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James M Daubenspeck, John Sanford, and Prescott Atkinson

University of Alabama at Birmingham

Abstract

DNA, RNA, Protein, the central dogma of biology is straightforward, simple, and implies that one gene equals one function. However, proteins can perform multiple functions by changing their location, interactions, shape, or oligomeric state. These additional functions are essential in organisms with minimal genomes and this process is still active in JCVI-syn3.0. Multifunction or “Moonlighting” proteins are ubiquitous, found in every domain of life. In bacteria, it is common for cytoplasmic enzymes to traffic to the membrane for an additional role, with most of these secondary functions being unknown. The mechanism for targeting these proteins to the surface is not well understood. In mycoplasmas, a rhamnophospholipid is utilized to link the protein to the membrane and may act as a signal for transport. In JCVI-syn3.0 rhamnose synthesis had been knocked out resulting in syn3.0 utilizing mannose for this molecule, highlighting the essentiality of this pathway and suggesting that rhamnose synthesis in the parent goes through mannose. Utilizing high resolution mass spectrometry, we have identified in excess of 100 proteins from the syn3.0 proteome that inhabit the membrane and have multiple functions. The highly conserved cytoplasmic proteins Enolase, DnaK, and EF-Tu have this modification and have been shown to be on the surface of JCVI-syn3.0. While a 21% increase in the size of the proteome is significant, I would suggest that as many as 50% of the proteins in JCVI-syn3.0 have multiple functions and are simply below our detection limit.

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John W. Sanford¹, James Mobley, PhD², Kevin Dybvig, PhD¹, T. Prescott Atkinson, MD, PhD¹, James Daubenspeck, PhD¹,

Department of Pediatrics¹ and Department of Anesthesiology and Perioperative Medicine², University of Alabama at Birmingham, Birmingham, AL

Abstract

Protein moonlighting is a phenomenon observed in all kingdoms of life where proteins perform additional functions beyond their canonical role. Many ancient proteins involved in indispensable metabolic pathways in the cytoplasm, such as glycolysis and translation, are found to localize to the cell surface, where they perform cryptic secondary functions which are difficult to discern using conventional molecular biology techniques. Organisms in the genus Mycoplasma are attractive models for studying protein moonlighting due to their minute genomes, often <1 megabase pairs in length. Furthermore, JCVI-Syn3A’s near-minimal gene set allows further insight into which metabolic pathways are indispensable for bacteria, even when grown in axenic culture.

We hypothesized that protein moonlighting previously observed in Mycoplasma pulmonis is conserved in other mycoplasmas, including JCVI-Syn3A. To test this, we performed high-resolution liquid chromatography-tandem mass spectrometry on ultracentrifuged membrane material to generate proteomic datasets in Mycoplasma mycoides subsp. capri, JCVI-Syn1.0, and JCVI-Syn3A. After digesting the membrane material through in-solution trypsin/Lys-C digestion, we generated mass spectra using both collision-induced dissociation and higher-energy collisional dissociation in Orbitrap mass analyzers.

Our data indicate proteins known to moonlight on the cell surface in other bacteria (e.g., enolase and EF-Tu) are found on the surface of JCVI-Syn3A. These data also expand the list of potential moonlighting proteins to include much of JCVI-Syn3A’s proteome. Conservatively restraining our putative surface proteins to include only proteins shown to be glycosylated with the putative sugar phosphate anchor (see James Daubenspeck’s talk) yields >50 moonlighting proteins, along with proteins known to localize to the surface.

Our JCVI-Syn3A data align with our current understanding of the dynamic cell surface landscape of mycoplasmas. We are currently performing surface shearing assays to increase the confidence of our putative identifications and account for the nebulous distinction between the mycoplasma surfaceome and mycoplasma secretome, which has recently been conceived as a unified ‘releasome’. Many moonlighting proteins conserved in pathogenic bacteria such as Staphylococcus aureus and Bacillus cereus are believed to function as virulence factors, however the presence of moonlighting in the non-pathogenic JCVI-Syn3A implies a more fundamental role in bacterial physiology. Further work on protein moonlighting will yield information on currently unknown fundamental processes in biology and increase accuracy of whole cell models.

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Nika Estiri, John Glass, and Tae Seok Moon

J. Craig Venter Institute

Abstract

The synthetic minimal cell JCVI-syn3.0B provides a uniquely simplified platform to investigate the fundamental biology of cellular aging. We combine a surface-capture system—anchoring individual “mother” cells while continuously removing their daughters—with integrated multi-omics analyses, including genomics, transcriptomics, metabolomics, and proteomics, to profile cells across their entire lifespan. This design enables direct comparison of senescent and young cells at multiple molecular layers, revealing how targeted genomic alterations influence gene expression, metabolic state, and protein composition over time. Coupled with advanced mathematical modeling, our approach identifies conserved aging pathways and offers a powerful, scalable strategy to accelerate the discovery of interventions that may modulate aging across species.

Impact Statement: This minimal-cell platform integrates real-time single-cell tracking with multi-omics to explore universal mechanisms of aging, paving the way for targeted interventions to extend healthy lifespan.

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Wichittra Arai, Masaki Mizutani, Shigeyuki Kakizawa

Molecular Biosystems Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

Abstract

Previously, we developed serum- and albumin-free media in which the minimal cell could grow well. Here, a synthetic defined medium was constructed based on this formulation. Undefined components of SP4 medium (fresh yeast extract solution, Mycoplasma broth base [PPLO powder], tryptone, and Yeastolate) were removed, while peptone was retained, and mixtures of amino acids, vitamins, and nucleobases were added. As a result, JCVI-syn3B grew well, whereas JCVI-syn3.0 exhibited slow growth. When peptone was excluded, no growth was observed even when all 20 amino acids were sufficiently supplied. To confirm the effect of peptone, synthetic peptides (chemically synthesized, purified, and custom-made peptides) were added to the medium instead of peptone, and robust growth of JCVI-syn3B was observed. These findings suggested that the minimal cell requires polymerized peptides as a nutritional source in addition to singular amino acids. It is expected that this defined medium will be used for various applications.

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Yavuz Yabuseven

J. Craig Venter Institute

Abstract

Mycoplasmas are notoriously difficult to count because of their small size and tendency to form clumps. Here, we evaluated the performance of BactoBox, an impedance flow cytometer, for monitoring the growth of Mycoplasma mycoides (GM12) and the minimal cell JCVI-syn3B. Measurements obtained with BactoBox were benchmarked against two gold-standard methods for mycoplasma enumeration—PicoGreen DNA quantification and color-changing unit (CCU) assays—at defined time points. BactoBox provided reliable counts during the first 48 hours of culture, enabling enumeration within minutes, compared to days with conventional methods. Beyond rapid counting, impedance data revealed a previously unrecognized phenotype linked to culture aging, marked by systematic shifts in electrical properties (phase angle distribution). This expands the spectrum of phenotypic traits observable in JCVI-syn3B and JCVI-syn1.0. Together, our results establish BactoBox as a practical tool for rapid mycoplasma enumeration across growth phases and highlight a novel electro-phenotype with potential value for laboratories studying synthetic and minimal cells.

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Jérémy Gagnon, Dominick Matteau, Simon Jeanneau, Anthony Duval, Julien Faure-Lévesque, David Jaramillo Salazar, Antoine Castonguay, Pierre-Étienne Jacques, and Sébastien Rodrigue

Université de Sherbrooke, Québec, Canada

Abstract

Mesoplasma florum, a fast-growing and non-pathogenic Mollicute, offers a streamlined platform for exploring minimal cellular life and advancing synthetic genomics. To enable precise experimental control, we developed a serum-free growth medium (CMRL-AT) and performed RNA-seq analysis to characterize transcriptional responses under defined versus complex conditions.

In parallel, we are developing a robust, low-cost, and modular method for assembling the wild-type genome of M. florum, laying the groundwork for future construction of a minimal genome. This approach will integrate high-density transposon mutagenesis (HDTM) data to identify non-essential genes and refine genome annotation. By cross-referencing HDTM results with curated databases, experimental datasets, and theoretical, we aim to rationally design a minimal genome optimized for growth and engineering.

Together, these efforts position M. florum as a versatile chassis for synthetic biology, bridging transcriptomic profiling, functional genomics, and genome design to illuminate the core principles of cellular life.

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Steven Bonn, Angela Gronenborn

University of Pittsburgh, Pittsburgh, PA United States of America

Abstract

Many genes encoding essential and quasi-essential proteins in the minimal cell genome do not yet have a specific annotation. We seek to use structural methods to elucidate the roles of some of these proteins. JCVISYN3A_0873 is highly conserved across Mycoplasma, and we predict that this protein is a single domain SH3-like protein, an anomaly for SH3-like proteins, which are known only as domains in larger proteins. We plan to carry out a more complete structural and biophysical characterization of 0873 to determine if it truly is an example of a free SH3, and we hope to identify interaction partners.

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Zachary Krieger, Cara Hull, Alessandro Coradini, Ian Ehrenreich

University of Southern California

Abstract

Minimal cells contain substantially reduced genomes, supported only by an essential set of genes required for survival and proliferation. A eukaryotic cell with a minimal set of genes can provide insight into the fundamental genomic components required for eukaryotic life. Generating such a minimal eukaryotic cell is challenging due to multiple biological and technical reasons. Here, we demonstrate a new method, Minimal or Streamlined Architectures of Individual Chromosomes (MoSAIC), that can be used to experimentally eliminate many non-adjacent dispensable regions of a chromosome in the budding yeast Saccharomyces cerevisiae. MoSAIC involves recombination between a focal chromosome and synthetic DNA fragments containing known essential genes from that chromosome. The output of MoSAIC is a panel of euploid cells that contain substantially reduced, if not minimal, focal chromosomes. I will describe our ongoing work to generate a minimal version of S. cerevisiae Chromosome I using MoSAIC. I will also discuss insights into how MoSAIC can be used to potentially generate cells in which all chromosomes have been minimized.

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SANTOS JÚNIOR, M. N.; GOMES SAMPAIO, B. A.; CAMPOS, G. B.; BASTOS, B. L.; FREIRE, M.; GLASS, J.; BITTENCOURT, D.M.C.; RECH, E.; MARQUES, L. M.

Abstract

This study evaluated the immunomodulatory effects of the synthetic genome of Mycoplasma mycoides subsp. capri (Mmc) strains on caprine peripheral blood mononuclear cells (PBMCs). PBMC cultures (1×10⁶ cells/mL) from clinically healthy goats were exposed for 6–24 h to JCVI-syn1.0, JCVI-syn3A, JCVI-Syn3⸫mch179-180, JCVI-Syn3⸫mch179-186, and a wild-type strain. Cell viability was measured by alamarBlue™, nitric oxide (NO) by the Griess assay, cytokines (IL-1β, TNF-α, IL-4) by ELISA, and immune cell phenotypes and lymphoproliferation by flow cytometry. Synthetic strains significantly inhibited PBMC proliferation compared with controls, except JCVI-Syn3⸫mch179-180 and JCVI-Syn3⸫mch179-186 at 12 h, and all strains induced NO production at all time points. IL-1β was consistently upregulated at 6 and 12 h, with JCVI-syn1.0 eliciting the strongest early response; TNF-α was initially triggered by the wild-type strain and subsequently by synthetic strains at later intervals, while IL-4 remained low until 24 h, when it increased in all strains except JCVI-syn3A. Flow cytometry revealed early CD14⁺ cell activation by JCVI-syn1.0 and the wild-type strain at 12 h, whereas JCVI-syn3A displayed a delayed response at 18 h. CD21⁺ B-cell frequencies increased across all strains at both time points, indicating robust B-cell activation. Lymphoproliferation analysis revealed that all strains, except JCVI-syn1.0, stimulated T-cell proliferation without cytotoxic effects. Together, these data provide a framework to dissect host–pathogen interactions and the immunological consequences of minimized bacterial genomes. An ongoing in vivo challenge is being conducted by intranasal inoculation of goats with both synthetic and wild-type strains to assess bacterial load, cytokine expression, and histopathological outcomes. These results indicate that synthetic Mycoplasma mycoides subsp. capri strains can modulate both innate and adaptive immune responses in a strain- and time-dependent manner. Differential cytokine production, NO induction, and activation of CD14⁺ and CD21⁺ cells suggest potential for dissecting host–pathogen interactions and guiding the design of immunomodulatory strategies. Ongoing in vivo studies will clarify their impact on immune responses and pathogenesis in goats.

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Daniel Nucifora

Western University

Abstract

Cloning, modification, and transplantation of whole genomes provide a powerful means to engineer bacterial strains, as demonstrated by the creation of the JCVI minimal cell.Currently, genome transplantation is limited to a handful of species within the class Mollicutes. We propose extending this technology to the related species Acholeplasma laidlawii, which, unlike most other Mollicutes, uses a standard genetic code and does not require serum for growth. These features could make A. laidlawii a more versatile platform for creating synthetic cells. To this end, we have selected two strains, PG-8A and 8195, to serve as a donor and recipient, respectively, for genome transplantation. We have established new genetic tools for A. laidlawii 8195, including an optimized electroporation protocol, multihost shuttle plasmids, and genomic integration of Tn5 transposons. We have also evolved a derivative strain with significantly higher plasmid transformation efficiency than wild type. Using these tools, we restored homologous recombination in A. laidlawii 8195 and created a strain with a yeast-vector insertion, allowing us to transfer the 1.5 Mb genome to Saccharomyces cerevisiae. We are now focusing our efforts on developing a working genome transplantation protocol for A. laidlawii, which is the final step before we can create synthetic Acholeplasma cells.

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Vinod Kumar Gupta

Chaitanya Science and Arts College (Autonomous)

Abstract

The exploration of minimal cellular systems to uncover life’s fundamental principles is a key scientific frontier. This work presents “Jeewanu,”¹ an abiogenically formed, protocell-like model synthesized via solar-induced photochemical reactions in a sterilised aqueous mixture of inorganic and organic substances. These self-organizing microstructures exhibit growth, budding, and metabolic features including ATPase-like activity, detected by labelled substrate assays. Jeewanu models provide an experimental platform to study prebiotic pathways from chemistry to living systems, bridging gaps between synthetic minimal cells and origin-of-life protocell constructs. By integrating photochemical synthesis with functional analyses, this approach offers new insights into early cellular evolution and the emergence of metabolism. This contribution complements minimal cell research by addressing the abiotic chemical foundations potentially preceding genetic minimalism, enriching understanding of cell design from both top-down and bottom-up perspectives.

Reference: 1. Bahadur, K. and Ranganyaki, S. (1970) J. Brit. Interplanetary Soc., 23, 813-829

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Yuge Feng, Junbiao Dai, Chunbo Lou

Abstract

We explore whether the E. coli proteome can function on a 19-amino-acid alphabet by systematically reassigning isoleucine codons to valine. Our design loop couples four constraints in one multi-objective programme: (i) residue-residue dependencies inferred from multi-species co-evolution to preserve structural couplings; (ii) sequence “viability” from protein language models to avoid out-of-distribution changes; (iii) codon-level usage metrics to maintain expression economy; and (iv) structure-aware risk screens integrating modern predictors with lightweight stability checks. We prioritise coordinated substitution sets at catalytic loops, cores and interfaces, rather than isolated edits, and validate accessibility using an adaptation assay (barcoded pools under T4 phage–host pressure) alongside an orthogonal wobble tRNA/aaRS layer that buffers historically Ile-sensitive positions. Early results indicate that coordinated designs reduce destabilisation at functional loops while preserving packing across essential complexes, with misreading risk localised to surface–core belts. The approach reframes genome rewriting as AI-constrained, evolution-validated optimisation. We anticipate that these principles — simplifying encodings while keeping cells evolvable — will be of direct interest to the minimal-cell community.

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Gesse Roure, Vishal S. Sivasankar, and Roseanna N. Zia

Department of Mechanical and Aerospace Engineering, University of Missouri, Columbia

Abstract

Nucleoid compaction in bacterial cells has been attributed to cytoplasmic crowding, supercoiling effects, and the action of nucleoid-associated proteins (NAPs). In most bacteria, including E. coli, these mechanisms condense the nucleoid to a smaller volume within the cell, excluding most ribosomes to the surrounding cytoplasm. In contrast, the nucleoid in Mycoplasmas, including the Mycoplasma-derived synthetic cell JCVI-Syn3A, spans the entire cell, with ribosomes distributed throughout. Recent models of Syn3A representing only DNA and ribosomes (both charge neutral) instantiated the experimentally-observed expanded nucleoid and ribosome distribution. However, we found that this configuration becomes dynamically unstable, giving way to a compacted nucleoid that expels ribosomes to the periphery, suggesting the need for a more detailed model. Speculation emerging from recent studies of Syn3A suggests that its lower concentration of NAPs underlie its expanded nucleoid. We are interested in this genotype to-`physiotype'-to-phenotype implication: that coupled transcription, translation, and nucleoid remodeling lead to different phenotypical outcomes. We developed a coarse-grained computational model of Syn3A, physically and explicitly representing ribosomes, cytoplasmic proteins, and a sequence-accurate chromosome with physiological distributions of size, charge, and relative molecular abundance. An interplay between Brownian dynamics, DNA stiffness (both inherent and NAP-enhanced), and electrostatic charge lead naturally to a stable molecular distribution. We find that an interplay between inherent and induced DNA stiffness, heterogeneous mesh size, and crowding enhances nucleoid compaction and ribosome expulsion via a competition between entropic and enthalpic forces.  In contrast, electrostatic interactions and size-polydispersity counteract these effects and expand the nucleoid. In particular, Syn3A's atypically high abundance of positively-charged proteins shields ribosomes' negative charge, allowing them to interpenetrate the nucleoid. Finally, we observe condensate formation arising from electrostatic interactions, giving potential implications on transcription and translation rates.

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Enguang Fu and Zan Luthey-Schulten

University of Illinois at Urbana-Champaign

Abstract

Macromolecular complexes in the genetically minimized bacterium, JCVI-syn3A, support gene expression (RNA polymerase, ribosome, degradosome), metabolism (ABC transporters, ATP synthase) and chromosome dynamics. In this work, we further incorporate the assembly of 21 unique macromolecular complexes into the existing whole-cell kinetic model of Syn3A. The synthesis and translocation of protein subunits in membrane complexes occur through distinct pathways. A range of 2D association rates on cell membrane were considered to guarantee a high yield of assembly given the existing time scales of the gene expression with derived formula to rationalize the choice. By alleviating the undesired kinetically trapped intermediates in ATP synthase assembly, the efficiency was improved, while the heterogeneity of subunit synthesis at the single-cell level greatly undermines it. The assembly of RNA polymerase, ribosome, and degradosome influence the speed and efficiency of protein synthesis. Collectively, this model predicted time-dependent cellular behaviors consistent with experiments. A deep learning analysis of the simulated time-dependent concentrations of 148 intracellular metabolites allowed the construction of a trajectory tree from which three distinct metabolic phenotypes were identified in the population of 100 cells considered. At last, we want to briefly discuss extending the simulation to cover multiple cell cycles.

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Alejandro Serrano Sánchez¹, Diego Laxalde Fernández², Marina de la Fuente García¹, Saúl Ares García1³, John Glass⁴, Germán Rivas Caballero², James F. Pelletier^1,3

¹ Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain; ² Centro de Investigaciones Biológicas Margarita Salas (CIB-CSIC), Madrid, Spain; ³ Grupo Interdisciplinar de Sistemas Complejos (GISC), Madrid, Spain; ⁴ J. Craig Venter Institute (JCVI), La Jolla, CA, USA

Abstract

Many bacterial species regulate cell division and cellular size; however, it remains unclear how cellular growth and fitness depend on cell division and cellular size, in part due to their complex genetic bases in natural bacteria. JCVI-syn3B provides a simplified model system but retains highly conserved genes that participate in cell division, such as the bacterial tubulin homolog ftsZ. To study relationships between cellular growth and size, we are measuring cellular growth rates by attenuation spectroscopy, replication of colony forming units, and accumulation of genomic DNA; and we are estimating cellular size distributions by confocal microscopy. A strain lacking ftsZ and adjacent genes grows faster than JCVI-syn3B. To characterize the metabolic burden of protein expression, we are engineering inducible promoters for JCVI-syn3B. To estimate the metabolic cost of GTP hydrolysis by FtsZ, we have performed biochemical studies with purified FtsZ. The strain lacking ftsZ exhibits a similar cellular size distribution as JCVI-syn3B, suggesting FtsZ-independent mechanisms for cell division.

Collaboration supported by the Programa Fundamentos 2022 of the Fundación BBVA.

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Mert Bozoflu, Jan A. Stevens, Bart M.H. Bruininks, Chelsea M. Brown, Siewert J. Marrink

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands

Abstract

The JCVI-syn3A minimal cell offers a unique platform to study how membranes encode the physical principles of life. Here we present large-scale molecular dynamics simulations of the complete Syn3A cell envelope, comprising ~0.7 billion coarse-grained particles and more than 4,400 membrane proteins. Using the Martini 3 force field and integrative modeling workflows, we constructed vesicle systems with either an experimentally validated five-lipid composition or a simplified two-lipid system proposed to support minimal cell growth. Microsecond-scale simulations reveal composition-dependent differences in protein clustering and lipid enrichment, demonstrating how membrane chemistry governs collective organization at the cellular scale. These simulations represent one of the most comprehensive minimal cell models to date and provide a framework for connecting molecular interactions to emergent cellular properties.

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Zumra Seidel¹, Nacyra Assad-Garcia², Vanya Paralanov², Feilun Wu², Olivia Chao², Elizabeth A. Strychalski³, Eugenia Romenstova³, Tyler Goshia¹, John I. Glass¹

¹ J. Craig Venter Institute, San Diego, CA 92037, USA; ²J. Craig Venter Institute, Rockville, MD 20850, USA; ³National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899, USA

Abstract

Whole genome transplantation (WGT) enables the replacement of a recipient chromosome with a donor genome, reprogramming the cell to adopt a new genetic identity. This strategy has the potential to redefine synthetic biology by making entire genomes accessible to precise manipulation. The process has been demonstrated only between closely related Mycoplasma species, most notably the transfer of Mycoplasma mycoides genomes into Mycoplasma capricolum. However, efforts to extend genome transplantation to other bacteria have been hindered by homologous recombination between donor and recipient genomes, which generates false positives and prevents full genome replacement. Here, we show that this barrier can be reduced by inactivating the recipient genome with the DNA crosslinking agent Mitomycin C. Treatment with Mitomycin C irreversibly crosslinks the recipient chromosome, blocking replication and rendering the genome nonfunctional, while leaving transcriptional and translational machinery temporarily intact. This creates a “recipient cell with inactivated genome” in which an intact donor genome can establish itself. Using this approach, transplantation assays yielded viable transplants, demonstrating that genome transplantation remains feasible with Mitomycin C treatment. These results demonstrate the potential of this strategy to expand genome transplantation beyond the Mycoides clade, reducing the risk of false positives from donor–recipient homologous recombination. By enabling genome transplantation across a broader range of organisms, this approach has the potential to expand genome transplantation to a wider range of organisms, advancing efforts toward building programmable cells for diverse applications in biotechnology, medicine, and vaccine development.

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Ronald Rodriguez, John Glass

J. Craig Venter Institute

Abstract

The construction of living cells with minimized genomes required development of methodologies that have been extremely valuable to synthetic biologists performing grand scale genome engineering. JCVI-Syn 3.0 (Syn 3.0) is a minimal bacterial cell derived from Mycoplasma mycoides containing only 473 genes and is currently   being utilized as a model system to study the basic principles of life. Syn 3.0 can replicate its genome and undergo cell division, despite the absence of genes thought to be necessary for normal cell division. Syn 3.0 cell division generates irregularly shaped cells and filamentous structures through an unknown mechanism. Normal cell division can be restored by the addition of seven non-essential genes, including cell division genes ftsZ, sepF, and five genes with unknown functions. To develop a better understanding of cell division in Syn 3.0, we will examine the cellular localization of FtsZ in combination with the origin of DNA replication using super-resolution fluorescence microscopy. Bacterial histone-like protein (HupA) is a high-copy essential protein in natural bacteria. HupA copy number per cell decreased from approximately 6000 to 28 in our minimal cell, presumably due to deletion of a non-essential gene (gpsA) located directly upstream of hupA. Surprisingly, insertion of gpsA back into the genome followed by whole-genome transplantation did not restore the expression levels of hupA, suggesting that factors required for regulating hupA expression remain to be discovered and is discussed. Our planned minimal cell experiments should expand our understanding of minimal cell physiology and potentially provide insight into what primordial cells may have looked like before the emergence of modern cell division mechanisms and chromatin in bacteria. Additionally, cell-based assays for attempting to understand the mechanism of whole-genome transplantation are discussed. 

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Lea D. F. Kloss, Martin J. Lercher

Institute for Computer Science & Department of Biology, Heinrich Heine University Düsseldorf, Germany

Abstract

Bacterial populations evolving independently under identical selective pressures can follow similar or divergent evolutionary trajectories. Our aim is to analyze the repeatability of phenotypic evolution under highly controlled experimental conditions. This is achieved by using a high-throughput automated system that enables the evolution of a large number of replicate populations in exponential growth with individually tuned dilutions and real-time fitness tracking. Additionally, we will compare predictions of growth rate and resource allocation from genome-scale kinetic balanced growth models (GBA) with the experimental results of multi-omics and growth rates. Since complex regulation in organisms like E. coli complicates interpretation, we are using the JCVI minimal cells as a model organism with reduced regulatory complexity. We will evolve the cells in three different environments, which alter enzyme kinetics and reshape the fitness landscape: in standard growth conditions (37 °C, pH 7); at a lower temperature of 30 °C; and at a reduced pH of 5.7. The latter is of interest because lactate fermentation strongly acidifies the medium, contributing to stress and thus evolution in stationary phase. Besides JCVI-syn3B, we will additionally evolve JCVI-syn3B + pdhAB, which allows acetate fermentation to be re-established during evolution.