1st Minimal Cell Workshop

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Andras Cook¹ and David Bianchi²

¹ J. Craig Venter Institute; ² University of Illinois (Luthey-Schulten Group)

Abstract

The near-minimal organism Mycoplasma mycoides JCVI-syn3 (syn3) is valuable for studying the very basics of life. It may also be useful to build metabolic or signal transduction pathways into, because it is less likely to contain interfering pathways than other model organisms like E. coli. Drawbacks of syn3 include a slow growth rate, morphologically irregular cells and the need for a complex growth medium containing many ingredients. syn3 has previously been improved to syn3A, which, with the addition of 19 genes present in wildtype M. mycoides but not in syn3, forms cells that are morphologically similar to wild type M. mycoides. These cells appear much more robust and have a higher growth rate. Here we further increase the tractability of this near-minimal organism by improving its metabolism: We find 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 syn3A cells can either secrete their pyruvate or convert it into lactate while producing NAD+ and then secrete the lactate. With the reintroduction of the E1 pyruvate dehydrogenase subunit, the cells also have the option to make acetyl phosphate from the pyruvate, which is a substrate for an ATP synthase that transfers the phosphate from the acetate to an ADP. Using FBA models, we predict that including this reaction should decrease the doubling time from 96 to 61 min. We test this experimentally and find that the doubling time decreases from 103 to 86 min.

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Abstract

With 493 genes and 452 protein coding genes the synthetically engineered J. Craig Venter Institute (JCVI) Syn3A Minimal Cell provides a unique platform to study the fundamental processes of a living cell. In addition to determining the production of membrane components (phosphatidylglycerol, glycolipids, transmembrane proteins etc.) required for the cell to double from the expected cell biomass, we have developed a lipid biomass based on lipidomic measurements obtained by the Saenz Research Group (TU-Dresden). After developing these expected “production goals” of the cellular economy, we have constructed a dynamic kinetic model linking lipid metabolism and membrane protein insertion via the Sec system to cell growth utilizing theoretical and experimentally determined values for lipid headgroup membrane surface area contribution. Simulating this model via multi-scale simulation methodologies that we have developed allows us to predict the variation in cell doubling times for populations of hundreds of cells, ranging from 90-110 minutes, that are determined by and responsive to other cellular processes such as gene expression and metabolism of key moieties, such as CTP used in phospholipid synthesis and UTP used in glycolipid synthesis.

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Abstract

Whole-cell modeling requires the combination of multiple simulation methods and importantly the results of many experimental techniques to inform dynamics as well as cell architecture. We constructed a whole-cell kinetic model of JCVI-syn3A using parameters obtained from single molecule experiments and kinetic parameter databases such as BRENDA. A spatial model of Syn3A was constructed from cryo-electron tomograms including transcription of all 493 genes and translation and degradation of all 452 mRNAs. From the mRNA diffusion and degradation reactions occurring at membrane-bound degradosomes, we predict the whole distribution of mRNA half-lives in Syn3A. A well-stirred version of the model includes multiple DNA replication initiation and elongation events per cell cycle, which agrees with qPCR experiments and was observed to help maintain constant nucleotide pools in metabolism. These novel simulations track the exact ATP use of each reaction in the simulation, accounting for all significant energetic costs in Syn3A.

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Abstract

To achieve a whole cell cycle in the whole cell computational model of JCVI-syn3A it is necessary to model its growth. From our lab’s metabolic model, we have a growth rate for JCVI-syn3A based on synthesis and incorporation of lipids and membrane proteins. Cryo-electron tomograms give the location of the cell’s ribosomes near the beginning and near the end of its growth: snapshots of the cell shortly after division and again when it’s volume has approximately doubled. From this experimental data, we simulate growth by updating membrane size and ribosome and membrane protein positions.

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Bittencourt, Daniela M de C^{1, 2, 3}; Brown, David²; Wise, Kim²; Rech, Elíbio^{1, 3}; Freire, Marcelo²; Glass, John²

¹ Embrapa Genetic Resources and Biotechnology, DF, Brazil; ² J Craig Venter Institute, La Jolla-CA, USA; ³ National Institute of Science and Technology in Synthetic Biology (INCT - BioSyn), DF, Brazil

Abstract

As the smallest free growing organism, it has been suggested that Mycoplasmas should be used as a blueprint to evaluate the genes necessary for life. In 2010, a 1.1 Mb synthetic genome based on the genome of Mycoplasma mycoides was used to create the strain JCVI-Syn1.0. The strain contains almost all the genes from the wild type mycoides. A few years latter (2016), JCVI has reported the minimal cell JCVI-Syn 3.0. Several strains of intermediate genome size were obtained until the M. mycoides genome was minimized to 531 kb. While it is likely that significantly minimized strains lack the capacity to infect mammalian cultures this study demonstrates the minimal cell JCVI-syn3.0 and its derivatives cannot survive in mammalian cell cultures. We have developed an infectivity assay to evaluate the capacity of M. mycoides strains JCVI-syn1.0, JCVI-syn3.0 along with other synthetic M. mycoides strains to survive and grow in HEK293 and HeLa cells culture. We identified a cluster of eight candidate genes (MMSYN1-179 to MMSYN1-186) for the persistence of M. mycoides in mammalian cell cultures and evaluate their contribution for the mechanism behind M. mycoides capacity to infect mammalian cells and trigger an immunological response. In addition to allowing a better understanding of bacterial pathogenesis, identification of virulence factors in M. mycoides may also allow the design of new potential antimicrobials and vaccines.

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Abstract

The bacterial transcriptome, composed of rRNA, tRNA, mRNA, and sRNA, governs expression level of the proteome throughout the cell. The transcriptome evolves from transcription events governed by probabilistic interactions within the cell. The boundaries of the transcriptional events are defined by the genome architecture defined as the local arrangement along the bacterial genome of genetic features identified via sequence motifs (promoters, gene coding regions, and transcription termination sites). The local arrangement of these sequence motifs form operon like regions, transcription units, directly controlling the expression of encoded RNA species. Using well known characteristics of these motifs, their locations were predicted computationally, and integrated together to outline transcription units within the genomes of Syn1 and Syn3A. The computational predictions agreed quite well experimental operon identification using Oxford Nanopore Technologies long read native RNA sequencing. In my talk, I will discuss ongoing work to unravel other impacts the Syn1 and Syn3A genome architectures has on the transcriptome, and any potential impact on cellular functions in the two organisms.

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Shigeyuki Kakizawa^1,2, Fumiko Nishiumi³, Itaru Yanagihara³, John I. Glass², Yo Suzuki²

¹ National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8560, Japan; ² Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, United States; ³ Department of Developmental Medicine, Research Institute, Women's and Children's Hospital, Izumi-city, Osaka 594-1101, Japan

Abstract

We observed Syn3.0 strains expressing several fluorescent proteins under microscopy, and found that some giant cells engulf smaller cells, and some giant cells fused each other. Most giant cells are continuously expressing fluorescent proteins for a long time. These results suggested that borderline between cells is ambiguous in the minimal cell. The relationships between the minimal cell and early life will be discussed.

In addition, we successfully reconstructed the attachment ability and pathogenicity of Ureaplasma parvum using the minimal cell. After expression of an Ureaplasma parvum surface membrane lipoprotein multiple banded antigen (MBA) in Syn3.0B, the cells could attach to HeLa cells. After expression of the MBA plus an U. parvum vacuolating factor (UpVF) in Syn3.0B, the cells could attach to HeLa cell and induce cell death.

Reference

Nishiumi, F., Y. Kawai, Y. Nakura, M. Yoshimura, H. N. Wu, M. Hamaguchi, S. Kakizawa, Y. Suzuki, J. I. Glass and I. Yanagihara (2021). "Blockade of endoplasmic reticulum stress-induced cell death by Ureaplasma parvum vacuolating factor." Cell Microbiol: e13392.

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Roy Moger-Reischer¹, John I. Glass², Kim S. Wise², Lijie Sun^2,3, Daniella Bittencourt^2,4, Michael Lynch⁵, Jay T. Lennon¹

¹ Department of Biology, Indiana University, Bloomington, IN 47405, USA; ² J. Craig Venter Institute, La Jolla, CA 92037, USA; ³ Sorrento Therapeutics, Inc., San Diego, CA 92121 USA; ⁴ Embrapa Genetic Resources and Biotechnology, Brasília, 70770-917, Brazil; ⁵ Arizona State University, Tempe, AZ 85287, USA

Abstract

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life. Here, we report on how a synthetically constructed minimal cell contends with the forces of evolution compared to a non-minimized cell from which it was derived. Genome streamlining was costly, but 80% of fitness was regained in 2000 generations. Although selection acted upon divergent sets of mutations, the rates of adaptation in the minimal and non-minimal cell were equivalent. The only apparent constraint of minimization involved epistatic interactions that inhibited the evolution of cell size. Together, our findings demonstrate the power of natural selection to rapidly optimize fitness in the simplest autonomous organism, with implications for the evolution of cellular complexity.

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Abstract

The bacterial strain JCVI-syn3.0 stands as the first example of a ‘synthetic’ organism, possessing a minimized version of the Mycoplasma mycoides genome that was chemically synthesized in vitro. Here we report the experimental evolution of a syn3.0-derived strain. Ten independent replicates were evolved for several hundred generations, leading to growth rate improvements of >15%. Endpoint strains possessed an average of 8 mutations composed of indels and SNPs, with a pronounced C/G-<A/T transversion bias. Multiple genes were repeated mutational targets across the independent lineages, including phase variable lipoprotein activation, 5 distinct nonsynonymous substitutions in the same membrane transporter protein, and inactivation of an uncharacterized gene. RNA-seq analysis showed how these strains evolved by upregulating ribosomal proteins and downregulating various DNA and RNA related proteins. This work establishes the suitability of synthetic, minimal strains for laboratory evolution, providing a means to optimize strain growth characteristics and elucidate gene functionality.

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John I. Glass⁴, Drago Haas¹, Antje M. Thamm², Jiayi Sun², Lili Huang³, Lijie Sun⁴, Guillaume A.W. Beaudoin², Kim S. Wise⁴, Claudia Lerma-Ortiz², Steven D. Bruner⁵, Marian Breuer⁶, Zaida Luthey-Schulten⁷, Jiusheng Lin⁸, Mark A. Wilson⁸, Greg Brown⁹, Alexander F. Yakunin⁹, Inna Kurilyak¹⁰, Jacob Folz¹⁰, Oliver Fiehn¹⁰, Andrew D. Hanson², Christopher S. Henry^{11, 12}, and Valérie de Crécy-Lagard^{1, 13}

¹ Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA; ² Horticultural Sciences Department, University of Florida, Gainesville, FL 3261, USA; ³ Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611; ⁴ J. Craig Venter Institute, La Jolla, CA 92037, USA; ⁵ Chemistry Department, University of Florida, Gainesville, FL 32611, USA; ⁶ Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, 6200 MD Maastricht; ⁷ Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL; ⁸ Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, NE; ⁹ Department of Chemical Engineering and Applied Chemistry, University of Toronto; ¹⁰ West Coast Metabolomics Center, UC Davis; ¹¹ Data Science and Learning, Argonne National Laboratory, Argonne; ¹² Consortium for Advanced Science and Engineering, The University of Chicago; ¹³ University of Florida Genetics Institute

Abstract

Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes with known metabolite damage repair or preemption functions were identified and experimentally validated, including 5-formyltetrahydrofolate cyclo-ligase, CoA disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase family domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage-control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal.

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Abstract

In recent years, the genomes of several organisms have been reported to be synthesized and functioning. They are constructed by linking chemically synthesized short DNA fragments in E. coli or yeast and gradually increasing their size to build a giant genome. Since current genetic engineering does not have the technology to amplify large genome-level DNA, amplification and linkage must be performed in the organism. Therefore, we focused on the RCR method¹, which can amplify large genome-level DNA in vitro, and attempted to amplify and edit the syn3.0 genome in vitro. We also show that the in vitro amplified syn3.0 genome can be used for transplantation. These results strongly suggest that genomes can be generated in vitro in the same way as plasmids.

Refrences

¹ Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle. Su'etsugu M, Takada H, Katayama T, Tsujimoto H. Nucleic Acids Res. 2017 Nov 16;45(20):11525-11534.

Funding

This work was supported by the JST CREST, Japan (JPMJCR18S6) to K.V.T., START (JPMJST1816) to K.V.T., and the MEXT Scientific Research on Innovative Areas, ‘Chemistry for multimolecular crowding biosystem’ (JP17H06355) to K.V.T.

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De Oliveira, Marco^{1, 2, 3}, BITTENCOURT, Daniela ^{2, 3}, SUZUKI, Yo⁴, GLASS, John⁴, RECH, Elibio^2,3

University of Brasília (UnB), DF, Brazil¹; Embrapa Genetic Resources and Biotechnology, DF, Brazil²; National Institute of Science and Technology in Synthetic Biology (INCT - BioSyn), DF, Brazil³; J Craig Venter Institute, CA, United States of America⁴

Abstract

The identification of the set of essential and quasi-essential genes for the assembly of the minimal cell approximation Mycoplasma mycoides JCVI-syn3B put us closer to understanding the basic molecular requirements to life. The transposon bombardment methodology used allows the screening of every gene in the cell through transposon insertion and gene disruption, although only one loss-of-function event can be observed in a single cell. However, genetic redundancy and functional interactions between genes makes it interesting to evaluate gene essentiality in a broader context of gene clusters. We propose that serine-integrases acting on defined groups of genes can work as such systems. We first evaluated the use of two serine-integrases (INT9 and INT13) as genetic switches capable of turning the expression of a reporter gene on by flipping its DNA sequence upon induction in the synthetic minimal cell Mycoplasma mycoides JCVI-Syn3.0. Cells were transformed with a plasmid containing one integrase gene under control of a Tetracycline inducible promoter as well as the mCherry gene in a reverse orientation relative to its promoter, therefore silenced, flanked by the att sites of that integrase. All the plasmids also had a Cre/loxP system to allow insertion on a landing pad present in the Syn-3B genome and a selection marker. Following incubation with tetracycline, we measured mCherry signal intensity in a microplate reader. Results show that INT9 activation led to a significant increase in fluorescence intensity, with no observed signal in non-induced groups, and the expression of the flipped reporter gene continued after tetracycline removal. INT13 induction did not result in apparent expression of mCherry, although reporter inhibition in one of the positive controls suggests an influence of the sites rather than INT13 ineffectiveness. Preliminary results obtained with INT2 also indicate no recombinase activity upon induction, but further investigation is necessary, especially regarding vectors design. Our next step will be the insertion of INT9 att sites flanking multiple endogenous genes of syn-3B while improving INT2 and INT13 systems. Considering the field of synthetic biology, these integrases can also be considered valuable tools for the assembly of a synthetic regulatory network in minimal and synthetic cells in a bottom-up approach.

Acknowledgements

This research was funded by National Institute of Science and Technology in Synthetic Biology, National Council for Scientific and Technological Development, and Research Support Foundation of the Federal District, Brazil.

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Keoni Gandall, Drw Endy (Stanford University)

Abstract

FreeGenes has synthesized the majority of proteins in JCVI-Syn3A with an optimized E.coli codon table, all compatible with MoClo assembly. We'll talk about how to get and use these genes in your experiments.

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Veronika Scholz, Magdalena Rakwalska-Bange, Roland Lill

Institute of Cytobiology, SYNMIKRO Center for Synthetic Microbiology, Philipps-University Marburg, Karl-von-Frisch-Str. 14, 35043 Marburg, Germany. Email: lill@staff.uni-marburg.de, sachsenh@staff.uni-marburg.de.

Abstract

Iron-sulfur (Fe/S) clusters are essential cofactors for a myriad of protein functions. However, Fe/S proteins cannot be employed in the JCVI minimal bacterial cell since Mycoplasma mycoides has lost the Fe/S cluster biogenesis machinery as it evolved towards an Fe/S protein independent lifestyle.

In this work we aim to develop a functional Fe/S cluster biosynthesis pathway in JCVI minimal cells. Through genome mining of related Fe/S protein dependent Mycoplasma species, genes that are likely part of a Fe/S cluster biogenesis machinery and are likely to be expressed in the minimal cells were identified. A well-studied, Fe/S dependent enzyme provides a sensitive readout for functionality and evolution of the synthetic Fe/S cluster biosynthesis system.

Engineering of a functional Fe/S cluster biosynthesis system may provide a better understanding of the basic components required for a minimal Fe/S cluster biosynthesis system in vivo and can serve as a platform for expression of Fe/S cluster dependent proteins in minimal cells.

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Abstract

The Synthetic Biology and Bioenergy group at the J. Craig Venter Institute has constructed a free-living bacterial cell that operates with only 473 genes (¹). This simple cell makes the complete understanding of a cellular system a realistic goal for the first time. A challenge for achieving this goal is that there are 149 genes that are only poorly characterized in the genome of this organism. The main objective of this project is to establish a high-throughput method to characterize these genes. For this purpose, we established conditional attenuation of gene expression via CRISPRi in the minimal cell^2,3 and also established a streamlined method to construct a genome-wide library. We are in an exciting phase to complete the construction of the library and initiate the systematic characterization of phenotypes.

Reference

¹ Hutchison, C. A. et al. Design and synthesis of a minimal bacterial genome. Science 351, aad6253 (2016).

² Qi, L. S. et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152, 1173–1183 (2013).

³ Mariscal, A. M. et al. Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells. ACS Synth. Biol. 7, 1538–1552 (2018).

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Abstract

Antibiotic resistance is a growing problem, but bacteria can evade antibiotic treatment via tolerance and persistence. Antibiotic persisters are a small subpopulation of bacteria that tolerate antibiotics due to a physiologically dormant state. Hence, persistence is considered a major contributor to the evolution of antibiotic-resistant and relapsing infections. Here, we used the synthetically developed minimal cell Mycoplasma mycoides JCVI-Syn3B to examine essential mechanisms of antibiotic survival. The minimal cell contains only 473 genes, and most genes are essential. Its reduced complexity helps to reveal hidden phenomenon and fundamental biological principles can be explored because of less redundancy and feedback between systems compared to natural cells. We found that Syn3B evolves antibiotic resistance to different types of antibiotics expeditiously. The minimal cell also tolerates and persists against multiple antibiotics. It contains a few already identified persister-related genes, although lacking many systems previously linked to persistence (e.g. toxin-antitoxin systems, ribosome hibernation genes).

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Sébastien RODRIGUE (Université de Sherbrooke, Canada)

Abstract

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~800kb), fast growth rate as well as the possibility to perform whole-genome cloning and transplantation with this organism. We recently reported a detailed characterization of M. florum describing several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. Using this experimental data as a foundation, we also developed a genome-scale model for M. florum that guides genome engineering projects in this simple organism.

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James F. Pelletier^1,2, Lijie Sun³, Kim S. Wise³, Nacyra Assad-Garcia³, Bogumil J. Karas⁴, Thomas J. Deerinck⁵, Mark H. Ellisman⁵, Andreas Mershin¹, Neil Gershenfeld¹, Ray-Yuan Chuang³, John I. Glass³, Elizabeth A. Strychalski²

¹ Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; ² National Institute of Standards and Technology, Gaithersburg, MD 20899, USA; ³ J. Craig Venter Institute, La Jolla, CA 92037, USA; ⁴ Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada; ⁵ National Center for Microscopy and Imaging Research, University of California-San Diego, La Jolla, CA 92037, USA

Abstract

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.

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John Sanford, BS; James Daubenspeck, PhD; Kevin Dybvig, PhD; T. Prescott Atkinson, MD, PhD Department of Pediatrics, University of Alabama at Birmingham, USA.

Abstract

Surface proteolipid glycosylation has been reported in numerous species across the genus Mycoplasma. There appear to be two types of glycosylation events on Mycoplasma surface proteolipids: a characterized hexosylation system that stochastically scavenges oligosaccharides from the environment (Type I glycosylation), and an uncharacterized glycosylation system that assumedly donates an oligosaccharide through a non-canonical glycosyltransferase (Type II glycosylation). We have been unable to find evidence of a transposon knockout for the Type I glycosylation system in every library we have tested, which we think is due to an essential function – likely protection from secreted proteases. Interestingly, the Mycoplasma mycoides cluster appears to have a different Type II glycosylation profile than human or murine Mycoplasma species. By investigating glycosylation through glycoprotein stained SDS-PAGE, gas chromatography-mass spectrometry, and high-resolution mass spectrometry in Mycoplasma genitalium and JCVI-syn3, we can investigate glycobiology at the cell surface in the context of minimal self-replicating life.

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Bittencourt^3,4, Christopher P. Kempes⁵, Kim S. Wise³, Hans A. Heus¹, Wilhelm T. S. Huck¹, Katarzyna P. Adamala², John I. Glass³

¹ Institute for Molecules and Materials, Radboud University; ² Department of Genetics, Cell Biology and Development, University of Minnesota; ³ J. Craig Venter Institute; ⁴ Brazilian Agriculture Research Corporation; and ⁵ Santa Fe Institute

Abstract

Cell-free expression (CFE) systems are one of the main platforms for building a synthetic cell. A major drawback is the orthogonality of cell-free systems across species.

In this paper, we describe our attempts to optimize a Mycoplasma bacterium-based CFE system. Creating a new CFE protocol has been proven to be surprisingly complex. Unexpected ribonuclease activity was found in both wild-type Mycoplasma capricolum (Mcap) and JCVI-syn3A (Syn3A) lysates. Current databases of Mcap and Syn3A genomes were unable to point out specific surface associated nucleases, therefore specific inhibition or depletion was not possible. This work provides information and possible roadmap for future attempts at engineering mycoplasma based CFE.

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Hana Kiyama¹, Shigeyuki Kakizawa², Makoto Miyata¹

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

Abstract

We have been working on Mycoplasma mobile gliding, Mycoplasma pneumoniae gliding, and Spiroplasma swimming since 1997. In the current project, we are trying to reconstruct these motility systems to clarify their structures, mechanisms and evolutional origins. Reconstruction of Spiroplasma swimming was succeeded in Syn3.0B by expressing seven genes including fibril and mreBs. The detailed analyses are ongoing.

Funding

Supported by Japan Science and Technology Agency (JST), CREST (Grant Number JPMJCR19S5)

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Yu Atsumi, PhD¹, Yo Suzuki, PhD²

¹ SyntheticGestalt, Business Development Department; ² J. Craig Venter Institute

Abstract

SyntheticGestalt has developed an AI Discovery System on JCVI data to develop AI-enhanced metabolic models, running numerous simulations to identify metabolic changes that optimize compound production. Then, using JCVI-syn3.0 as the foundation, Dr. Suzuki and his team generate these conditions by altering media components or engineering genetic changes. The refined model will save valuable time and resources, allowing Dr. Suzuki to quickly identify exactly how to set the environment and the genetic content of the organism to maximize target bioproduction and minimize byproducts. Ultimately, the combined team aims to develop a series of safe, efficient cellular micro-factories for producing a wide variety of compounds, spanning chemicals, cosmetics, foods, fuels, materials and pharmaceuticals, through the metabolic engineering of JCVI-syn3.0.

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Abstract

A recent major breakthrough in the field of synthetic biology was the creation of the artificial synthetic organism JCVI-syn3.A. With only 452 protein-coding genes, it is the organism with the smallest viable genome up-to-date. However, the number of proteins with unknown functions is clear evidence that further studies need to be carried out. With the most recent efforts to shed light into the veil of the unknown, we have put all current data together and organized it in a simple-to-read manner in our latest database - SynWiki (http://synwiki.uni-goettingen.de). SynWiki is based on the relational model and gives access to published information about the genes and proteins of JCVI-syn3.A. To gain a better understanding of the functions of the genes and proteins of the artificial bacteria, we provide interactive maps of protein-protein networks, list of potential protein homologs and offer the possibility to peruse expression data. We want SynWiki to become an important support platform with the necessary tools for the synthetic biology community to further expand the current understanding of this organism.

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Adam Belsom¹, Neil Singh², Jörg Stülke², Juri Rappsilber^{1, 3}

¹ Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany; ² Department of General Microbiology, Institute of Microbiology and Genetics, GZMB, Georg-August-University Göttingen, Göttingen, Germany; ³ Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom

Abstract

We are applying advanced crosslinking mass spectrometry methods to capture protein-protein interactions in JCVI-syn3A.

The approach leverages chemical reactions to translate proximity information in (and between) proteins into covalent bonds, which can be read out by mass spectrometry and database search, in the form of crosslinked peptides.^{1, 2}

We have synthesized and developed a cell-permeable, enrichable crosslinker reagent that provides a greater depth of analysis inside cells, by allowing us to home in on the crosslinked peptides of interest.

So far, we have identified >20,000 unique crosslinks across 383 proteins in JCVI-syn3A. This includes >6,000 unique crosslinks between 178 proteins, building a fascinating picture of the protein-protein interactions in JCVI-syn3A.

References

¹ O'Reilly, F. J.; Rappsilber, J., Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology. Nat Struct Mol Biol 2018, 25 (11), 1000-1008; ² O'Reilly, F. J.; Xue, L.; Graziadei, A.; Sinn, L.; Lenz, S.; Tegunov, D.; Blötz, C.; Singh, N.; Hagen, W. J. H.; Cramer, P.; Stülke, J.; Mahamid, J.; Rappsilber, J., In-cell architecture of an actively transcribing-translating expressome. Science 2020, 369 (6503), 554-557.

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Neil Singh¹, Adam Belsom², Aleksander Chernev³, Henning Urlaub³, Juri Rappsilber², Jörg Stülke¹

¹ General Microbiology, Göttingen University, Germany; ² Institute for Biotechnology, Bioanalytics, TU Berlin, Germany; ³ Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Germany

Abstract

To map the whole cell protein-protein interactome of Syn3A, we developed a novel crosslinking pipeline. Additionally, whole cell UV crosslinking allowed us to identify probable RNA-interacting proteins. 3578 heteromeric protein-protein links were identified, including links within known complexes and a novel complex of three unknown lipoproteins. A further 23 proteins of unknown function containing at least 2 crosslinks were also found. Separately, UV crosslinking enriched 124 proteins as hypothetical RNA binding proteins. Of 9 uncharacterized enriched proteins 6 have RNA localization data, while 11 others are not enriched but an RNA interacting residue site was identified. Contextual information from XL-MS experiments enable characterization of unknown Syn3A proteins. These maps are valuable for elucidation of hitherto undescribed essential functions.

Keywords: XL-MS, Protein-protein interactions, Protein-RNA interactions, uncharacterized proteins

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Isaac Justice, PhD Student (Saenz Group), B-CUBE Center for Molecular Bioengineering, Dresden, Germany

Abstract

An essential feature of cellular life is the presence of cell membranes. These membranes share a number of conserved features including selective permeability to extracellular chemicals, mechanical robustness against environmental conditions, and the maintenance of strictly constrained microenvironments in which membrane proteins can properly function. Additionally, all membranes share the feature of being composed primarily of lipids--a diverse category of macromolecule which share the key feature of being insoluble in water. The self-assembling nature of lipids is likely one reason why they have become the building blocks of cell membranes, but another crucial feature is the remarkable diversity of lipid monomers compared to the other major classes of biological macromolecules (AA = 20, NA = 4). Lipid diversity gives living membranes their ability to maintain their properties in the face of changing environmental conditions by dynamically and responsively remodeling the lipid composition of their membrane. I have developed a lipid diet which limits the remodeling capabilities of M. mycoides and JCVI-Syn3, allowing us to probe the relationship between membrane complexity and the functionality of various essential membrane properties.

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Nataliya Safronova, and James Saenz — Technische Universität Dresden

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Fatema Zahra M. Rashid and Remus T. Dame (rtdame@chem.leidenuniv.nl) — Leiden University, Leiden, The Netherlands

Abstract

The chromosome is the blue-print of life. Transcription of the genes encoded within this macromolecule is not only dependent on the genomic sequence and transcription factors that bind to the genome, it is also influenced by the three-dimensional structure of the macromolecule. Chromosome structure affects the accessibility of the genome, permits the co-regulation of loci that lie in physical proximity, and physically insulates segments of the genome from each other. In bacteria, archaea, and eukaryotes, the features of the three-dimensional structure of the chromosome that allow co-regulation and insulation manifest as loops and as domains — physically insulated sections of the chromosome that exhibit self-interaction. We use Chromosome conformation capture-based techniques to examine the three-dimensional structure of the JCVI-Syn3A chromosome. We study the occurrence of loops and domains in the minimal chromosome, and assess the significance of this organisation and its interplay with gene expression. Preliminary studies show the presence of four loops in the chromosome, but are inconclusive regarding the occurrence of domains.

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Abstract

New measurement methods and tools are required to realize the routine ability to design and build safe, well-characterized, and predictive function into biological systems for a desired purpose. The Cellular Engineering Group at the National Institute of Standards and Technology (NIST) works to provide this foundation of measurement assurance needed for biological control in engineering biology. To this end, we will discuss recent progress in the Group in building living measurement systems, such as biomolecular circuits and cells, engineered to sense and respond in programmed ways. Minimal cells, in particular, offer an intriguing opportunity to advance both measurements and capabilities for engineering biological function.

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Abstract

Minimal cells maintain their life activity only with a minimal amount of genes. Although the whole regulatory machinery is much simpler than natural species, the details of sub-cellular structures and biomolecular distributions are hindered by the small size of the minimal cells. We are investigating the structural details of key proteins inside the JCVI-Syn3A/B cells by using single-molecule localization microscopy. In this presentation, I will share experimental procedures, results, and problems to be addressed to discuss the future direction of this research.

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Abstract

For a bacterial cell to organize and complete cell division, they must recruit and control multiple proteins at the right time and place. A genomically minimal cell offers a rare opportunity to study the essential physiological components to complete this cytokinesis. In this study, we aim to optimize and develop a platform to live cell timelapse imaging to monitor growth in real-time of JCVI-syn 3A. Thus far, we have developed a platform to monitor cell clump growth in real time, with a doubling time of clump area of ~9 hours. Our current efforts focus on developing a 3D method to monitor Syn3A volume through time.

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Clyde A. Hutchison III, Chuck Merryman, Lijie Sun , Kim Wise, Hamilton O. Smith, Yo Suzuki, John I. Glass, and J. Craig Venter (J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla CA, USA); Troy Brier, David Bianchi, Zane Thornburg, and Zaida Luthey-Schulten (Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana IL, USA)

Abstract

In 2016 we reported the design and synthesis of the JCVI-syn3.0 genome. This chemically synthesized 531 Kbp genome contains 438 protein-coding genes and 35 genes for non-coding RNAs. When installed by genome transplantation it produces a cell that grows in rich medium with a doubling time of 2 hours. Annotations of its genes were classified into five ascending levels of confidence: unknown, generic, putative, probable, and equivalog, based mainly upon bioinformatic analysis. Surprisingly, 65 of the genes were of unknown function and 84 genes had only generic functional assignments. Since publication of these results in 2016 a number of groups have reported additional analysis of these 149 genes of unclear function. We review these reports here, along with additional analysis that allows the assignment of putative or probable functions to 4 of the unknowns and 45 of the generics. Among the genes of unknown function, we identified 59 encoding membrane-spanning proteins and 11 predicting lipoprotein motifs for membrane anchorage. JCVI-syn3.0 now has 59 unknown and 41 generic genes that still require precise functional annotation. Plans for further annotation and genome reduction are discussed, along with the implications of our results for a complete understanding of the genetic requirements for life. JCVI-syn3.0, and related near-minimal strains such as JCVI-syn3A, are the simplest and also the most completely characterized genomes that support independently replicating cellular life. As such, they are attractive experimental organisms for defining the basic principles of life.