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Daniel Nucifora

Western University

Abstract

Cloning, modification, and transplantation of whole bacterial genomes enable an almost unmatched ability to engineer strains, as demonstrated by the creation of the JCVI minimal cell. Currently, genome transplantation is only possible for bacteria in the class Mollicutes, specifically for species in the Spiroplasma phylogenetic group. Here, we propose Acholeplasma laidlawii, a Mollicute that uses a standard genetic code and does not require any serum for growth, as a new platform for creating synthetic cells. Towards this goal, we have selected two A. laidlawii strains, PG-8A and 8195, to serve as a donor and recipient, respectively, for genome transplantation. We have established a growing genetic toolbox for A. laidlawii, including a new electroporation protocol, an oriC-based plasmid, transformation with transposons, and exogenous gene expression. We have also evolved a strain with greatly improved DNA uptake via electroporation. Using these genetic tools, we restored homologous recombination in A. laidlawii 8195, allowing us to integrate yeast elements into the genome of this strain for cloning in Saccharomyces cerevisiae. We next plan to establish a working genome transplantation protocol for A. laidlawii, which is the last step before it will be possible to create synthetic Acholeplasma strains.

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Jan A. Stevens¹, Mert Bozoflu¹, Linus Grünewald² , Melanie König¹, Fabian Grünewald², Siewert-Jan Marrink¹

¹ Molecular Dynamics Group (GBB), Groningen, The Netherlands; ² Heidelberg Institute for Theoretical Studies, Heidelberg, Germany

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 modeling methods and computational infrastructures, the study of mesoscopic, multi-component systems has become more attainable. However, the intricacies involved in setting up MD simulations for these systems remain daunting, requiring the integration of diverse data from both experimental and in silico sources.

Here we present how the coarse-grained Martini force field and its associated tools, form an ideal ecosystem for facilitating a integrative modeling pipeline. Employing a CG resolution, typically representing four heavy atoms by one CG bead, significantly reduces the computational cost inherent in simulating large-scale MD models. Furthermore, a key feature of the force field is its universality, which allows us to create CG models of all major biological components and construct complete cellular environments.

The Martini force field's capabilities are showcased in an ongoing effort to simulate a genetically minimal cell: JCVI-syn3A. We constructed the first near-atomistic MD model of a cell based on data from kinetic models, Cryo-Electron Tomograms, and omics experiments. Studying entire cells under the computational microscope will allow us to look into a wide range of problems, ranging from drug design to understanding the internal organization of cellular environments.

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Essa Ahsan Khan^a, Christian RÅNuckert-Reed^b, Gurvinder Singh Dahiya^c, Lisa Tietze^a, Maxime Fages-Lartaud^a, Tobias Busche^b, JÅNorn Kalinowski^b, Victoria Shingler^d, Rahmi Lale^a*

^a Department of Biotechnology and Food Science, Faculty of Natural Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; ^b Bielefeld University, Center for Biotechnology (CeBiTec), Technology Platform Genomics, Bielefeld, Germany; ^c Syngens AS, Trondheim, Norway; ^d Department of Molecular Biology, Ume˚a University, Ume˚a, Sweden; * Presenting author (rahmi.lale@ntnu.no)

Abstract

The variable sigma (σ) subunit of the bacterial RNA polymerase holoenzyme determines promoter specificity and facilitates open complex formation during transcription initiation. Understanding σ-factor binding sequences is therefore crucial for deciphering bacterial gene regulation. We have devised a high-throughput approach that utilizes an extensive library of 4.5 million DNA templates to provide artificial promoters and 5′ UTR sequences for σ- factor DNA binding motif discovery. This method combines the generation of extensive DNA libraries, in vitro transcription, RNA aptamer selection, and deep DNA and RNA sequencing. It allows direct assessment of promoter activity, identification of transcription start sites, and quantification of promoter strength based on mRNA production levels. Using this method, we determined the DNA binding sequences of 17 sigma factors from three bacterial species: Bacillus subtilis, Escherichia coli, and Pseudomonas putida. From the library of 4.5 million DNA templates, we identified approximately 2.5 million functional RNA sequences across these σ-factors. In this presentation, I will introduce our approach for high-resolution mapping of σ-factor-DNA binding sequences in the aforementioned three bacterial species. Additionally, I will share our efforts in updating annotations for the regulatory sequences in the host genomes. I will conclude my presentation with our ongoing work on developing deep learning-based promoter prediction tools and outline our future plans.

Acknowledgement

We acknowledge the funding from The Research Council of Norway (grant no. 316129); NTNU-Discovery program; NTNU-Biotechnology, Enabling Technologies Program (personal PhD stipend to L.T.); Faculty of Natural Sciences at NTNU (personal PhD stipend to M.FL.).

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John W. Sanford¹, James Daubenspeck¹, James Mobley², Kevin Dybvig¹, T. Prescott Atkinson¹

¹ Department of Pediatrics and ² Department of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL 35294

Abstract

Protein glycosylation has been reported in all forms of life. Previous work from our group showed mycoplasmas scavenge hexoses from exogenous oligosaccharides to glycosylate surface proteins at serine, threonine, tyrosine, asparagine, and glutamine residues without utilizing a consensus sequence as seen in canonical glycosylation systems. We screened Mycoplasma genitalium (Mgen), Mycoplasma mycoides subsp. capri, and JCVI-Syn3A (Syn3A) for glycosylation using a periodic acid-based staining of whole cell lysates separated on polyacrylamide gels. After detecting evidence of glycosylation in all three organisms, we determined the glycosyl donor to be predominately glucose derived from our SP4 growth medium supplemented with maltose, a disaccharide of glucose. To identify glycosylation sites in these organisms, we performed high-resolution mass spectrometry on in-solution digested membrane materials. We detected hexoses attached to Syn3A surface proteins as well as housekeeping proteins that are known moonlight on the surface of mycoplasma cells such as EF-Tu. We have also determined that a population of a single peptide sequence can be glycosylated at different amino acid sites, further demonstrating a wide degree of variability in the mycoplasma hexosylation system. Furthermore, we have detected glycosylation of aspartic acid and glutamic acid residues, expanding the pool of known glycosyl acceptors in bacteria to include the acidic amino acids. It is not known what enzyme(s) catalyze this glycosylation reaction. We hypothesize that this hexosyltransferase is among the genes annotated with an unknown function, as this glycosylation reaction is remarkably distinct from traditional glycosylation systems. We are currently investigating how the identity of the glycosyl donor impacts protein glycosylation by supplementing conditioned SP4 with differing disaccharides. Finally, since this hexosylation system is active in both the naturally streamlined genome of Mgen and the synthetically reduced genome of Syn3A, we are planning to screen JCVI-Syn3.0 for glycosylation, which will give strong evidence of this hexosylation system being either an essential or quasi-essential function for mycoplasmas and potentially narrow the candidate hexosyltransferase gene(s).

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James M. Daubenspeck and Prescott Atkinson

University of Alabama at Birmingham

Abstract

Moonlighting proteins have been identified in all domains of life. These multi-function proteins are essential for genome reduced organisms like Mycoplasma genitalium 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, 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 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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Ha Ngoc Anh Nguyen, James Saenz

B CUBE, Technische Universität Dresden

Abstract

Cells use 10s to 100s of coordinated lipid species to perform cellular functions, creating a complexity that eludes complete understanding. To tackle this problem, we characterize the lipidome of two simple organisms: Mesoplasma florum and JCVI-syn3B. Their limited modeling capacity resulted in a reductive lipidome, with most of the lipidome made of mere 7 species. However, despite their similarity, these organisms showcased two different membrane systems and adaptation strategies. Taken together, we present new insight on how minimal membranes work, and how we can strategize a minimized functional membrane system.

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Edwin Ortega Arzola

The University of Edinburgh

Abstract

Drawing from recent findings on the minimum energy required to synthesize a cell, this presentation explores the correlation of minimal bacterial cells to optimize metabolic pathways for efficient resource management. We aim to highlight the missing parts of the puzzle needed to work on efficient biological systems. Applications include developing biofactories (self-sustaining systems) capable of producing essential resources such as oxygen and water while recycling waste and using thermodynamic principles to minimize energy consumption. These innovations have potential applications in creating autonomous biological systems and promoting sustainability in environments that demand high resource efficiency.

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Mariana Mathias Conroy Araujo^1,2, Lukas Brayan Gomes de Andrade², John I. Glass³, Daniela Matias de C. Bittencourt², Elibio Rech²

¹Univerisidade de Brasília, Instituto de Ciências Biológicas, Programa de pós-graduação em Biologia Molecular, Brasilia/DF, Brazil; ²Embrapa Genetic Resources and Biotechnology / National Institute of Science and Technology - Synthetic Biology, Brasilia/DF, Brazil; ³The J. Craig Venter Institute, La Jolla/CA, USA

Abstract

Mycoplasmas, and their derived cells (SynCells JCVI-Syn1.0 and JCVI-Syn3B), possess limited molecular mechanisms for inducing mutations, necessitating new approaches for precise gene modification and functional studies. In response to this challenge, we propose utilizing CRISPR-based systems for targeted gene editing, offering higher precision and efficiency than traditional transposon-based mutagenesis methods. Our research has focused on employing the dCas9 mutagenesis system, as described by Ipoutcha et al. (2022). Specifically, we have used an inducible dCas9 with a cytosine deaminase base editor system to explore guided mutagenesis in JCVI-Syn1.0. We have designed single guide RNAs (sgRNAs) to direct the base editor to introduce a stop codon (TAA) by altering specific amino acid codons. We are investigating potential applications including targeting the ∆ gene, which encodes a ribosomal RNA methyltransferase, that was used as proof of concept in other works with SynCells. Disruption of this gene can confer resistance to the antibiotic kasugamycin. Current efforts include evaluating dCas9 expression through RT-qPCR and assessing base changes via dPCR with specific probes for the TAA mutation, enabling quantification of mutation frequencies. Further, we are exploring alternative mutagenesis approaches by introducing native Cas proteins, in combination with the Mycoplasma capricolum RecA system. We plan to test both Cas9 and Cas12 for their expression and capability to induce genomic modifications in synthetic Mycoplasma cells. These efforts aim to establish a versatile CRISPR-based toolkit for precise genome engineering in SynCells.

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Wichittra Arai, Masaki Mizutani, Minoru Moriyama, Yoko Hagiwara, Takema Fukatsu, Shigeyuki Kakizawa

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

Abstract

Pathogenic bacteria often require nutrients from their hosts, and some of them require serum in their cultivation medium. Mycoplasmas typically lack lipid biosynthesis systems and depend on serum-derived lipids for growth. However, serum-containing media are difficult to use for the secretome analysis due to their high protein content, such as albumin. In addition, serum is usually expensive and has lot-to-lot variation, and reducing serum usage is also associated with enhancing animal welfare.

Here, we developed serum-free and albumin-free media for Mycoplasmas. We added three types of lipids and two types of lipid carriers as alternatives to serum. This new media supported the growth of the minimal cells JCVI-syn3.0, JCVI-syn3B, JCVI-syn1.0, Mycoplasma pneumoniae, M. gallisepticum, M. synoviae, Spiroplasma chrysopicola, and S. eriocheiris. Similar growth speeds of JCVI-syn1.0 and JCVI-syn3B were observed both in SP4-FBS (fetal bovine serum) and serum-free SP4 media. We also tested colony formation on solid media and recovery from -80°C freeze stocks, obtaining positive results. Proteomics with mass spectrometry and RNA-seq analysis were conducted on JCVI-syn3B cells cultivated in both the original serum-containing SP4-FBS medium and the serum-free medium. The results indicated that the expression levels of most genes remained largely unchanged at both RNA and protein levels. This suggests that the newly developed serum-free medium could potentially serve as a viable alternative to the original serum-containing medium. These newly developed media would be useful for research, detection, and vaccine production for those mycoplasmas.

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Valerio G. Giacobelli

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

Abstract

All extant living cells synthesize proteins using a set of 20 canonical amino acids. However, research into the origins of life suggests that earlier cells may have utilized a more limited set of amino acids before the establishment of the Central Dogma. This notion is particularly compelling in contemporary biology, where each of the 20 amino acids plays a unique and seemingly indispensable role.

Our research indicates that functional proteins with stable conformations can be constructed using approximately half of the current amino acid set. However, it remains uncertain whether entire metabolic pathways or a biological system can operate with this reduced amino acid repertoire.

Towards this aim, we are evolving adaptive evolution and genome engineering strategies in the Syn3B.

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Laura Heinen^1,3, Marco van den Noort¹, Martin S. King², Edmund R.S. Kunji² and Bert Poolman¹

¹ Department of Biochemistry, University of Groningen, The Netherlands; ² Medical Research Council Mitochondrial Biology Unit, Cambridge, UK; ³ DWI – Leibniz-Institute for Interactive Materials, Aachen, Germany

Abstract

Living systems depend on a continuous input of energy for growth, replication, and information processing. Cells breakdown nutrients or use light to generate energy in the form of ion gradients or ATP. However, to install sustained fueling pathways in synthetic systems is challenging. Inspired by endosymbionts that rely on the host cell for their nutrients, we introduce the concept of cross-feeding between synthetic vesicles that can exchange ATP and ADP across their membranes. One population of vesicles produces and exports ATP, while a second population of vesicles takes up ATP to fuel energy-consuming reactions. The produced ADP feeds back to the first vesicles. The vesicles are a new platform technology to fuel ATP-dependent processes in a sustained fashion, with wide applications in synthetic cells and nanoreactors. Fundamentally, the vesicles enable studying non-equilibrium processes in an energy-controlled environment and promote the development and understanding of constructing life-like metabolic systems.

Based on Nat Nanotech, in press

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Miyer F. Patiño-Ruiz, Zaid Ramdhan Anshari, Bauke Gaastra, Dirk J. Slotboom and Bert Poolman

Department of Biochemistry, University of Groningen, The Netherlands

Abstract

Cellular homeostasis depends on the supply of metabolic energy in the form of ATP and electrochemical ion gradients. The construction of synthetic cells requires a constant supply of energy to drive membrane transport and metabolism. Here, we provide synthetic cells with long-lasting metabolic energy in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution with the transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. We couple the accumulation of lactose to a metabolic network for the generation of intermediates of the glycolytic and pentose phosphate pathways. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, paving the way for the development of more complex synthetic systems.

Based on Nat Commun, in press (2024)

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Jelmer Coenradij, Eleonora Bailoni, and Bert Poolman

Department of Biochemistry, University of Groningen, The Netherlands

Abstract

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove external components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameter by fluorescence readout. Our membrane protein-containing vesicles of 100–400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy. The vesicle systems are used to synthesize lipids, which is driven by an out-of-equilibrium reaction network for ATP productio

Based on J. Vis Exp doi: 10.3791/66627 (2024) and ACS Synth Biol 13: 1549 (2024). doi: 10.1021/acssynbio.4c00073.

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Hajar Estiri, John Glass, and Tae Seok Moon

Abstract

Aging, a complex process affecting all living organisms, is characterized by genetic, metabolic, and environmental interactions. However, the exact molecular mechanisms driving aging remain elusive, particularly at the cellular level. By utilizing the minimal genome bacterium (i.e., JCVI-syn3.0 synthetic minimal bacterial cell), which carries only the genes essential for life, we can systematically investigate the core biological functions that contribute to aging, many of which may be conserved across species. In this talk, we will discuss our project that leverages JCVI's expertise in omics, synthetic biology, single-cell analysis, and modeling to discover the fundamental and universal mechanisms of aging and potentially contribute to longevity research in an innovative way.

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Jana Oertel¹, Natalya Safronova², James Saenz², Karim Fahmy^1,3

¹ Helmholtz-Zentrum Dresden – Rossendorf, Inst. Resource Ecology, Biophysics Department, Bautzner Landstrasse 400, 01328 Dresden; ² Technische Universität Dresden, B CUBE – Center for Molecular Bioengineering, Tatzberg 41, 01307 Dresden, Germany; ³ Technische Universität Dresden, BIOTEC – Biotechnology Center (BIOTEC), Tatzberg 47/49, 01307 Dresden, Germany

Abstract

We investigate the consequences of defined lipid compositions of the plasma membrane on the metabolic activity of minimal cells. Using isothermal microcalorimetry (IMC) as a real time monitor of the growth and energy dissipation in batch cultures of minimal cells, we show that different lipid diets seriously affect biomass and heat production. We have developed novel analytical tools based on an extended calorimetric Monod equation (ECME) covering growth far beyond exponential phases. The ECME allows dissecting the heat release into enthalpy contributions from either growth or lipid-related cell biochemistry. The key finding is a severe influence of the lipidome on the balance between biomass formation and cell division rate. As a consequence, the heat measurements predict a lipidome-dependent alteration of the cell volume which we confirm by differential light scattering. The combination of IMC with lipidome manipulation opens entirely novel metabolic studies based on the surprisingly robust heat flow signals from minimal cells. The quantitative analysis of such curves is greatly facilitated by the lack of metabolic pathway diversity.

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James F. Pelletier, Lijie Sun, Kim S. Wise, Nacyra Assad-Garcia, Bogumil J. Karas, Thomas J. Deerinck, Mark H. Ellisman, Andreas Mershin, Neil Gershenfeld, Ray-Yuan Chuang, Saúl Ares, John I. Glass, and Elizabeth A. Strychalski

Abstract

The genomically minimal cell JCVI-syn3A offers a simplified model system to study cell division in the absence of a peptidoglycan cell wall. JCVI-syn3A exhibits binary fission, resulting in round cells less than one micron in diameter. Deletion of some genes of unknown function perturbs cell division, generating a fraction of large cells greater than several microns in diameter. JCVI-syn3A retains the highly conserved gene ftsZ, which encodes a bacterial tubulin homolog that can assemble into a constricting ring at the division site in most bacteria. Surprisingly, deletion of ftsZ does not produce large cells, suggesting other forces contribute to constriction during cell division in JCVI-syn3A. As a reference, physical models to describe shape transformations in vesicles can predict spontaneous constriction in the absence of a constricting ring, for certain values of the surface-area-to-volume ratio and the preferred membrane curvature. We are characterizing how FtsZ may act in the biophysical context of a membrane curved by other forces. This physical view provides a quantitative framework to compare mechanisms of cell division in top-down genomically minimal cells and bottom-up synthetic cells.

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Sage Glass¹, Andras Cook¹, David Bianchi², Marian Breuer², Kim Wise¹, Zan Luthey-Schulten², John Glass¹

¹J. Craig Venter Institute; ²University of Illinois at Urbana-Champaign

Abstract

The organism JCVI-syn3A (syn3A) is a bacterium derived from Mycoplasma mycoides with a reduced synthetic genome that is only 543 kb in size. In 2019, Breuer et al. published a metabolic map of this organism, and in 2022 Thornburg et al. published an updated map to account for newer insights. Using this map, we observe that in syn3A, the pyruvate dehydrogenase complex is missing its E1 subunit, which is coded for by pdhA and pdhB in wild type M. mycoides. Lacking this enzyme, the cells can either secrete their pyruvate or convert it to lactate while producing NAD⁺ and secrete lactate. With the reintroduction of the pyruvate dehydrogenase subunit E1, the cells also have the option to make acetyl phosphate from the pyruvate, which is a substrate for acetate kinase, which transfers the phosphate from acetyl phosphate to ADP, thereby generating ATP. In an FBA model, using constraints that rely on measurements in M. pneumoniae, the inclusion of this reaction decreases the doubling time from 96 to 61 min. We test this experimentally and find that the doubling time decreases from 103 to 86 min. We then make some adjustments to the constraints in the FBA model so that it fits our observed change in growth rate.

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Manoel Neres Santos Junior¹, Beatriz Almeida Sampaio¹, Guilherme Barreto Campos¹, Bruno Lopes Bastos¹, Marcelo Freire², John Glass², Daniela Matias de Carvalho Bittencourt³, Elibio Rech³, Lucas Miranda Marques¹

¹ Multidisciplinary Health Institute, Federal University of Bahia, Brazil; ² John Craig Venter Institute - West Coast Campus, JCVI/La Jolla, United States 3 EMBRAPA Genetic Resources and Biotechnology, Brazil

Abstract

Concurrently, synthetic genome bacterial strains have broadened the scope for investigating aggression and defense mechanisms in this domain. This study aimed to assess the microorganism-host interaction and the immunomodulation of the synthetic genome Mycoplasma mycoides subsp. capri (Mmc) strains in cell culture. Four previously characterized synthetic strains were used: JCVI-syn1.0 has a synthetic genome almost identical to wild-type Mmc. JCVI-syn3A, JCVI-Syn3⸫mch179-180, and JCVI-Syn3⸫mch179-186 possess a second copy of the rRNA operon, lack an efflux protein-encoding gene, and have protein-coding genes that were reintroduced into the JCVI-syn3.0 genome, which makes the morphologies and growth rates of these minimized strains more similar to those of wild-type Mmc. Additionally, a wild-type Mmc strain was included. These strains were used to infect cultures of peripheral blood mononuclear cells (PBMCs - 1x106 cells/mL) from goats. Following the assessment of cell viability using alamarBlue™, cytokine production (IL-1, TNF-α, and IL-4) was examined via ELISA. Nitric oxide (NO) levels were evaluated using the Griess assay. Inoculated cultures were exposed to varied bacterial concentrations for 6-24 hours. Viability testing revealed that all synthetic strains, except JCVI-Syn3⸫mch179-180 and JCVI-Syn3⸫mch179-186 at 12 hours, significantly inhibited PBMC proliferation (p<0.05) compared to controls. All tested strains could increase NO production at all analyzed infection time points. All strains induced IL-1β at 6 and 12 hours, with JCVI-syn1.0 showing higher induction at 6 hours (1x104 and 1x105 cells/mL) and 12 hours (only 1x105 cells/mL). The wild-type strain induced significant TNF-α production at 6 hours (1x104 and 1x105 cells/mL) compared to controls and synthetic strains. After 12 hours, JCVI-syn3A and JCVI-Syn3⸫mch179-186 strains also induced TNF-α production. At 18 hours, all treatments with synthetic strains at 1x105 cells/mL stimulated TNF-α production. IL-4 production lacked significance at 6-18 hours but increased significantly after 24 hours for all strains except JCVI-syn3A. This study demonstrates synthetic Mmc strains' potential in modulating the host-pathogen interaction and immune response in cell cultures. They inhibit PBMC proliferation and induce pro-inflammatory cytokine production. Further studies, including animal models, are crucial for understanding these mechanisms and developing therapeutic strategies against Mmc and related pathogens.

Financial Support

E.L.R. is supported by Embrapa Genetic Resources and Biotechnology/National Institute of Science and Technology in Synthetic Biology, National Council for Scientific and Technological Development (465603/2014-9), Research Support Foundation of the Federal District (0193.001.262/2017).

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Mallak Ali

Chan-Zuckerburg Imaging Institute

Abstract

Cryoelectron tomography has emerged as a high-resolution structural biology technique enabling the study of biological macromolecules in their native cellular context. A common approach for studying cells in situ is to use focused ion-beam milling (FIB-milling) to carve a thin slice out of the cell (<200 nm) to use for tilt series data collection. However, this approach is expensive and time-consuming in the context of rapid testing of image processing pipelines. Minicells have been reported to be thin enough for imaging without FIB-milling (ref), yet providing a crowded in situ environment suitable for developing cryoET tools. In collaboration with John Glass’ lab at JCVI, we are using the Syn 3A minimal genome cells for the rapid development of tomography processing pipelines including on-the-fly tilt series alignment, particle picking, and ab initio 3D refinement. In particular, Syn 3A is full of large identifiable ribosomes making it practical for developing on-the-fly in situ picking algorithms. We present a large dataset of Syn 3A minicells and ribosome picks generated using the tools developed at CZII.

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Rridhisha Kumar and Yo Suzuki

J. Craig Venter Institute

Abstract

A recent study established the in vitro functionality of a synthetic carbon fixation cycle containing four enzymes (PYC, OAH, ACS, PFOR), titled the POAP pathway. We propose to demonstrate the functionality of this pathway in vivo, using the JCVI Syn3B Minimal Cell as a biological chassis. To achieve this, we developed two 2-Enzyme Plasmid Designs, allowing us to create minimal cell strains that contain half of the POAP pathway each, which will then be pieced together to build the full cycle. Ongoing work focuses on testing the efficacy of these strains by using Gas Chromatography-Mass Spectrometry (GC-MS) to quantify successful enzyme activity including the perturbations of the substrate and product levels between the original and transformed strains. We anticipate our findings will be a starting point for using the minimal cell as a screening platform to analyze the effects of engineered proteins on basic biological functions. Additionally, we aim to develop a pipeline to engineer a full metabolic pathway into a viable organism from the ground up.

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Lea D. F. Kloss and Martin J. Lercher

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

Abstract

Independent, replicate bacterial populations that evolve under identical selective pressures can follow either similar or distinct phenotypic evolutionary trajectories. It remains to be elucidated whether different adaptive pathways indicate local fitness optima or alternative paths towards the same fitness optimum. We aim to analyze phenotypic variation across multiple replicate populations during adaptive evolution in continuous exponential growth under well-defined and constant environmental conditions. Multi-omics measurements at multiple time points during the experiment will allow us to trace parallel and divergent evolutionary trajectories. These data will be used to evaluate patterns of adaptation and to infer the repeatability of phenotypic evolution. Furthermore, we will evaluate the predictability of evolution by comparing the experimentally collected data to calculated predictions of growth rate and resource allocation derived from evolutionary simulations with a genome-scale kinetic balanced growth model (GBA).

A functional understanding of these issues is hampered by the complex regulatory systems of model organisms such as E. coli. It is therefore advantageous to use an organism with minimal regulatory elements, for which the JCVI minimal cells present a unique and interesting opportunity. We are currently investigating the feasibility of our experimental design using the minimal cell as a model organism. In particular, we are testing the ability of the minimal cell to be evolved in a robotics system that provides growth measurements and culture dilutions within a specified time interval. This platform would enable cultures to remain in exponential phase and facilitate the observation of fitness changes in real time. Additionally, we are deciding on what selection pressure to apply to best address our research questions.

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Zumra Seidel

J. Craig Venter Institute

Abstract

Whole genome transplantation (WGT) involves installing synthetic genomes into bacterial cells to construct cells with synthetic genomes. This method has been successfully applied only to mycoplasmas, which lack a cell wall and have a cholesterol-phospholipid membrane. The JCVI WGT protocol uses polyethylene glycol (PEG) to alter membrane fluidity and calcium chloride (CaCl₂) to optimize electrostatic interactions. However, we do not know exactly how WGT works. WGT relies on PEG mediated alteration of recipient cell membranes to either enable uptake of the donor genome or to promote the fusion of multiple bacterial cells around a donor genome. By understanding the mechanism of WGT we hope to be able to extend this technology for the installation of whole genomes or chromosome into other bacteria and eukaryotic cells, which could revolutionize the field of synthetic biology.

We hypothesize that the WGT procedure involves the integration of cell fusion, facilitated by modifying recipient cell membranes with PEG and CaCl₂. To test this hypothesis, we will utilize microfluidic devices that allow real-time imaging of the fusion process between donor genomes and recipient cells. This research aims to enhance the efficiency and reproducibility of WGT and advance its application for non-mycoplasma bacteria, starting with Gram-negative E. coli and Gram-positive Streptococcus thermophilus.

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Daniela Matias de C. Bittencourt^1,2, Marco Oliveira¹, Raquel Sampaio¹, Mariana Mathias Conroy Araujo^1,3, Igor Santos^1,3, Pedro Henrique Burgel^1,3, Lukas Brian G. de Andrade¹, Stephan Niele¹, John I. Glass², Elibio Rech^1,3

¹ Embrapa Genetic Resources and Biotechnology / National Institute of Science and Technology - Synthetic Biology, Parque Estação Biológica, PqEB, Av. W5 Norte (final) Caixa Postal 02372 – Brasília, DF – CEP, 70770-917; ²  The J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA; ³ Univerisidade de Brasília, Instituto de Ciências Biológicas, Programa de pós-graduação em Biologia Molecular, Brasilia/DF, Brazil

Abstract

JCVI-Syn3B is a cell that encodes the essential genes of Mycoplasma mycoides subspecies capri. Its synthetic genome has less than 500 genes, the majority of which are essential. The study and development of minimal genomes serve as a basis for better understanding the structural and functional organization of the genome and, consequently, how to manipulate it to perform specific functions in response to external stimuli. Therefore, the development of synthetic cells and effective tools for their assembly are pivotal for refined genetic control and the birth of novel processes and products. In this study we report the development of chromosome-free cells based on the minimal cell JCVI-Syn3B, as a prototype for the design of synthetic cells capable of acting as biosensors and performing specific functions in response to external stimuli. To this end, we engineered a genetic circuit using serine integrases (INT9) to initiate the expression of the endonuclease I-Ceul, which, in concert with native endonucleases, eradicates the host cell genome. I-Ceul targets a unique 26 base pair sequence (5'-TAACTATAACGGTCCTAAGGTAGCGA-3'), ubiquitous in bacterial genomes and situated within the conserved 23S rRNA rrl gene. The absence of a chromosome turns SimCell_JCVI-syn3B uncapable to replicate. This strategy promises to foster the innovation of secure, cost-effective biological systems for diverse sectors such as agriculture, industry, and healthcare, and offers an alternative technique for preparing recipients of synthetic genomes.