2nd Minimal Cell Workshop

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Isaac Justice and James Saenz

TU-Dresden

Abstract

Living membranes are complex systems, and cells invest many resources in regulating their composition and organization. Specific lipid species alter membrane functionality in tightly regulated ways, and membrane properties in turn control the functionality of membrane proteins. Robust homeoviscous adaptation (the ability to change membrane lipid composition in response to environmental perturbations in order to maintain membrane properties) makes cells more fit, and can be achieved with access to a large suite of possible membrane lipids with unique properties, as well as highly complex regulatory networks capable of tightly controlling membrane remodeling in response to environmental perturbations. However, the complexity of membrane lipidomics makes it difficult to isolate changes in specific lipid species, as well as overarching remodeling machinery. To understand the design principles of membrane remodeling, I work with Mycoplasma mycoides and its synthetic analog JCVI-Syn3. M. mycoides is a mammalian pathogen with one of the simplest known genomes. Among the properties that make it useful as a simple membrane model system are its lack of a cell wall, which makes the plasma membrane easy to access, and its reliance on its host for lipid uptake, which leaves it without almost all of its lipid synthesis capability. Because M. mycoides cannot synthesize its own acyl chains or head groups, we have unparalleled control over its membrane lipid composition based on the diet we provide it with. Using this control, I have designed specific diets, based on synthetic lipids that cannot be modified by M. mycoides phospholipases, that have pushed the M. mycoides and JCVI-Syn3 membranes to a simplicity of only 4 membrane lipids--the simplest reported living membrane--while retaining its viability. Using the diether membrane as a static backbone, I developed an assay where individual lipid species can be introduced to the membrane, and their remodeling can be quantified. This system allows me to directly probe what happens when homeoviscous adaptation is disrupted, as well as to identify a link between membrane lipidome composition, complexity, and cell fitness.

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James F. Pelletier^1,2, John I. Glass³, and Elizabeth A. Strychalski^4,*

¹ Centro Nacional de Biotecnología, 28049 Madrid, Spain; ² Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; ³ J. Craig Venter Institute, La Jolla, CA 92037, USA; ⁴ National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

Abstract

In JCVI-syn3A, cell division depends on multiple genes of known and unknown biomolecular function. To help hypothesize possible functions for these genes, we consider a physical description of cellular mechanics, which can provide an interlayer between gene function and cell division. We review current knowledge of genes in JCVI-syn3A contributing to two physical parameters relevant to cell division, namely, the surface-area-to-volume ratio and membrane curvature. This physical view of JCVI-syn3A may inform the attribution of gene function during cell division and other conserved physiological processes.

Funding

J.F.P. was supported by a Fannie and John Hertz Foundation Fellowship, the Mitchison laboratory at Harvard Medical School, the Fakhri laboratory at the Massachusetts Institute of Technology, and an International Human Frontier Science Program Organization (HFSPO) Cross-Disciplinary Fellowship LT000901/2021-C. J.I.G. was supported by National Science Foundation grants MCB 1840320, MCB 1818344, and MCB 1840301. E.A.S. was supported by the National Institute of Standards and Technology: certain commercial equipment, instruments, or materials are identified in this talk to foster understanding; such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the stated purpose.

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Hana Kiyama¹, Shigeyuki Kakizawa², Yuya Sasajima¹, Yuhei O Tahara¹, Daichi Takahashi¹, Makoto Miyata¹

¹ Osaka Metropolitan University, Japan; ² National Institute of Advanced Industrial Science and Technology (AIST) Japan

Abstract

In the last year’s workshop, we reported Spiroplsma swimming reconstructed in JCVI-syn3B, by inducing seven genes including fibril and five classes of mreB. This year, we searched for the smallest gene set for swimming. Surprisingly, sets of only two classes of MreBs were sufficient to confer the swimming, suggesting that Spiroplasma swimming may be originated from MreB differentiation based on accumulated mutations (Kiyama, H., et al. bioRxiv, 2021). To discuss the mechanism and evolution of Spiroplasma swimming, we are analyzing effects of randomly induced MreB mutations on cell shape and swimming.

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Miriam Bregman

University of Illinois – Urbana-Champaign

Abstract

Eukaryotic organelles, like mitochondria and chloroplast, evolved from free-living bacteria that were internalized within a host cell (archaeal or eukaryotic) and retained as endosymbionts (i.e., symbionts within a cell). Through a series of evolutionary transformations, these bacterial endosymbionts were transformed into host cell-organelles. We have minimal understanding of how this remarkable evolutionary transformation occurred; this remains one of the most outstanding unknowns in the evolution of life. A key feature central to this transformation is the extensive minimization of the endosymbiont genome due to loss of non-essential genes and transfer of genes to the host nucleus; thereby only a small fraction of the endosymbiont genome was retained in the newly formed organelle. Inspired by these evolutionary events, we are engineering artificial endosymbiosis between extant free-living bacteria and eukaryotic cells with a goal to engineer, evolve and transform the engineered endosymbiotic bacteria into novel synthetic organelles. We have previously engineered cyanobacteria and E. coli endosymbionts in yeasts where the endosymbionts perform organelle-like functions (e.g., bioenergetics) for the host yeast cell. For our efforts with JCVI, we are investigating if we can engineer JCVI’s minimal cells (which already lack non-essential genes) as endosymbionts within yeasts. If successful, we believe that this platform will presents us with an unprecedented opportunity to study and explore synthetic organelle generation and organelle evolution starting from an already minimal endosymbiont genome. Therefore, combining our expertise in directing endosymbiosis with JCVI’s ability to generate and manipulate minimal cells, our first step will be to engineer endosymbiosis between minimal cell mutants and yeast cells by creating metabolic intercedences between the endosymbiont and the host. Based on the evolutionary premise, we will then explore several strategies, including but not limited to metabolic coupling, further genome minimization amongst others, to engineer, evolve and transform the minimal cell-derived endosymbionts into an artificial, synthetic organelles. Such studies are expected to provide molecular insights related to the transformation of bacteria into organelles and also open up a new frontier in synthetic biology.

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Zane R. Thornburg¹, David Bianchi¹, Troy Brier¹, Benjamin Gilbert¹, Tyler Earnest¹, Marcelo Melo¹, Nataliya Safronova², James Saenz², Vinson Lam³, Elizabeth Villa³, Jiwoong Kwon⁴, Taekjip Ha⁴, Andras Cook⁵, Kim S. Wise⁵, Clyde A. Hutchison III⁵, Hamilton O. Smith⁵, John I. Glass⁵, Zaida Luthey-Schulten¹

¹ Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL; ² B CUBE Center for Molecular Bioengineering, Technische Universität Dresden, Germany; ³ University of California San Diego, La Jolla, CA; ⁴ Johns Hopkins University, Baltimore, MD; ⁵ J Craig Venter Institute, La Jolla, CA

Abstract

I will present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. The WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior. The 3D model is being extended to include cell division with geometries influenced by fluorescent imaging and cryo-ET of dividing Syn3A cells.

Reference: Thornburg et al., “Fundamental behaviors emerge from simulations of a living minimal cell”, Cell, 2022. DOI: 10.1016/j.cell.2021.12.025

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Abstract

Whole-cell models (WCMs) of Syn3A have been developed by combining a kinetic models of the essential metabolism and genetic information processing. For the spatially resolved WCM of Syn3A, we reconstructed cell geometries of Syn3A, including the ribosome distribution, from cryo-electron tomography and generated ensembles of self-avoiding circular chromosome configurations on a lattice, which were constrained by the cell geometry and ribosomes. These configurations of the DNA were static within any individual simulations of the WCM and their heterogeneous crowding influenced the diffusion of biological macromolecules, such as RNA polymerases (RNAP) and mRNA. A continuum model of Syn3A’s chromosome has now been developed to model the dynamics of the chromosome’s organization over the course of a complete cell cycle, including both DNA replication and cell division. Within the continuum model, the DNA is modeled as a stiff homopolymer with a torsional stiffness at a resolution of 10 bp per monomer. Brownian dynamics simulations using this improved model will allow for changes in the accessibility of genes to RNAPs, kinetics-based replication to govern the processive motion of replication forks, and segregation of daughter chromosomes during cell division. While Syn3A lacks the complex systems known to orchestrate DNA segregation and division in bacteria, its genome does code for SMC protein complexes (SMC-scpAB), which may facilitate entropic segregation of chromosomes by DNA compaction through local looping. Additionally, the computational model permits simulation of chromosomes in nontrivial replication states, whose presence is evident from qPCR experiments and WCM simulations. Work is ongoing on coupling the spatially-resolved WCM simulations to Brownian dynamics simulations of the chromosome and ribosomes.

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Abstract

Stochastic gene expression begins with the repeated transcription of DNA by RNA polymerase (RNAP) to create the various functional forms (rRNA, tRNA, mRNA, and sRNA) of the bacterial transcriptome. A transcriptional event can be reduced to a three stage mechanism: (1) transcription initiation, the association of RNAP to the DNA, (2) transcription elongation, the 1-dimensional diffusion of RNAP along the DNA, and (3) transcription termination, the disassociation of RNAP from the DNA. The initiation and termination of transcriptional events are outlined by the genome architecture defined as the local arrangement of genetic features along the circular bacterial genome identified via sequence motifs (promoters, Shine-Dalgarno, gene open-reading frames, and transcription termination sites, etc.). The local arrangement of these sequence motifs form operon-like regions, known as transcriptional units, directly controlling the expression of encoded RNA species. The stochastic activity between RNAP and the genetic features of a transcription unit results in differential transcription forming RNA isoforms, RNA transcripts from a similar region of the genome encoding different information. Locations of the genetic features that define the transcription unit boundaries were predicted using structural and bioinformatic analysis, and then integrated to predict transcriptional units within JCVI-syn1.0. The theoretical predictions agree well with observations of expression obtained using Oxford Nanopore Technologies Direct RNA sequencing. In this talk I will discuss how the comparison of the two methods has begun to resolve the distribution of RNA isoform complexity within key genomic regions and provide new insight into transcriptional activity within Syn1.0.

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Daniela Matias de C. Bittencourt, Marco Oliveira, Raquel Sampaio, Mariana Mathias, Elibio Rech, John Glass

EMBRAPA Genetic Resources and Biotechnology / EMBRAPA Genetic Resources and Biotechnology / National Institute of Science and Technology - Synthetic Biology, Brasilia, DF, Brazil

Abstract

The minimal cell JCVI-Syn3B is an attractive host for the manipulation of gene activity in order to uncover some of the currently unknown basic biological processes in Mycoplasmas. Furthermore, the development of genetic regulatory parts will provide the foundation for the creation of new, even more sophisticated bioprocess control devices, bolstering the viability of JCVI-Syn3B as a biotechnology platform. Aiming to that, we are testing and optimizing a variety of induction systems and genetic tools to create more complex behaviors and tighter biological process control in the minimal cell. Recently, we have demonstrated effective control of the RNA polymerase flux using an integrase (INT9) to catalyze unidirectional inversion of regulatory DNA sequences to turn on-off a reporter gene in JCVI-Syn3B. Now, we are testing the INT9, under the control of the tetracycline inducible promoter, to precise activate the expression of the endonuclease I-Ceul. The endonuclease I-Ceul recognizes a specific 26 bases pairs sequence (5'-TAACTATAACGGTCCTAAGGTAGCGA-3') present in most bacterial genomes and encoded within the conserved 23S rRNA rrl gene. Once expressed, the I-Ceul has the capacity to induce the full degradation of the bacterial genome, allowing the development of non-replicable functional synthetic cells. For the functional analysis of the new cell (SimCell_JCVI-syn3B), a genetic construct with the complete arginine deaminase metabolic pathway will be used. A third construction containing a variation of the CRISPR-Cas9 system, CRISPRi, will also be implemented to repress the expression of an essential gene in JCVI-Syn3B, in order to purify SimCell_JCVI-syn3B from any remaining parent cell. Engineered Mycoplasma pulmonis inducible promoters have also been tested to regulate the expression of genes in JCVI-Syn3B, seeking the activation of the genetic constructions independently. Once validated and optimized, these can be excellent tools to study minimal genomes as well for the development of novel biological processes for different biotechnological applications.

Grants: FAP-DF (no. 0193.001.262/2017 and no. 0193-00000229/2021-21), CNPq (no.465603/2014-9)

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Samuel Miravet-Valverde¹, Daniel Shawn¹, Rocco Mazzolini², Carlos Piñero², Luis Serrano^2,3,4, and Maria Lluch-Senar^2,5

¹ Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; ² Pulmobiotics ltd, Dr. Aiguader 88, Barcelona 08003, Spain; ³ Universitat Pompeu Fabra (UPF), Barcelona 08002, Spain; ⁴ ICREA, Pg. Lluís Companys 23, Barcelona 08010, Spain; ⁵ Basic Sciences Department, Faculty of Medicine and Health Sciences Universitat Internacional de Catalunya, 08195 Sant Cugat del Vallès, Spain

Abstract

To obtain a minimal cell often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deep-sequencing (TnSeq) to identify insertion points and gene essentiality. However, several aspects like genome annotation, environmental conditions or epistatic interactions can cause unforeseen changes in essentiality maps. Here, we present bioinformatic and experimental approaches to annotate genomes and to define essentiality maps. We designed two bioinformatic tools (FASTQINS and ANUBIS) to explore facts and artifacts in transposon sequencing and essentiality studies. Also, ProTInSeq and LoxTnSeq are two new experimental “-omic” approaches to refine genome annotations and to generate large genome reduction, respectively. ProTInSeq technique is based on transposons engineered to have a positive or negative protein selection marker expressed when the transposon is inserted in-frame into a protein-coding gene. In the genome-reduced bacterium Mycoplasma pneumoniae, ProTInSeq identified 80% of known expressed proteins and 153 novel small ORF-encoded proteins (SEPs; ≤100 aa) that represent up to 18% of this minimal bacterium’s proteome. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to M. pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions.

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Carlos Piñero-Lambea^1,*, Eva Garcia-Ramallo¹, Samuel Miravet-Verde², Raul Burgos², Margherita Scarpa¹, Luis Serrano^2,3,4, and Maria Lluch-Senar^1,5*

¹ Pulmobiotics ltd, Dr. Aiguader 88, Barcelona 08003, Spain; ² Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; ³ Universitat Pompeu Fabra (UPF), Barcelona 08002, Spain; ⁴ ICREA, Pg. Lluís Companys 23, Barcelona 08010, Spain; ⁵ Basic Sciences Department, Faculty of Medicine and Health Sciences Universitat Internacional de Catalunya, 08195 Sant Cugat del Vallès, Spain

Abstract

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.

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Christoph Elfmann, Tiago Pedreira, and Jörg Stülke

Department of General Microbiology, University of Göttingen, Grisebachstr. 8, 37077 Göttingen

Abstract

Model organism databases are of crucial importance for biological research. By combining information from different domains of knowledge, they represent vital tools for the development of research hypotheses. Here we present two Mycoplasma databases: SynWiki for the artificial minimal organism Mycoplasma mycoides JCVI-syn3a, and MycoWiki for the pathogen Mycoplasma pneumoniae (1, 2). These platforms host a mixture of manually curated expert knowledge and imported data sets from relevant publications and other resources. The data is organized around the dedicated web pages for individual genes and proteins, which give an overview over functional annotation, gene products, neighboring genes, and other properties. Interactive browsers are a highlight of our websites. They allow users to explore the genome, protein-protein interactions, and structures in an intuitive way. SynWiki and MycoWiki are open for contributions by the research community, and we hope that our databases can be an asset to the investigation of Mycoplasma.

¹ Pedreira et al. (2022) SynWiki: functional annotation of the first artificial organism Mycoplasma mycoides JCVI-syn3A. Protein Sci. 31: 54-62.

² Elfmann et al. (2022) MycoWiki: functional annotation of the minimal model organism Mycoplasma pneumoniae. Front. Microbiol. 13: 935066.

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Anthony Duval

Universite de Sherbrooke

Abstract

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~800kb), rapid growth rate, and the possibility of whole-genome cloning and transplantation with this organism. We have recently developed a semi-defined growth medium for M. florum in which growth depends on the addition of a sugar molecule for energy production. Different carbon sources were investigated and the importance of several components of the medium was evaluated. The final medium composition supports rapid growth rates and high cell concentrations. We then performed high-density transposon mutagenesis to study the essentiality of genes in cultures grown with different carbon sources, which allows us to identify genes involved in their uptake and metabolism. Overall, our work provides an interesting strategy to infer a function for yet uncharacterized genes in this organism.

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David S. Goodsell

Center for Computational Structural Biology, Scripps Research Institute

Abstract

Data from genomics, proteomics, structural biology and cryo-electron microscopy are integrated into a structural illustration of a cross section through an entire JCVI-syn3.0 minimal cell. The illustration is designed with several goals: to inspire excitement in science, to depict the underlying scientific results accurately, and to be feasible in traditional media. Design choices to achieve these goals include reduction of visual complexity with simplified representations, use of orthographic projection to retain scale relationships, and an approach to color that highlights functional compartments of the cell. Given that this simple cell provides an attractive laboratory for exploring the central processes needed for life, several functional narratives are included in the illustration, including division of the cell and the first depiction of an entire cellular proteome. The illustration lays the foundation for 3D molecular modeling of this cell.

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Carole Lartigue and Pascal Sirand-Pugnet

UMR BFP INRAE, Université de Bordeaux, team Mollicutes

Abstract

Due to the lack of efficient recombination in most species, production of mutant strains and genome engineering of mycoplasmas and other mollicutes has long been limited. More than a decade ago, the first successful cloning of a mycoplasma genome in yeast and subsequent back transplantation into a recipient cell opened undreamed possibilities of bacterial genome manipulations. From that time, 20 natural genomes from 15 species of mollicutes have been cloned in yeast, using various protocols as the newly released CreasPy-cloning and CreasPy-Fusion methods. Genome transplantation remains the main bottleneck and is currently available for seven species, related to the so-called Mycoplasma mycoides cluster. Our current work aims to identify the main barriers limiting the expansion of genome transplantation to other species. In parallel, several new genetic tools have been recently developed for gene targeting directly in mycoplasma cells. These include an exogenous recombination RecET system, a CRISPR base editor and a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology.

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Department of Physics, University of California at San Diego (Laboratory of Suckjoon Jun)

Abstract

One of the primary goals in our lab is to understand the control of growth and the cell cycle in bacteria. Very recently, we started experiments to grow minimal cells in the microfluidic mother machine developed in our lab. In this talk, I will show very preliminary data on the growth of Syn3.0 in the mother machine.

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David Bianchi

University of Illinois Urbana-Champaign (Luthey-Schulten Group)

Abstract

Recently, we presented a whole-cell kinetic model of the genetically minimal bacterium JCVI-syn3A that described the coupled metabolic and genetic information processes and predicted behaviors emerging from the interactions among these networks. JCVI-syn3A is a genetically reduced bacterial cell that has the fewest number and smallest fraction of genes of unclear function, with approximately 90 of its 452 protein-coding genes (that is less than 20%) unannotated. Further characterization of unclear JCVI-syn3A genes strengthens the robustness and predictive power of cell modeling efforts and can lead to a deeper understanding of biophysical processes and pathways at the cell scale. Here, we apply computational analyses to elucidate the functions of the products of several essential but previously uncharacterized genes involved in integral cellular processes, particularly those directly affecting cell growth, division, and morphology. We also suggest directed wet-lab experiments informed by our analyses to further understand these “missing puzzle pieces” that are an essential part of the mosaic of biological interactions present in JCVI-syn3A. Our workflow leverages evolutionary sequence analysis, protein structure prediction, interactomics, and genome architecture to determine upgraded annotations. Additionally, we apply the structure prediction analysis component of our work to all 452 protein coding genes in JCVI-syn3A to expedite future functional annotation studies as well as the inverse mapping of the cell state to more physical models requiring all-atom or coarse-grained representations for all JCVI-syn3A proteins.