 Mb) and low GC content (~ 31-32%). This low GC content makes A. laidlawii a potentially useful host for the cloning and maintenance of low-GC DNA that can be difficult to clone in conventional bacterial systems. In particular, this research explores A. laidlawii as a host for cloning and maintaining Phytoplasma genome fragments, as well as chloroplast genomes from Phaeodactylum tricornutum and Solanum tuberosum. Phytoplasmas are important plant pathogens that remain challenging to study and develop treatments for, partly due to the inability to efficiently grow them in axenic cultures. Cloning Phytoplasma genomic DNA in A. laidlawii provides an alternative approach for studying their genomes and gene function. Beyond its potential as a cloning chassis, A. laidlawii may also serve as a vehicle for DNA delivery to eukaryotic cells through cell fusion. Cell fusion enables the transfer of large DNA constructs and whole genomes. Previous research demonstrated the transfer of the ~1.5-Mb A. laidlawii genome to Saccharomyces cerevisiae. Building on this work, this research aims to establish polyethylene glycol (PEG)-mediated cell fusion between A. laidlawii and selected prokaryotic and eukaryotic species.

Olivia Bulka鹿*, Cl茅mence Frioux鹿, Sylvain Prigent^2,3

*Presenting author

鹿Inria, University of Bordeaux, Talence, France

虏University of Bordeaux, INRAE, BFP, Villenave d鈥橭rnon, France

鲁Bordeaux Metabolome, MetaboHUB, INRAE, Villenave d鈥橭rnon, France

Abstract

Many environmental bacteria remain uncultivated, often due to a lack of understanding about their growth requirements. For example, the Mollicutes class includes plant pathogens like Spiroplasma and Phytoplasma that decimate fruit trees each year, but their study is limited by the inability to cultivate them in defined lab media. While Phytoplasmas are uncultivable in any known lab media, most Spiroplasmas are only cultivable in undefined media including serum and other complex mixtures of metabolites, complicating controlled growth and metabolism studies. A promising cultivation strategy is to integrate high-throughput 鈥渃ulturomics鈥� via microfluidics with meta-omics, informed by initial metabolic modelling predictions.

Here, we present a genome-scale metabolic model of S. citri for prediction of defined minimal media. The metabolic network comprises 200 metabolic genes, 641 reactions, and 507 metabolites. A draft network was reconstructed from the S. citri genome, which was curated with orthology-based annotation and incorporation of biochemical data reported in literature. The biomass reaction (representing the cell composition) was tailored to Spiroplasma through integration of in-house and published experimental data. Using this metabolic model, we compared growth of S. citri in established media previously designed for Mollicutes, and use initial results of de novo medium prediction to contrast S. citri with other well-studied Mollicutes (涩里番-Syn3, Mesoplasma florum, and Mycoplasma pneumoniae). This metabolic model is a first step in an interdisciplinary effort to cultivate S. citri in defined media, with experimental validation still to come.

Peter L眉ttge Jordal

Affiliation: SBT Instruments, Symfonivej 37, 2730 Herlev, Denmark. http://www.sbtinstruments.com/

Abstract

Mycoplasma-like cultures are hard to track. The cells are tiny and scatter little light. As a result, OD and McFarland readings are unsuited. Colour-changing units (CCUs) take days, while PCR-and fluorescence-based methods are laborious and do not produce real-time insights.

BactoBox庐 is a label-free flow cytometer. It uses impedance flow cytometry to count single cells by their electrical signature. No stains. No incubation. Results come in real time.

This talk explains the technology. It then shows three ways BactoBox gives insight into minimal cell cultures:

1. Cell concentration in real time. Know how many cells you have before you inoculate, passage or harvest.

2. Growth phase. Pair concentration with CIZE, an impedance-based cell-size estimate, to tell lag, exponential and stationary phase apart.

3. Contamination in overnight cultures. Spot unexpected cell populations before they ruin an experiment.

The result is reproducible inocula, across runs.

Jos茅 Manuel Blasco Losa鹿, Alejandro Serrano S谩nchez^1,2, Marina de la Fuente Garc铆a鹿, James F. Pelletier^1,3

鹿 Centro Nacional de Biotecnolog铆a (CNB-CSIC), Madrid, Spain

虏 Universidad Aut贸noma de Madrid (UAM), Madrid, Spain

鲁 Grupo Interdisciplinar de Sistemas Complejos (GISC), Madrid, Spain

Abstract

The physical chemistry of the cytoplasm influences key physiological processes, such as cell size regulation and cellular metabolism. The structural, genomic, and metabolic simplicity of the minimal cell 涩里番-syn3B make it an ideal system to study mechanisms that regulate physicochemical properties, such as the osmolarity, pH, and membrane potential. Our goal is to adapt a quantitative coarse-grained framework based on the classical Tosteson and Hoffman pump-leak model, which predicts the flux of each ion due to electrochemical gradients as well as specific ion pumps. We are experimentally measuring model parameters to make quantitative predictions such as the volume response to osmotic perturbations and the ATP cost of maintaining ion homeostasis.

This work was supported by the Fundaci贸n Occident, the Programa Fundamentos of the Fundaci贸n BBVA, the Programa de Atracci贸n de Talento 鈥淐茅sar Nombela鈥� of the Comunidad de Madrid, and the Programa de Generaci贸n de Conocimento of the Spanish Agencia Estatal de Investigaci贸n.

Marina de la Fuente Garc铆a鹿, Jos茅 Manuel Blasco Losa鹿, Alejandro Serrano S谩nchez^1,2, James F. Pelletier^1,3

鹿 Centro Nacional de Biotecnolog铆a (CNB-CSIC), Madrid, Spain

虏 Universidad Aut贸noma de Madrid (UAM), Madrid, Spain

鲁 Grupo Interdisciplinar de Sistemas Complejos (GISC), Madrid, Spain

Abstract

With the smallest known genome, proteome, transcriptome, metabolome, etc., the genomically minimal cell 涩里番-syn3A has provided a simplified model system for whole-cell models (WCMs), such as the models developed by the Luthey-Schulten group. WCMs of 涩里番-syn3A have simulated thousands of coupled reactions that describe metabolism, transcription, translation, and other essential processes. Most metabolic reactions have been modeled by the reversible Michaelis-Menten equation, which depends on substrate and product concentrations. Allosteric modulation by activators or inhibitors has not yet been incorporated but could contribute to metabolic regulation in 涩里番-syn3A.

Here, we adapt the existing state-of-the-art dynamical metabolic model (Fu et al., 2025) to include allosteric regulation of two glycolytic enzymes, phosphofructokinase and pyruvate kinase, predicted to maintain high ATP levels in mammalian glycolysis (Choe et al., 2025). In the absence of allosteric regulation, the model predicts sharp transitions between three metabolic states with respect to changes in external glucose. Allosteric regulation softens these transitions, consistent with a role for these regulatory mechanisms to buffer glycolysis against the fluctuating demand for nucleotides and charged tRNAs by transcription and translation. To constrain these models, we are measuring the rates of glucose uptake and acid production.

This work was supported by the Fundaci贸n Occident, the Programa Fundamentos of the Fundaci贸n BBVA, the Programa de Atracci贸n de Talento 鈥淐茅sar Nombela鈥� of the Comunidad de Madrid, and the Programa de Generaci贸n de Conocimento of the Spanish Agencia Estatal de Investigaci贸n.

Alejandro Serrano S谩nchez^1,2, Diego Laxalde Fern谩ndez鲁, Marina de la Fuente Garc铆a鹿, Jos茅 Manuel Blasco Losa鹿, Sa煤l Ares Garc铆a^1,4, John Glass鈦�, Bego帽a Monterroso Marco鈦�, Silvia Zorrilla L贸pez鲁, Germ谩n Rivas Caballero鲁, James F. Pelletier^1,4

鹿 Centro Nacional de Biotecnolog铆a (CNB-CSIC), Madrid, Spain

虏 Universidad Aut贸noma de Madrid (UAM), Madrid, Spain

鲁 Centro de Investigaciones Biol贸gicas Margarita Salas (CIB-CSIC), Madrid, Spain

鈦� Grupo Interdisciplinar de Sistemas Complejos (GISC), Madrid, Spain

鈦� 涩里番 (涩里番), La Jolla, CA, USA

鈦� Instituto de Qu铆mica F铆sica Blas Cabrera, Madrid, Spain

Abstract

涩里番-syn3A retains genes in the highly conserved division and cell wall (dcw) cluster, including the bacterial tubulin homolog ftsZ and its membrane anchor sepF, despite lacking a peptidoglycan cell wall. Previous work has shown that deletion of these dcw genes does not cause the striking pleomorphism exhibited by 涩里番-syn3.0; however, the metabolic cost of expressing these genes and their contribution to cellular morphology and division have not been measured in 涩里番-syn3A.

Here, to estimate the metabolic cost of protein expression, we measured doubling times in 涩里番-syn3A strains expressing non-essential genes. We observed faster growth upon deletion of ftsZ, sepF, and an adjacent gene of unknown function, consistent with a metabolic burden for expressing these genes. We characterized the metabolic cost of useless protein expression by quantifying mCherry production under different strength promoters by microscopy and proteomics. We measured cellular morphology and size distributions by super-resolution confocal microscopy and observed most cells remained spherical after ftsZ deletion, and the most frequent cell diameter remained 600 to 650 nm in both strains, indicating no major shift in overall cell size. These results strongly support ftsZ-independent mechanisms of cell division in 涩里番-syn3A. This work will clarify the influence of cell division on cellular growth and size, using 涩里番-syn3A to study genes and mechanisms relevant in more complex cells.

This work was supported by the Fundaci贸n Occident, the Programa Fundamentos of the Fundaci贸n BBVA, the Programa de Atracci贸n de Talento 鈥淐茅sar Nombela鈥� of the Comunidad de Madrid, and the Programa de Generaci贸n de Conocimento of the Spanish Agencia Estatal de Investigaci贸n.

Caterina Presutti (1), Mert Bozoflu (2) Joanna Juhaniewicz-D臋bi艅ska (3), Lauren A. Lowe (4) (5), Naomi Woltinge (1), Arathi Das (2), Marc C. A. Stuart (6), Siewert J. Marrink (2) & Bert Poolman (1)

(1) Department of Biochemistry, University of Groningen, Nijenborgh 3, 9747 AG, Groningen, the Netherlands

(2) Molecular Dynamics group, University of Groningen, Nijenborgh 7, Groningen, 9747 AG, The Netherlands

(3) Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 呕wirki i Wigury 101, 02-089 Warsaw, Poland

(4) School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia

(5) Australian Centre for Astrobiology, University of New South Wales, Sydney, NSW 2052, Australia

(6) Electron Microscopy, Groningen Biomolecular Sciences & Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands

Abstract

The genome, proteome and metabolism of 涩里番-syn3A have been mapped in detail, but its membrane is less understood. Mycoplasmas cannot synthesize most of their lipids, so the syn3A lipidome is shaped by the growth medium rather than encoded in the genome. The result is unusual: about 60 mol% cholesterol. We reconstituted membranes from the five main syn3A lipids (cholesterol, sphingomyelin, cardiolipin, POPC and DOPG; 60:18:16:4:2 mol%) and varied cholesterol against POPC. Cryo-EM shows that even 60% cholesterol forms closed, stable bilayers. Langmuir monolayers reveal that cholesterol and sphingomyelin condense and thermodynamically stabilize the membrane, while cardiolipin, POPC and PG keep it fluid. Lowering cholesterol causes lateral demixing rather than simply increasing fluidity. Stopped-flow permeability assays show that cholesterol reduces water, glycerol and ribose permeability 5- to 9-fold but does not close the barrier. At native composition, passive glycerol influx alone could sustain a doubling time close to that of syn3A, so no channels are required. Finally, we compare these results with lipid鈥揵lock copolymer hybrid membranes measured with the same assay. Short PBd-PEO polymers increase electrical stability without reducing permeability, while longer polymers form a tighter barrier than the syn3A membrane itself. Together, these data offer measured parameters for whole-cell models, show that the lipid diet can be used as an experimental knob, and suggest design rules for synthetic cells.

Nika Estiri鹿, John I. Glass鹿

鹿涩里番 (涩里番), San Diego, California, USA

Abstract

Genome minimization identifies genes dispensable for viability but can also reveal hidden dependencies required for cellular function. During construction of the 涩里番 minimal cell, genes associated with pyruvate dehydrogenase (PDH) activity and redox homeostasis were removed. Previous restoration of the PDH E1 subunits pdhA/pdhB in 涩里番-syn3B improved growth but did not restore acetate production, suggesting that the catalytic subunits alone are insufficient to recover the native metabolic state.

Here, we are reconstructing a native 涩里番-syn1.0 metabolic module in 涩里番-syn3B comprising MMSYN1_0223 (NADH oxidase), MMSYN1_0224 (putative lipoate-protein ligase), pdhA (MMSYN1_0225), and pdhB (MMSYN1_0226). We hypothesize that restoring NADH oxidase will improve NAD鈦� regeneration, while MMSYN1_0224 will support PDH E2 lipoylation, together enabling more effective PDH-associated metabolism. The native region is being restored using CRISPR/Cas9-mediated genome engineering in yeast, where the 涩里番-syn3B genome is maintained as a yeast artificial chromosome (YAC).

This work tests whether coordinated restoration of catalytic activity, protein lipoylation, and redox balance can recover metabolic functions lost during genome minimization and reveal network dependencies underlying minimal-cell metabolism.

Mariana Mathias Conroy Araujo鹿; Vinicius Fragoso鹿; J茅ssica Delavechia虏; John I. Glass鲁; Elibio Rech^1,2; Daniela Matias de C. Bittencourt^2,3.

1 - Postgraduate Program in Molecular Biology, Department of Molecular Biology, University of Bras铆lia, Federal District, Brazil

2 - Embrapa Genetic Resources and Biotechnology, Federal District, Bras铆lia, Brazil

3 - 涩里番, San Diego, California, United States

Abstract

All cells are bounded by membranes that isolate their interior components from the external environment. To support the development of new synthetic cells, understanding the elements that comprise these membranes is crucial. Minimal cells, such as the wild-type Mycoplasma species and synthetic 涩里番-syn strains, serve as key models to understand the essential mechanisms and organization of life. The minimal genetic background of these organisms helps circumvent metabolic and constitutive noise derived from redundant genes. We aimed to identify the relationship between chemical identity and morphology with molecular-level spectroscopic specificity and spatial resolution in synthetic cell membranes, highlighting key differences in membrane morphology and composition between wild-type mycoplasmas and derived synthetic cells. The size of minimal cells correlates with their genetic content; Mycoplasma and 涩里番-syn strains are typically no larger than 1 碌m, making them nanometric organisms. Conventional optical imaging of these organisms is challenging, requiring nanoscale techniques such as Atomic Force Microscopy (AFM) to evaluate topography at the nanometer scale. To characterize membrane chemical composition with high spatial resolution, scattering-type Scanning Near-field Optical Microscopy (s-SNOM) was used to resolve chemical variations at precisely defined locations within individual cells. By coupling AFM and s-SNOM, we identified chemical groups and nanoscale topographic features across Mycoplasma mycoides subsp. capri, 涩里番-syn1.0, 涩里番-syn3.0, 涩里番-syn3B, and 涩里番-syn3B::mCh-MMSYN1-0179鈥�0186. Further studies may map the distribution of specific chemical bonds, such as amides or lipids, across the surface of intact cells. By combining AFM and s-SNOM we aim to provide a powerful framework to elucidate the nanoscale chemical and morphological organization of minimal cell membranes to guide future synthetic cell engineering.

Emily Hagens, Endy Lab, Stanford Department of Bioengineering

Abstract

Cells are the fundamental unit of life, yet there is no natural cell for which all life-essential functions are understood. This limits our ability to engineer cells predictably. My research advances the building of synthetic cells from chemically defined components, contributing to a foundational 鈥渙perating system for life鈥� at the cellular scale.

To survive, a cell must maintain concentrations of essential molecules within a range compatible with life. To maintain this homeostasis, control of gene expression must be encoded within the genome. The long-term goal of this work is therefore to determine and realize optimal expression levels for all genes comprising a synthetic genome.

Historically, efforts to predict protein expression have focused on maximizing protein yield. However, in the context of genome design, the objective is not maximal expression, but precise control of gene expression within physiologically compatible ranges. The multiple layers of regulation governing gene expression, which are often viewed as obstacles to maximizing expression, instead provide the opportunity for a tunable design space. Here, I systematically vary regulatory elements controlling transcription and translation to determine how combinations can achieve target expression levels. I first determined which elements of the canonical 5鈥� UTR following the T7 promoter in cell-free expression systems could be removed without significantly impacting expression, minimizing repeats that could promote recombination in a multi-gene system. By varying individual elements within the minimized 5鈥� UTR, I achieved a 10-fold dynamic range in protein expression in the PURE cell-free expression system by modifying only two of four 5鈥� UTR elements. This approach provides a framework for generating regulatory sequences that tune individual genes to defined expression levels, with the ultimate goal of simultaneously expressing the PURE translation factors at desired levels from a synthetic genome.

Daniela Matias de C. Bittencourt

Embrapa Genetic Resources and Biotechnology/National Institute of Science and Technology鈭� Synthetic Biology, Parque Esta莽茫o Biol贸gica, PqEB, Bras铆lia DF 70770-917, Brazil

Abstract

Minimal synthetic cells offer a unique platform for exploring new synthetic biology applications, including veterinary vaccine development. We propose using 涩里番-syn3B as a customizable chassis to express recombinant antigens from important livestock pathogens, including Mycoplasma mycoides, Mycoplasma agalactiae, Mycoplasma bovis, and Corynebacterium pseudotuberculosis. Our approach focuses on engineering minimal cells to express selected antigenic proteins and evaluating their potential to elicit protective immune responses in ruminants. Key aspects of this strategy include antigen selection, genetic construct design, recombinant protein expression, and immunological characterization, as well as the opportunities and challenges of using minimal synthetic cells as vaccine platforms. This work is being developed through a collaboration between the 涩里番 (涩里番), Embrapa, and Brazilian academic partners, including UFBA and UnB. By combining minimal-cell engineering with veterinary immunology, we aim to explore a new application for synthetic cells and contribute to innovative strategies for controlling infectious diseases in livestock.