3rd Minimal Cell Workshop

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Vishal Sivasankar¹, Gesse Roure¹, Chunyang Lu¹, Ronald Rodriguez², John Glass², Roseanna Zia¹

¹ Department of Mechanical and Aerospace Engineering, University of Missouri, MO, USA; ² J. Craig Venter Institute, La Jolla, CA, USA

Abstract

The DNA-centric view of life focuses on understanding the building blocks of life with the goal of capitalizing on this “blueprint” to unlock the workings of the organism. Even though we have access to an extensive database of genomes and proteomes of a living cell, it can be challenging to understand the cellular behavior and diseases that arise from the collective behavior of several genes which in turn depends on local effects and environmental conditions. The importance of physical organization and dynamics of the cellular constituents in regulating the cellular processes is now widely accepted. As such, new models probe cellular behavior from a physico-chemical perspective wherein one views cells as a system of biomolecular constituents that are governed by physical and thermodynamic forces. To study these ideas, we have built a mesoscopic physics-based model that spans the molecular to the microscopic scale and captures the functions of the whole cell. Our investigations of spatial organization and physical dynamics of E. coli has revealed that speedup of translation elongation in faster growing bacteria is driven directly and nontrivially by colloidal-scale stoichiometric crowding. We achieved this insight by detailed modeling of colloidal scale physics in a physically-resolved computational cell that captures the effects of confinement and biochemical and physical interactions between the interacting constituent biomolecules. The choice to model E. coli was driven partly by the abundance of experimental data available in the literature. But we believe the most fundamental insights about basic cellular processes can be gleaned by study of the so-called minimal cell, the JCVI-Syn3A. Our group is constructing a physically- and chemically-resolved in silico model of the minimal cell based on our existing platform. A key goal for our group is to rally the experimental community to develop a database for the minimal cell sufficient to construct our computational model. To that end, we discuss the success of our existing E. coli model, the database acquisition, analysis, and implementation that supported it, and present a request to workshop attendees to collaborate in generating similar data for the new model.

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Mone Mimura¹, Hana Kiyama¹, Shingo Kato², Yuya Sasajima¹, Atsuko Uenoyama¹, Shigeyuki Kakizawa³, André Antunes⁴, Tomoko Miyata⁵, Fumiaki Makino⁵, Keiichi Namba⁵, Makoto Miyata^{1, 6}

¹ Grad. Sch. Sci., Osaka Metropolitan Univ., Japan; ² RIKEN BRC., JCM., Japan; ³ Bioproduction Res. Inst., AIST., Japan; ⁴ Macau Univ. of Sci. and Tech., China; ⁵ Osaka Univ., Japan; ⁶ OCARINA, Osaka Metropolitan Univ., Japan

Abstract

Previously, our group reconstructed Spiroplasma swimming with helicity switching in syn3B by expressing a pair of bacterial actins, MreBs (Kiyama H et al., Sci Adv 2022). In the present study, we focused on MreB proteins derived from Haloplasma contractile which phylogenetically derived from around the root of all Mycoplasmas (Phylum Mycoplasmatota). Seven MreB proteins from Haloplasma (HMreB) are not closely related to those of Spiroplasma and divided into three groups. We succeeded to reconstruct movements in syn3B by expressing MreB pairs from different groups. Notably, the combination of HMreB1 and HMreB2 showed Haloplasma-like coiling motility. These results suggest that more than one types of MreB dependent motility were developed in Mycoplasma evolution.

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Yoshiki TANAKA¹,Hana KIYAMA¹, Makoto MIYATA^{1, 2}

¹ Grad. Sch. Sci., Osaka Metropolitan Univ. Japan; ² OCARINA, Osaka Metropolitan Univ. Japan

Abstract

Previously, our group reconstituted the helicity shifting swimming of Spiroplasma in syn3B by expressing a pair of Spiroplasma MreBs, bacterial actin protein (Kiyama H et al., Sci Adv 2022). To clarify the mechanism, we traced the behavior of MreB5 by mCherry fusion. syn3B is advantageous for cell biology because of its cell dimension and easy transformation and limited number of large complexes. MreB5 fluorescence was always observed in helical and moving parts of the cell. A22, an inhibitor for MreB polymerization stopped the cell movement and straightened the helix. Fluorescence recovery after photobleaching (FRAP) showed no significant difference in MreB replacement between moving and non-moving cells. These observations suggest that the formation and reversal of the helix involves ATP dependent subunit displacements, but not obvious subunit replacements.

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Haruka Yuasa¹, Yuya Sasajima¹, Hana Kiyama¹, Daichi Takahashi¹, Takuma Toyonaga^{1, 2}, Tomoko Miyata³, Fumiaki Makino³, Keiichi Namba³, Makoto Miyata^{1, 2}

¹ Osaka Metropolitan Univ., Grad. Sch. Sci., Osaka Metropolitan Univ., Japan; ² OCARINA, Osaka Metropolitan Univ., Japan; ³ Osaka Univ., Grad. Sch. Front. Biosci., Japan

Abstract

Syn3B is advantageous also for structural studies because of limited number of complexes and the small cell size. Previously, our group reconstituted Spiroplasmal helicity switching swimming by expressing two different classes of bacterial actin, MreB in syn3B (Kiyama H et al., Sci Adv 2022). In the present study, we focus on the helicity switching mechanism, which is not found in other systems of actin superfamily proteins. We visualized MreB filaments in the swimming syn3B, by using cryo electron tomography. A sheet-like structure was visualized with some attachments to the cell membrane. The pitch of the MreB monomers was approximately 5 nm. Our next focus is to identify two types of MreB proteins with better image quality.

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Manoel Neres Santos-Junior

University of Bahia. Brazil

Abstract

The Mycoplasma genus encompasses agents that infect a wide range of hosts. This genus includes Mycoplasma mycoides subsp. capri (Mmc), which causes pneumonia, mastitis, arthritis, keratitis, and septicemia in goats. Genomic analysis, immunomodulation assays, molecular cloning of recombinant antigens, and gene editing techniques have significantly contributed to our understanding of the pathogenesis caused by Mollicutes. Thus, this study aims to evaluate the microorganism-host interaction and mechanisms of immunomodulation of strains with a synthetic genome of Mmc in cell culture and a goat animal model. For this purpose, the strains JCVI-syn1.0, JCVI-syn3A, JCVI-Syn3⸫mch179-181, and JCVI-Syn3⸫mch179-186, as well as Mmc wild strain, will be used to infect primary cultures of peripheral blood mononuclear cells (PBMC). After assessing cell viability using the alamarBlue™ Cell Viability Reagent (to determine bacterial load and PBMC culture duration), we will examine lymphoproliferation capacity, induction of apoptosis, and expression of surface markers (CD3, CD4, CD8, CD21, and CD45RO). The production of cytokines (IL-1, TNF-α, INF-γ, and IL-4) will also be evaluated through ELISA tests. Hydrogen peroxide (H2O2) and Nitric oxide (NO) levels in PBMC cultures will be measured using the Amplex Red hydrogen peroxide/peroxidase kit (Thermo Fisher Scientific) and the Griess assay, respectively. The strains will also be analyzed in an animal infection model in goats. The animals will undergo tracheal inoculation with synthetic Mmc. Over two months, samples will be collected at 15-day intervals to monitor the infection through serology (IgG), culture, and PCR. During this period, the animals will be monitored, clinically examined, and assessed for biochemical markers of inflammation. After euthanasia, lung tissue samples will be collected and analyzed for histopathology, surface markers, gene expression, and NO and H2O2 production. Therefore, we anticipate exploring different aspects of the interaction between the synthetic Mmc genome and the goat host.

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Zachary Krieger, Cara Hull, Alessandro Coradini, Ian Ehrenreich

University of Southern California

Abstract

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 library of euploid cells that contain substantially reduced, if not minimal, focal chromosomes. I will describe our work to generate a minimal version of S. cerevisiae Chromosome using MOSAIC. I will also discuss insights into multiple minimal chromosome states and how MOSAIC can be used to potentially generate cells in which all chromosomes have been minimized.

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Keoni Gandall

Nanala, LLC

Abstract

Abstract: I am building a 61 codon JCVI-Syn3a. I'll be presenting on how I am accomplishing this for <$10,000, and how I hope to construct additional minimal genomes, such as a 59 codon JCVI-Syn3A, in the near future.

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Ioana M. Gherman^{1, *}, Joshua Rees-Garbutt^{1, 3}, Wei Pang^2†, Zahraa S. Abdallah^1†, Thomas E. Gorochowski^{3, 4†}, Claire S. Grierson^{3, 4†}, Lucia Marucci^1,4†

¹ Department of Engineering Mathematics, University of Bristol; ² School of Mathematical and Computer Sciences, Heriot-Watt University, Edinburgh, EH14 4AS, UK; ³ School of Biological Sciences, University of Bristol, 24 Tyndall Avenue, Bristol BS8 1TQ, UK; ⁴ BrisEngBio, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK; ∗ Presenting and corresponding author; † These authors should be considered joint senior authors

Abstract

Whole-cell models are mathematical models designed to capture the function of all genes and core processes within a cell. Whole-cell modelling has been explored for over a decade, but only two partially complete models have been published to date, for bacteria Mycoplasma genitalium and Escherichia coli. These models have been successfully used for applications such as minimal genome design, identifying novel functionalities of a biological system and guiding experimental pipelines. Here we show how machine learning can be used to reduce the computational complexity of whole-cell models for applications such as minimal genome design. The results demonstrate that using machine learning algorithms together with the data produced by whole-cell models, it is possible to speed up simulations by up to 16 times and to generate a reduced E. coli genome with 39% of the in silico modelled genes removed. Furthermore, through this approach, we uncover interesting dynamics of the cell that would be nearly impossible to assess with other experimental or computational methods, such as the relationship between gene deletions and specific macromolecular masses. We anticipate that such interdisciplinary approaches may hold the key to making whole-cell modeling more accessible to the wider community, by reducing their computational burden and uncovering the added value of the information that they provide.

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Bogumil Karas

University of Western Ontario

Abstract

Assembling synthetic bacterial genomes in yeast and genome transplantation has enabled an unmatched level of bacterial strain engineering, giving rise to cells with minimal and chemically synthetic genomes. However, this technology is currently limited to members of the Spiroplasma phylogenetic group, mostly Mycoplasmas, within the Mollicute class. Here, we propose developing these technologies for Acholeplasma laidlawii, which is phylogenetically distant from Mycoplasmas and, unlike most Mollicutes, uses a standard genetic code.

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Lea D. F. Kloss, Martin J. Lercher

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

Abstract

Independently adapting replicate populations evolving under the same selective pressure can show similar evolutionary trajectories with respect to their phenotypic traits. We aim to analyze phenotypic variation across multiple populations of bacterial cells during adaptive evolution in continuous exponential growth under well-defined and constant environmental conditions. Multi-omics measurements at multiple time points during the experiment may allow the identification of local and global fitness optima and the evolutionary paths towards them. These data will be used to evaluate patterns of adaptation and to infer the repeatability of phenotypic evolution. Furthermore, the predictability of evolution will be assessed by comparing the experimentally collected data with theoretical fitness predictions derived from a genome-scale kinetic model of balanced growth (GBA). We are currently investigating the feasibility of our experimental design concept using the minimal cell as a model organism. The minimal cell provides a unique opportunity for this type of study because the reduced genome greatly reduces the complexity of the system. Based on what is technically feasible, we are deciding what selection pressures to apply to best address our research questions.

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James M. Daubenspeck and Prescott Atkinson

University of Alabama at Birmingham

Abstract

Moonlighting proteins, a protein that has acquired a secondary function, have been identified in all domains of life. These multi-function proteins are essential for genome reduced organisms like JCVI-syn3A to expand their proteome. One of the more common examples is the enzyme enolase which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in glycolysis. In the bacterial cytoplasm enolase is a homo-octamer, but a small percentage is transported to the surface as a monomer where it can no longer dimerize and is thought to function as a receptor. Our data suggests that Mycoplasma species initiate the export of the moonlighting protein by attaching a rhamnose to the protein. In the case of enolase this occurs at the dimerization interface. The rhamnose residue is linked through a phospholipid and anchored to the membrane. Phospholipase D, an enzyme that cleaves the rhamnophospholipid linking proteins to the membrane surface, releases proteins from JCVI-syn3A suggesting that this system is like the one identified in other Mycoplasma species. Our analysis of post-translational modifications in JCVI-syn3A have identified moonlighting proteins that are modified by a single hexose. This hexosylation modification is limited to surface exposed proteins in Mycoplasma species. These data suggest that the moonlighting system is active in JCVI-syn3A and may be essential for growth.

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John W. Sanford¹, James Daubenspeck¹, Lissa Anderson², Jason Needham³, Kevin Dybvig¹, T. Prescott Atkinson¹

¹ Dept. of Pediatrics, University of Alabama at Birmingham; ² National High Magnetic Field Laboratory, Tallahassee, FL; ³ Dept. of Cell Biology and Molecular genetics, University of Maryland, College Park, MD

Abstract

Protein glycosylation is a ubiquitous process found across all forms of life. Previous work in our group determined a surface protein hexosylation system was active in the murine pathobionts Mycoplasma pulmonis and Mycoplasma arthritidis, as well as the human pathobiont Mycoplasma pneumoniae. This hexosylation process uses disaccharides or larger glycans as the activating sugar instead of the canonical UDP-nucleotide seen in most bacterial glycosylation systems. Furthermore, no peptide sequence motif was found for glycosylation sites, indicating it is a highly generalized and stochastic process. We have never found evidence of a complete glycosylation knockout in mycoplasmas, and the few annotated glycosyltransferases in mycoplasmas appear to be essential.

A currently unknown glycosyltransferase mediates this stochastic hexosylation system and we have hypothesized it is active in the synthetic near-minimal organism JCVI-syn3A (syn3A) based on periodic acid staining of ultracentrifuged membranes from the organism. We performed high-resolution mass spectrometry on in-solution digested peptides from syn3A membranes. Our results indicate this hexosylation system is active in syn3A and appears to be a dynamic process.

The presence of this hexosylation system in syn3A, in tandem with a dearth of glycosylation knockouts across the genus, strongly implies that this process is essential for mycoplasmas even without the selective pressure of the host immune system. While a stochastic glycosylation system would increase the antigenic variation of a population of mycoplasmas, it may also protect the organism from its own secreted proteases or play a role in another fundamental process for life.

Ultimately, the presence of glycosylation in syn3A strongly implies that glycosylation is a fundamentally essential process for mycoplasmas. Analyzing syn3A can allow for intriguing insight into how dynamic and chaotic the surface of cells can be. Furthermore, the heretofore uncharacterized mycoplasma protein glycosyltransferase may be a target for novel therapeutics against pathogenic mycoplasmas downstream of understanding the basic biology of the system.

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Daniela M de C. Bittencourt

EMBRAPA, Brazil

Abstract

Current advances in synthetic biology allow the engineering of bacteria cells, designed with specific signaling networks, built with novel programmable genetic parts, and explicitly created to handle mechanisms underlying disease and related organism events. Furthermore, the design, synthesis and construction of genomes from scratch open possibilities for engineering living cells for precise cell therapy. Recently, comparing the infectivity assay results from JCVI-syn1.0, JCVI-syn3A and several intermediate minimized genome strains, we verified that the genome minimization processes led JCVI-syn3A to lost its capacity to attach and multiply in co-culture with mammalian cells. Differently from JCVI-syn1.0, the minimal cell was practically invisible to neutrophil-like cells (dHL-60). We also identified a cluster of eight genes (MMSYN1-179 to MMSYN1-186) that were capable of returning JCVI-syn3B the capacity of attaching to HeLa cells and to induce a higher phagocytic index in dHL-60 than other synthetic cells. These studies suggest that the minimal cell can be designed to produce specific proteins to modulate cell behavior. For instance, JCVI-syn3A could be engineered to attach to the surface of cancer cells, in order to interfere and alter cellular pathways to trigger target cell death. Accordingly, we will evaluate different strategies in order to make the minimal cell capable of functioning as a bacteria-based cancer therapy. Overall, the application of the minimal cell as a chassis for cell therapies is an attractive direction to improve specificity and efficiency of drug delivery systems.

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Marco Oliveira

INCTBioSyn / Embrapa, Brazil

Abstract

Serine-integrases are Site-Specific Recombinases (SSR) capable of rearranging DNA regions without the need of any auxiliary endogenous factor or cellular machinery, a valuable feature when selecting molecular tools to work in the minimized molecular environment of Mycoplasma mycoides JCVI-Syn3B. The outcome from a serine-integrase recombination event is determined by the orientation of their recombination sites and can result in integration, inversion or excision of a specific DNA sequence. We have already successfully used inversion by serine-integrase Int9 to control gene expression in syn3B. To further expand integrase applications in the minimal cell, candidates will now be screened for their ability to integrate or excise target sequences. The ultimate goal is the assembly of an enhanced landing pad to be integrated into the syn3B genome. The proposed landing pad harbors orthogonal recombination sites for different integrases to allow multiple entry points through recombinase mediated integration, as well as flanking site pairs designed for removal of insertions.

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Klara Hlouchova

Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic

Abstract

All extant cells known to humankind build proteins from the same 20 coded amino acids. However, the study of origins of life implies that earlier cells functioned with a smaller alphabet, before the fixation of the Central Dogma. That is intriguing within today’s biology where each of the 20 amino acids occupies a unique and seemingly indispensable role.

Our work indicates that structured conformations and functional proteins can be constructed with about half of today’s alphabet, energetically less costly and available already through prebiotic synthesis. Whether whole biological systems can still work with a reduced amino acid alphabet remains to be seen.

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Benjamin R. Gilbert

Luthey-Schulten Group at UIUC

Abstract

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identified in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we calculate in silico chromosome contact maps that capture inter-daughter interactions as a function of the cell’s replication state.

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Troy A. Brier¹, Jay E. Cournoyer¹, Ben R. Gilbert¹, Zane R. Thornburg¹, Pratap Venepally², John I. Glass³, Angad P. Mehta¹, Zaida Luthey-Schulten⁴

¹ Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA; ² J. Craig Venter Institute, Rockville, MD, USA; ³ J. Craig Venter Institute, La Jolla, CA, USA; ⁴ Department of Chemistry, Department of Physics, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Abstract

Probabilistic interactions between cellular components dictate bacterial gene expression, a multi-step process where information encoded at the genome level is converted to the transcriptome level and then eventually the proteome level within a cell. In addition to its stochastic nature, bacterial gene ex-pression is differential meaning there are many distinct mechanisms generating the same gene ex-pression products. However, bacterial gene expression is highly robust, consistently generating pro-teins at proper stoichiometric ratios necessary for complex formation as well as cellular functionality for example proteins in a related metabolic pathway. A major factor leading to the differential aspect of gene expression is the complexity of the transcriptome. RNA transcripts encode many different functional forms of RNA (tRNA, rRNA, mRNA, etc), and are processed a variety of ways in the cell even among identical functional forms. The processing of the transcriptome occurs either at the transcrip-tional or post-transcriptional level. To understand the complexity of the transcriptome and its role in bacterial gene expression, computational and experimental technique have been employed. A bio-informatic method identifying transcription units from transcription initiation and termination motifs, was used to identify RNA isoforms, RNA transcripts evolved from a similar region of the genome with different genetic information. The RNA isoforms highlight possible co-expression events typically of genes encoding proteins involved in complexes or related metabolic function. Computational predic-tions were validated with Oxford Nanopore Technologies RNA sequencing, which detects native tran-scripts within a cell. Stochastic simulations were performed with varied gene expression patterns in-formed by the transcription unit predictions. In this talk I will discuss how we have characterized co-expression and provide a description of how varying the gene expression pattern impacts cellular pro-cesses such as the cell’s ability to robustly generate its protein at proper stoichiometric ratios.

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Jan A. Stevens, Siewert-Jan Marrink*

Molecular Dynamics Group (GBB), Groningen, The Netherlands

Keywords: Martini, Whole-cell Modeling, Molecular Dynamics, Computational Microscopy

Abstract

Molecular dynamics (MD) is a well-established simulation method that has successfully been applied to study a wide range of biomolecular processes. As a result of continuous improvements in both computational infrastructure and modeling methods, we currently observe that the study of mesoscopic, multi-component systems has become attainable. Utilizing the Martini coarse-grained forcefield and its accompanying tools1, we exemplify these advancements through constructing models of increasingly complex systems, extending to the scale of viruses2 and even cellular organelles such as the mitochondrion3. In this study, we further advance the complexity of biomolecular simulations by introducing an integrative modeling approach that demonstrates the feasibility of simulating a Martini model of the genetically minimal cell: JCVI-syn3A2,3. Although some components of the cell composition have been simplified, this model serves as a starting point for whole-cell simulations with molecular detail. Studying entire cells under the computational microscope will provide valuable insights into a wide range of problems, ranging from drug design to understanding the internal organization of cellular environments. This work represents a significant milestone in the ongoing pursuit of understanding the complexities of living systems through MD simulations.

References

S.J. Marrink et al. (2022), Two decades of Martini: Better beads, broader scope , WIREs Comput Mol Sci.

W. Pezeshkian et al. (2023), Molecular architecture and dynamics of SARS-CoV-2 envelope by integrative modeling, Structure.

W. Pezeshkian et al. (2020), Backmapping triangulated surfaces to coarse-grained membrane models, Nat. Commun.

M. Breuer et al. (2019), Essential metabolism for a minimal cell, eLife 8:e36842.

J.A. Stevens et al. (2023), Molecular dynamics simulation of an entire cell, Front. Chem.

*Corresponding author: s.j.marrink@rug.nl

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Evan Kalb and Kate Adamala

University of Minnesota

Abstract

Cell-free expression systems (CFESs) derived from non-model organisms present exciting opportunities for expanded protein and natural product syntheses. Minimal cells offer a particularly good chassis for CFES, due to their simple, well understood genomes, and the unique ability to re-engineer many essential parts of the cell metabolism. Despite their promise, these systems are challenged by relatively poor yields compared to traditional E. coli CFES. To develop new CFESs, reaction conditions are optimized by altering a swathe of parameters including harvesting conditions, lysis, energy substrates, and other crucial metabolic molecules. Often included in these optimization attempts is the addition of E. coli tRNA which exhibits varying levels of success. Rather than rely on the uncertain compatibility of E. coli tRNA with diverse translation machineries, we propose matching non-model CFESs to their native tRNAs pools. To do so, we will employ RNA extraction and fractionation methods to generate native tRNA pools for use in non-model CFESs. We anticipate our findings will underscore the importance of pairing native tRNAs to their respective translation machineries and narrow the gap between model and non-model CFESs.

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Andrei Sakai¹, Kate Adamala², John Glass³, Wilhelm Huck¹

¹ Radboud University; ² Universityof Minnesota; ³ J. Craig Venter Institute

Abstract

The genome-reduced JCVI-syn3A (Syn3A) is an interesting model for the construction of a minimal synthetic cell due to its minimal genome and metabolism. For that, we developed a cell-free expression (CFE) system based on Syn3A cell extracts which de novo synthesized proteins up to 350 nM. Next, we plan to build a synthetic protocell by assembling isolated Syn3A genomic DNA in liposomes containing the Syn3A CFE system. The complete reconstruction of Syn3A from its parts might reveal important design principles for building synthetic cells from the bottom up.

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Masaki Mizutani¹, Minoru Moriyama¹, Ryuichi Koga¹, Takema Fukatsu^{1, 2, 3}, Shigeyuki Kakizawa¹

¹ Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), JAPAN; ² Department of Biological Sciences, The University of Tokyo, JAPAN; ³ Graduate School of Life and Environmental Sciences, University of Tsukuba, JAPAN

Abstract

Recently, JCVI-syn3B has been used as a recipient to reproduce complex cellular functions, such as Spiroplasma swimming and Ureaplasma adhesion, by expressing several genes. In order to precisely reproduce the function of expressed proteins, the growth temperature of transformant should be similar to that of original bacterium. In the present study, we performed adaptive laboratory evolution of JCVI-syn3B to lower temperatures. First, we measured the growth speed of original JCVI-syn3B strain in SP4 medium at 37°C and also at lower temperatures. The original strain grew 3‒4 times slower at 30°C than 37°C, and no significant growth was observed at 27°C. Next, we continuously cultivated JCVI-syn3B at 30°C. The strains passaged 20 times grew twice as fast as the original strain. Interestingly, the 20-passaged strains could also be culturable at 25°C. We analyzed the changes of individual protein expressions by mass spectrometry, and discuss the mechanisms for low temperature adaptation.

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James Pelletier^{1, 2}, Saúl Ares^{1, 2}, Germán Rivas³, John Glass⁴, Elizabeth Strychalski⁵

¹ Centro Nacional de Biotecnología (CNB), CSIC, 28049 Madrid, Spain; ² Grupo Interdisciplinar de Sistemas Complejos (GISC), Madrid, Spain; ³ Centro de Investigaciones Biológicas (CIB), CSIC, 28040 Madrid, Spain; ⁴ J. Craig Venter Institute, La Jolla, CA 92037, USA; ⁵ National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

Abstract

JCVI-syn3.0 and JCVI-syn3A offer simplified model systems to study bacterial cell division. They retain highly conserved genes involved in cell division and exhibit varied patterns of cell division relevant to diverse bacterial species. JCVI-syn3.0 exhibits pleomorphism, similar to ureaplasmas, cell wall-deficient bacteria, and some intracellular endosymbionts. JCVI-syn3A retains 19 more genes than JCVI-syn3.0 and displays significantly less variation in cellular size and shape, consistent with regulation of cell division, as in most bacterial species. Many of the genes required for cell division in JCVI-syn3A do not have a known biomolecular function. We are planning biochemical experiments to investigate the biomolecular function two genes, 0527 and 0033. Deletion of 0527 from JCVI-syn3A increases variation in cellular size and shape. 0527 is highly conserved in bacteria and plant plastids, but we are aware of only one article on this protein family, which affected the accumulation of 23S rRNA (Yang et al., 2016). While the connection between cell division and 23S rRNA remains unknown, as a first step, we plan to test if the protein encoded by 0527 interacts directly with 23S rRNA. 0033 is essential and predicted to have a single N-terminal transmembrane domain and a large hydrophobic cavity exposed to solvent outside the cell. A recent study has described the structure and function of P116, encoded by MPN_213 in M. pneumoniae and distinguished by a similar large, extracellular hydrophobic cavity (Sprankel et al., 2023). P116 specifically extracts lipids such as cholesterol, sphingomyelin and phosphatidylcholine from the growth medium to the membrane. We have upgraded gene 0033 from unknown function to a putative homolog of MPN_213 and are considering experiments to test if 0033 has a similar function. We anticipate biochemical experiments with purified gene products will help connect top-down and bottom-up approaches to synthetic cells.

References

Sprankel, L., Vizarraga, D., Martin, J., Manger, S., Meier-Credo, J., Marcos, M., Julve, J., Rotllan, N., Scheffer, M.P., Escola-Gil, J.C., et al. (2023). Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas. Nat Struct Mol Biol 30, 321-329.

Yang, J., Suzuki, M., and McCarty, D.R. (2016). Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis. J Exp Bot 67, 5447-5460.

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Ronald Rodriguez

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 these non-essential gene products with high-resolution light 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 located directly upstream of hupA. We hypothesize this is why chromosome conformation capture studies of our minimal cell displays a lack of long-range chromosome interactions. Perhaps by dramatically reducing the copy number of HupA per cell, we have recapitulated a physiological state of primordial cells before the evolution of chromatin. We will restore the non-essential gene upstream of hupA and determine if our long-range chromosomal interactions date is analogous to what occurs in other bacteria. 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.

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Yan Zhe (Leo) Liu, Amit Klein, Yo Suzuki, John I. Glass

Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, CA

Abstract

Recently, a project at J. Craig Venter Institute aims to insert pyruvate:ferredoxin oxidoreductase (PFOR) into JCVI-syn3B to facilitate pyruvate synthesis using acetyl-CoA. One obstacle to PFOR implementation is that its pyruvate synthesis activity only becomes feasible at temperatures above 40°C, therefore making the growth conditions hostile to JCVI-syn3B. To overcome this, we attempted to evolve thermal tolerant JCVI-syn3B that can grow and be sustained at temperatures above 40°C. Using adaptive laboratory evolution, we have evolved lines of JCVI-syn3B that can now grow overnight at 44.2°C.

In the beginning, growth of JCVI-syn3B at different temperatures was gauged using a temperature gradient experiment. JCVI-syn3B showed significantly hindered growth at 42°C and no growth above 42°C. Using 42°C as the first bottleneck condition, lines of JCVI-syn3B were continually grown and propagated (1/11 passage ratio) at 42°C until overnight growth was achieved. The next bottleneck condition was determined with a new temperature gradient experiment, and this process was repeated for approximately 4 months until we achieved overnight growth of JCVI-syn3B at 44.2°C. In addition to obtaining lines of JCVI-syn3B that can facilitate PFOR pyruvate synthesis, we were also intrigued by the evolutionary changes responsible for thermal tolerance. Because heat can cause a myriad of adverse effects including protein degradation, cell membrane degradation, and DNA damage, evolution of thermal tolerance can cause diverse metabolic changes and reveal critical genes and mutations regulating heat stress.

Our first investigation of evolved JCVI-syn3B thermal tolerance is from a genetic basis. In collaboration with the Palsson Lab, we have extracted and sequenced gDNA of both ancestral JCVI-syn3B and evolved JCVI-syn3B to identify the genetic changes. From this, we discuss early analysis of mutations that may be responsible for JCVI-syn3B thermal tolerance and interesting phenotypic differences between evolved and ancestral JCVI-syn3B.

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Antony Duval

Université de Sherbrooke

Abstract

We report the development of CMRL-AT, a semi-defined growth medium custom-formulated for the near-minimal organism Mesoplasma florum. Transcriptomic analyses comparing cells cultivated in traditional ATCC1161 medium and CMRL-AT revealed significant metabolic constraints unique to each condition. In addition, the ability to use different carbon sources in CMRL-AT enabled us to refine the annotated functions of several genes. Finally, CMRL-AT opens new avenues for metabolic engineering and systems biology studies in M. florum.

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Simon Jeanneau

Université de Sherbrooke

Abstract

The study of gene essentiality is pivotal for understanding critical cellular functions under varied conditions. We performed transposon mutagenesis coupled with high-throughput sequencing (Tn-Seq) on the near-minimal bacterium Mesoplasma florum, grown in ATCC161 or semi-defined CMRL-AT medium. Using a time-course approach and hierarchical clustering, we categorized genes into distinct levels of essentiality. Our findings offer a comprehensive map of gene essentiality in M. florum, revealing which genes are strictly or conditionally essential, thereby yielding important insights for the design of a minimal genome.

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Jeremy Gagnon

Université de Sherbrooke

Abstract

The creation of minimal genomes serves as a powerful tool for unlocking the fundamental principles of biology. We present the initial stages of constructing a synthetic Mesoplasma florum genome through PCR amplification of genome sections, assembly in Saccharomyces cerevisiae, and subsequent transplantation into related M. florum strains or Mycoplasma capricolum. Our work serves as a foundational step towards more complex genome engineering projects, such as the creation of a minimal genome.