Meeting Proceedings from 4th Minimal Cell Workshop: Exploring JCVI Minimal Cell Fundamental Insights and Integrative Applications

Abstract

The annual Minimal Cell Workshop, hosted by the J. Craig Venter Institute (JCVI), is an international virtual seminar that brings together over 80 academic, industrial, and government laboratories. Researchers use the JCVI’s minimal bacterial cell platform to explore the principles of cellular life and integrate new chemical pathways. Since its creation in 2016, this platform has fostered global collaborations. The fourth workshop featured 26 talks on ongoing research with minimal cell strains, including JCVI-syn1.0, JCVI-syn3.0, JCVI-syn3A, and JCVI-syn3B. Topics included innovative imaging techniques like super-resolution microscopy and cryotomography, DNA replication assays, and minimal cell division. New approaches for ATP synthesis and the role of moonlighting proteins were also discussed. JCVI-syn3B applications explored its potential in understanding persistent pathogens and as an anticancer therapeutic. The workshop encouraged sharing techniques for cell culture and genetic manipulation, fostering collaboration and advancing efforts to develop genome-scale algorithms for understanding and manipulating cellular functions.

Introduction

In 2016, a team from the J. Craig Venter Institute (JCVI) published their groundbreaking work on the design, construction, and analysis of a minimal bacterial cell, JCVI-syn3.0¹. This cell, the simplest bacterium capable of autonomous growth in axenic culture, serves as a platform for exploring the fundamental principles of cellular life. Since then, over 90 research groups have utilized and studied the JCVI minimal cell and related strains (Figure ).

1.

Derivation and Lineage of JCVI Minimal Cells. JCVI-syn1.0 is a near wild type strain of Mycoplasma mycoides subspecies capri that has a synthetic genome. JCVI-syn3.0 is the most minimized strain. It has an unusual division phenotype, is fragile, and not easily handled in many laboratory operations. JCVI-syn3A encodes 19 nonessential genes not in JCVI-syn3.0. It divides like a normal bacterial cell and is not as fragile as JCVI-syn3.0. JCVI-syn3B is the same as JCVI-syn3A except that it contains a dual loxP landing pad that makes Cre recombinase mediated-insertion of new genes straightforward. ,

While the JCVI minimal cell offers a powerful platform for genome reduction and synthetic genome design, many participants also presented research grounded in different strategies and experimental chassisconstructing cell-like systems from defined molecular components, providing insights into constructing metabolic networks, energy management, and membrane synthesis in artificial cells, enriching the development of synthetic biology tools and expanding our understanding of cellular functions. These approaches complement each other by showing how genome-defined minimal cells inform synthetic cell assembly and how bottom-up platforms can test minimal functions in controlled environments. Bottom-up designs can ultimately enhance the JCVI minimal cell framework and other synthetic cell systems. Together, they bridge the gap between reductionist and constructive views of life.

Starting in 2021, members of this collaborative consortium launched a series of multiday online Minimal Cell Virtual Workshops. These workshops featured brief 12–15 min talks on research involving the JCVI minimal cell, as well as other minimal cell systems. This format has proven highly effective, attracting substantial participation from laboratories working on minimal cells and promoting cross-laboratory collaboration. All presentations are recorded and available without restriction at https://www.jcvi.org/events/minimal-cell-workshop.

What follows is a summary of the fourth Minimal Cell Workshop, held in September and October 2024.

Characterization of Minimal Cell: Exploring Cellular Fundamentals

James Pelletier (Centro Nacional de Biotecnología, Spain)

investigated mechanisms governing cell division in the minimal cell, focusing on JCVI-syn3.0, which divides without the FtsZ protein, the bacterial tubulin homologue that forms a Z-ring at the division site in most bacteria. That protein is a key player in most bacterial division processes. JCVI-syn3.0 forms irregularly shaped cells and can divide without both FtsZ and SepF, a protein that stabilizes FtsZ at the division site. Adding these genes did not restore normal cell shape, instead requiring five additional uncharacterized genes. , Pelletier applied biophysical models of membrane physics to explore cell shape and division, using microfluidic devices to study membrane fluidity and its relation to cell shape. Additionally, he worked on biochemically reconstituting FtsZ and related proteins, potentially revealing novel insights into cell division that are difficult to observe in natural systems.

Ronald Rodriguez (JCVI, United States)

aimed to validate computational models for DNA replication, chromatin structure, and central metabolism in minimal cells, − using assays for model confirmation. He discussed fluorescently labeling the DNA replication initiation protein DnaA to visualize DNA replication and cell division with super-resolution microscopy. He also examined a 300-fold reduction in bacterial histone-like protein expression in JCVI-syn3A, potentially caused by genome minimization, which may have led to a lack of chromatin, offering insights into primordial chromatin and cell division. , Finally, he explored predictions of a quasi-essential transaldolase required for a 2 h doubling time, despite being unannotated, and efforts to identify it via transposon mutagenesis and biochemical assays, potentially revealing a novel transaldolase and enhancing our understanding of minimal cell metabolism.

Tae Seok Moon (JCVI, United States)

explored cellular metabolism and aging, focusing on biological ″robustness″ and ATP’s role in maintaining organisms. His study of Escherichia coli showed that removing a kinase increased heat production and metabolic inefficiency due to ATP buildup. He also introduced a microbiota engineering tool to target specific bacteria in complex environments, aiding aging research. Building on this, Moon plans to use JCVI-syn3.0 in aging studies, incorporating advanced single-cell techniques like mother machine technology to track DNA, RNA, and metabolite dynamics.

Edwin Ortega Arzola (The University of Edinburgh, Scotland)

studied energy requirements for sustaining life, focusing on the ″minimum energy″ needed for endergonic reactions that synthesize biomolecules. His team published thermodynamic energy estimates for producing E. coli, mammalian cells, and JCVI-syn3A, showing simpler cells like JCVI-syn3A require less energy. They developed a public algorithm to model energy demands based on genome and protein sequences and growth temperature. Results revealed that while proteins are abundant, lipids are more energy-intensive to synthesize. Although the study excluded metabolic pathways, ongoing work aims to integrate these dynamics. These findings lay groundwork for engineering efficient biofactories and resource management systems.

Karim Fahmy (Technische Universität Dresden BIOTEC, Germany)

used heat flow measurements to study metabolism in JCVI-syn3B minimal cells, offering a systems biology perspective on cellular energetics. By measuring heat flowreflecting metabolic enthalpy changes (ΔH)he created a metabolic profile across the batch culture lifecycle. Using an extended Monod equation, he linked nutrient availability and biomass evolution to heat output. Results showed lipid composition impacted metabolism; for example, sphingomyelin in the diet reduced heat release compared to a two-fatty-acid diet. Dynamic light scattering validated heat flow-derived biomass predictions, showing minimal diets produce smaller cells. These insights advance the use of isothermal calorimetry to explore cellular machinery.

John Sanford (University of Alabama at Birmingham, United States)

focused on protein glycosylation in the minimal cell, using acid–based staining and mass spectrometry to identify glycosylated sites. − Although minimal cells lack genes tied to mammalian pathogenicity, cell surface glycosylationkey to mycoplasma pathogenicityremains. Sanford identified previously unknown glycosylation sites on EF-Tu at aspartic and glutamic acid residues, broadening the known scope of this modification. Future research will explore glycosylation’s essentiality, and identify a hexosyltransferase catalyzing this reaction in the minimal cell. These findings could enhance understanding of glycobiology across life domains.

Shigeyuki Kakizawa (National Institute of Advanced Industrial Science and Technology, Japan)

shared insights on developing serum- and albumin-free media for mycoplasmas, using the minimal cell as a design basis. A combination of 3–5 lipids with cyclodextrin and poly­(vinyl alcohol) (PVA) eliminated the need to bind the lipids to albumin as has been used in earlier serum free media. While growth rates were slower than with standard SP4 serum media, this formulation lowers costs, eliminates variability from animal-derived serum, and removes animal byproducts, making research more sustainable and accessible.

Expanding Minimal Cell Functionality: Genome Modification and Metabolic Enhancement

James Daubenspeck (University of Alabama at Birmingham, United States)

studied protein moonlighting, where a single protein performs multiple biological functions, a phenomenon seen across all life domains. , Many such proteins were cytoplasmic, surface-associated, or both. He highlighted cytoplasmic proteins like DnaK, Enolase, and EF-Tu, which associate with the minimal cell surface through a rhamnose modification that binds to membrane phospholipids. Future research will explore the roles of these surface-associated proteins and whether they differ from their annotated functions. These findings could significantly impact efforts to streamline natural genomes further.

Manoel Neres Santos (Federal University of Bahia, Brazil)

presented research on microorganism-host interactions between minimal cells and caprine cell cultures, focusing on immunomodulatory mechanisms of Mycoplasma mycoides and JCVI-syn cells. Initial experiments showed that only wild-type M. mycoides subsp. capri significantly inhibited goat peripheral blood mononuclear cell proliferation. He analyzed immune response modulation by measuring inflammatory markers, finding nitric oxide production across all strains, while markers like IL1β, TNFα, and IL4 varied in JCVI-syn3A at different time points, indicating potential immunomodulation. Future plans include transcriptomic analysis in goats, aiming to confirm whether these cells avoid provoking immune responses, with implications for vaccine development.

Sage Glass (JCVI, United States)

discussed efforts to validate computational models predicting the impact of restoring the pyruvate dehydrogenase enzymatic complex (PDHEC) in JCVI-syn3B. During JCVI-syn1.0 minimization, the pdhA and pdhB genes were removed, deactivating PDHEC. Models predicted that reintroducing these genes would restore PDHEC, enhance JCVI-syn3B growth, and resume acetate production. Inserting pdhA and pdhB into JCVI-syn3B’s dual loxP landing pads improved growth rate but did not restore acetate production. Glass described methods to measure acetate diffusion in modified cells and outlined future experiments to further test these predictions.

Rridhisha Kumar (JCVI, United States)

worked to integrate the POAP carbon fixation pathway into the JCVI-syn3B minimal cell, aiming to create the first synthetic in vivo carbon capture system. The POAP cycle, chosen for its efficiency, uses just four enzymes, pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase, to fix carbon dioxide at rates surpassing natural and synthetic pathways. Leveraging the minimal cell’s streamlined metabolism, the team inserted POAP enzymes into its genome and used GC-MS to assess metabolic impacts. They also thermally adapted the cell to 44.2 °C to support the pyruvate:ferredoxin oxidoreductase enzyme, which functions best above 40 °C. Current efforts focus on optimizing the pathway and validating its functionality via GC-MS.

Mariana Mathias Conroy Araujo (EMBRAPA, Brazil)

outlined her goal to enhance genome manipulation in JCVI-syn cells by leveraging CRISPR/Cas and recombinase proteins to address whole genome transplantation (WGT) challenges. Her strategy integrates Cas protein expression with repair systems, absent in some mycoplasmas like JCVI-syn cells, regulated by inducible promoters and integrase-based circuits. The research aims to develop Cas-expressing strains and refine the minimal cell by reducing its genome and investigating genes with unknown functions.

Keoni Gandall (Nanala, LLC, United States)

showcased efforts to reduce DNA synthesis costs and improve accessibility for large-scale synthetic gene assembly by developing a high-throughput pipeline. By optimizing oligo pool cloning, automated liquid handlers, and Flongle nanopore sequencing he created a cost-effective system capable of producing hundreds of plasmids weekly at a fraction of typical costs. He tackled challenges like high mutation rates and sequencing errors with robust quality control methods. Moving forward, Gandall aims to assemble a 60-codon JCVI-syn3A genome, scale up his workflow, live-stream plasmid assemblies, and refine oligo pools to lower mutation rates, advancing routine, affordable synthetic genome construction.

Computational Approach to Mapping Minimal Cell Functionality

Mallak Ali (Chan-Zuckerberg Imaging Institute, United States)

outlined the workflow and automation used to generate cryo-electron tomography (cryoET) images of JCVI-syn3A. Its small size and morphology eliminated the need for focused ion beam milling, accelerating tomography processing. Ali also introduced the CryoET Data Portal, an open platform by the Chan-Zuckerberg Imaging Institute for sharing and collaborating on annotated tomograms. Those images were recently posted.

Jen Stevens (University of Groningen, Netherlands)

developed molecular simulations of the JCVI-syn3A minimal cell using coarse-grained Martini models, which are high-resolution computational tools from the University of Groningen that simulate molecular interactions. − These models provide insights into cellular organization, helping experimentalists better understand cell behavior, structure, and dynamics. Stevens highlighted efforts to build these models based on chromosome dynamics and omics data.

Rahmi Lale (Norwegian University of Science and Technology, Norway)

presented the Sigma Factor DNA Binding Sequences (SiD) project, which investigates sigma factor binding motifs in promoters to understand transcriptional regulation. The team mapped binding sites to identify active promoters in organisms like JCVI-syn3.0, Bacillus subtilis, E. coli, and Pseudomonas putida. Using microfluidic droplets for in vitro transcription, they linked genotype to phenotype by analyzing transcriptional activity. Lale detailed the project pipeline and its applications, including deep learning-based promoter prediction tools.

Nguyen Ha Ngoc Anh (B Cube-Technische Universität Dresden, Germany)

analyzed lipid complexity and remodeling in the minimal cells JCVI-syn3B (493 genes) and Mesoplasma florum (720 genes). Despite sharing enzymatic pathways, the two species employed distinct lipid strategies to adapt to temperature changes. JCVI-syn3B’s imported lipids varied with cholesterol, sphingomyelin, and phosphatidylcholines (PC) and it altered the amounts of cardiolipin it synthesized. Mesoplasma florum’s imported lipids were stable, Instead, it dynamically regulated amounts of phosphatidylglycerol (PG) and diacylglycerol (DAG) it synthesized. JCVI-syn3B also showed headgroup-specific lipid remodeling, unlike M. florum, which appeared to use a simpler mechanism. The study highlights the complexity of minimal cell membranes and points to future research on the roles of CL and DAG in cell shape and division.

Valerio G. Giacobelli (Charles University in Prague, Czech Republic)

of Klara Hlouchová’s lab described plans to use the JCVI minimal cell as a model for studying adaptive evolution, focusing on replacing complex amino acids (basic, amides, aromatic, sulfur-containing) with simpler, early amino acids (hydrophobic, acidic, hydroxy). Giacobelli explained early amino acids are less energetically costly but may not support complex metabolic processes. The lab demonstrated that individual proteins could be composed entirely of simple amino acids without losing functionality. They plan to use bioinformatics tools to explore this approach in JCVI-syn3B, potentially advancing our understanding of amino acid evolution and the shift to complex proteins.

Lea Kloss (Heinrich Heine University, Germany)

explored the repeatability and predictability of adaptive laboratory evolution (ALE) in the JCVI-syn3B cell. By leveraging its reduced complexity, Kloss assessed whether evolutionary outcomes were consistent across replicates and modeled these processes with constraint-based metabolic models. She integrated nonlinear reaction kinetics, including Michaelis–Menten dynamics, into flux balance analysis for better accuracy, factoring in density constraints for metabolite and protein predictions. Initial robotic platform experiments under continuous growth faced challenges with condensation, optical density, and pipetting errors. Ultimately, the study aimed to refine growth models and enhance ALE as a tool for studying evolution and metabolism.

Building New Synthetic Cells: From Concept to Construction

Bert Poolman (University of Groningen, Netherlands)

presented a conceptual minimal cell network composed of membrane transporters, encapsulated enzymes, and passively diffusing small molecules. The system relied on arginine deamination and ATP/ADP antiporters for ATP generation, with ADP recycling stabilizing and enhancing ATP levels. − To minimize futile ATP hydrolysis and internal acidification, the study emphasized the need for ATP consumption by downstream processes. By coupling vesicles that exported ATP with those that imported ATP and secreted ADP, syntropic behavior emerged, extending reaction longevity. Future work aims to enable vesicle growth through lipid synthesis and mimic organelle function by encapsulating smaller vesicles and introducing electrochemical energy modules to support pH and ion homeostasis.

Miyer Patino-Ruiz (University of Groningen, Netherlands)

explored using proton motive force (PMF) to enable nutrient uptake and metabolic activity in synthetic cells. Using large unilamellar vesicles (LUVs) with a reconstituted L-malate decarboxylation pathway, the membrane potential probe DiSC3(5), and the pH probe pyranine, they demonstrated that the two-protein system of L-malate and L-lactate generated sufficient PMF to drive glutamate transport into vesicles, achieving a 150-fold accumulation to millimolar levels, suitable for synthetic cell metabolism. The same malate decarboxylation system, combined with a proton-lactose symporter, transported substantial amounts of lactose. Coupling PMF with a minimal metabolic pathway also enabled NADPH production. Future efforts focus on enhancing efficiency by proton-amino acid symporters, scaling up vesicle volumes for greater metabolic capacity, and integrating PMF conservation with ATP generation.

Jelmer Coenradij (University of Groningen, Netherlands)

described his investigation of the membrane volume and surface area needed to encapsulate soluble and membrane proteins. ,,, Models estimated that reconstituting the JCVI-syn3A membrane proteome at natural occupancy required vesicles of at least a 1 μm radius when including peripheral membrane proteins and free-floating lipids. Jelmer integrated Bert Poolman’s ATP-generating module into micron and submicron vesicles to observe single-cell pathway progression. For JCVI-syn3A-sized vesicles, the Arg/Orn antiporter was crucial for ATP production, but in larger vesicles, it was unnecessary due to amino acid permeability and ATP retention. Jelmer is now working on coupling ATP vesicle modules with membrane growth modules, focusing on 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol) synthesis from 3,4-Dihydroxyphenylalanine (with plans to encapsulate ATP-generating LUVs in giant unilamellar vesicles with phospholipid synthesis pathways.

Daniel Nucifora (Western University, Canada)

described his efforts to develop WGT for Acholeplasma laidlawii by leveraging insights from mycoplasma genome manipulation. He developed a stable transformation protocol and genetic toolbox for A. laidlawii, successfully cloning its genome into yeasta key step toward creating hybrid strains for phytoplasma research. By disrupting the toxic gene ACL_0177, he demonstrated the feasibility of integrating A. laidlawii and phytoplasma genomes, enabling advances in pathogen studies and genome engineering. His future work will optimize WGT and establish A. laidlawii as a versatile synthetic biology model for designing endosymbiotic and organelle-like systems.

Zumra Seidel (JCVI, United States)

described development of WGT for non-Mycoplasma bacteria, tackling challenges like genome degradation and endonuclease activity. Initial findings indicated that trypsin and mitomycin C treatments of recipient cells might facilitate successful genome transplantation for other bacteria. Her plan focused on intraspecies transplantation in model Gram-positive and Gram-negative bacteria, specifically Streptococcus thermophilus and E. coli, chosen for their small genomes and established transformation protocols. Plans included using microfluidic technology to automate and visualize the process, enhancing efficiency and reducing reliance on manual operations for synthetic cell creation.

Tyler Goshia (JCVI, United States)

outlined progress in building synthetic cells from nonliving components using mycoplasma parts. This work, initially presented by Andrei Sakai at the third Minimal Cell Workshop, focuses on combining a minimal genome with energy and replication machinery from Mycoplasma capricolum in a TXTL system. The components will be encapsulated in synthetic lipid liposomes mimicking M. capricolum membranes. Membrane-bound RNases are separated from the cytoplasmic extracts using nitrogen cavitation, and metabolite transport is enabled by incorporating transporter proteins into the liposome envelope via a plasmid. Two methods for introducing the minimal genome were discussed: WGT and a chemical transplantation technique using liposome fusion, developed by Kate Adamala. This fusion is triggered by complementary DNA molecules on liposome membranes. Key project challenges will likely include optimizing genome installation and fine-tuning transporters, osmotic pressure, and temperature during activation.

Daniela Bittencourt (EMBRAPA, Brazil)

described her lab’s approach to producing SimCellsgenomeless cells incapable of replication. She engineered JCVI-syn3B and JCVI-syn1.0 to inducibly express I-CeuI, an endonuclease targeting bacterial 23S rRNA genes. I-CeuI cleaves the genome, and endogenous exonucleases degrade the DNA, leaving functional cells without chromosomes. To prevent lethality from constitutive I-CeuI expression, its gene was placed under a tetracycline-inducible promoter within a genetic circuit for precise regulation. , She also introduced an alternative ATP-production pathway via arginine deaminase that enabled her to effectively monitor SimCell metabolic functionality after DNA degradation. Future work aims to purify genome-free cells, evaluate their viability, and use them as carriers for synthetic genomes.

Conclusion Remarks and Outlook toward Future

The fourth Minimal Cell Workshop highlighted progress in synthetic cell technology, starting with the JCVI minimal cell framework. This platform enables exploration of life’s fundamentals, such as metabolic systems, homeostasis, and reaction networks, while serving as a chassis for developing synthetic biology tools, and studying biological processes.

Simultaneously, bottom-up approaches focused on creating synthetic cells from scratch offer valuable lessons in designing metabolic networks, achieving homeostasis, and managing energy production through compartmentalized systems like vesicles. These systems aim to replicate biological functions, such as ATP production, in simplified environments, often employing lipid-based membranes and metabolic pathways, such as the arginine deiminase pathway, to power essential cellular processes. By integrating insights from both top-down (JCVI) and bottom-up approaches, we can foster the development of more complex, functional synthetic cells, driving forward advances in biotechnology and other applications.

Minimal cells provide a foundation for uncovering essential networks, expanding functionality through genome modifications, and enhancing metabolic capabilities. Computational models are being developed to guide synthetic cell design and uncover unexplored genomic processes. These advances pave the way for custom synthetic cells with novel functions, driving innovation in biotechnology and beyond.

Zumra Seidel, Rridhisha Kumar, Mariana Mathias Conroy Araujo, Sage Glass, Ronald Rodriguez, and Tyler Goshia summarized the presentations, with each author covering 3–5 talks. Zumra Seidel and Rridhisha Kumar created the figures. Zumra Seidel drafted the manuscript, including the introduction and conclusions sections. John I. Glass supervised the writing and did the final editing. All authors provided critical feedback, contributed to shaping the manuscript, and approved the final version.

The authors declare no competing financial interest.