Biology subjects

Glass, J. I.

Publications and source records attributed to Glass, J. I..

13 recordsLinked to original sources

bioRxiv2026

Full-length transcriptomics and proteomics reveal how genome minimization reshapes gene expression in synthetic bacteria

JCVI-syn1.0 (Syn1.0) and JCVI-syn3A (Syn3A), genetically synthetic and genome-reduced versions of the naturally occurring bacterium Mycoplasma mycoides, are landmark platforms for defining the gene set required for life, yet how their genomes are expressed at the RNA level remains uncharacterized. We combined full-length PacBio and native long-read RNA sequencing with short-read quantification and complementary proteomics to map transcription, RNA processing, and protein abundance in both cells. In Syn1.0, full-length sequencing resolved 459 operons encompassing 911 genes, revealing pervasive RNA processing with a strong 3' bias. Analysis of the division and cell-wall cluster showed how transcriptional context explained the restoration of genes required for normal cell division in Syn3A. Most antisense and intergenic transcription in Syn1.0 reflected low-level transcriptional noise arising from inherited mis-annotation, read-through, and synthetic sequences. Much of that transcription was lost in Syn3A after genome minimization. Reducing the genome unexpectedly altered the expression of several retained genes by deleting promoters, most prominently reducing expression of the nucleoid protein HupA and central-carbon enzymes. Meanwhile, one third of the coding mRNA pool was allocated to a single 21-gene ribosomal-protein operon, while several other ribosomal proteins had reduced transcript abundance, which possibly led to imbalanced ribosome assembly. Expression of RNA polymerase and central-carbon metabolism declined at both mRNA and protein levels whereas RNase Y degradosome abundance increased. These shifts in the synthetic evolution of Syn3A suggest plausible mechanisms for its reduced chromosome contacts and slower growth. Together, these results show that genome minimization alters not only gene content but also the transcriptional context and resource allocation of retained genes. The analysis and visualization that are shared via Jupyter Notebook provide the RNA-level foundation for whole-cell modeling of the minimal cell.

synthetic biology↗

bioRxiv2026

Selection-free whole genome transplantation revives dead microbes

We present a living, synthetic bacterial cell made by transplanting a complete genome into a dead cell. After killing Mycoplasma capricolum cells by chemically crosslinking their genome with Mitomycin C (MMC), we installed synthetic Mycoplasma mycoides genomes into the resulting dead cells using Whole Genome Transplantation (WGT)1,2. During WGT, a synthetic donor genome is placed into a recipient cell, thereby reprogramming that cell to adopt a new genetic identity3. WGT has only been demonstrated using species within one phylogenetic clade of Mollicutes bacteria4. A major barrier to expanding WGT to diverse bacterial species has been the inability to inactivate the recipient genome, leading to false positive transplants due to homologous recombination of antibiotic resistance markers from the donor genome into the recipient cell genome. Here, we address this key limitation by removing reliance on an antibiotic resistance marker to select for transplants; recipient cells are dead unless revived by the installation of a new genome. Our work demonstrates a general approach to fully inactivate the recipient cell genome, reports the first living synthetic bacterial cell constructed from non-living parts, and advances WGT for building engineered or synthetic cells for diverse applications.

synthetic biology↗

bioRxiv2025

Unraveling the Transcriptional Landscape within a Minimized Bacterium via Comparative Analysis

Stochastic nature of gene expression leads to the complex formation of the bacterial transcriptome and proteome. In contrast to typical transcriptome studies, we employ a near wild-type, Syn1.0, of the naturally genome-reduced Mycoplasmas, and the dramatically further genome-reduced JCVI-syn3A thus avoiding additional contributions from many non-essential cellular functions. To aid in profiling the transcriptional landscape within these bacteria, we present a bioinformatic analysis of the genetic sequence motifs implicated in modulating the stochastic gene expression events, coupled with genome-wide short-read (Illumina) and long-read (Oxford Nanopore Technologies and Pacfic Biosciences) RNA sequencing. The bioinformatic analysis coupled with information from structural studies assigns strengths of the Shine-Dalgarno signatures and identifies both transcription initiation and termination sites, leading to predictions of RNA isoforms in Syn1.0 (and related organisms). The long-read and short-read RNA sequencing characterized the predicted transcriptional activity, and the long-read methods provide direct insight into the RNA isoform complexity within Syn1.0. Comparison of the RNA sequencing results with that of the bioinformatic analysis highlights the inability of bioinformatics alone to capture the results of bacterial transcription without including effects of RNA degradation. This study emphasizes the need for comparative analysis and potential dangers of genome reduction, exemplified through the discovery of altered gene expression patterns of JCVI-syn1.0 and JCVI-syn3A, achieved via the union of our transcriptome study with their proteomics data. Analysis of the transcriptomics data sets through a Jupyter notebook allows any genomic region to be easily examined. Table of Content Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/681674v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@78a9e0org.highwire.dtl.DTLVardef@1d8c9fcorg.highwire.dtl.DTLVardef@1b4e129org.highwire.dtl.DTLVardef@2a8c3f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Rapid assembly of functional modules for generating human artificial chromosome constructs compatible with epigenetic centromere seeding

The ongoing development of human artificial chromosomes (HACs) will permit investigation into essential centromere processes and the means to deliver large genetic cargoes to target cells. Starting with large ([~]750 kb) yeast artificial chromosome (YAC)-based constructs limits the rampant multimerization that has complicated many prior types of HACs. Large YAC construction is accomplished using transformation-associated recombination (TAR) strategies that can become unwieldly when several functional modules are to be incorporated and tested. To address this issue, we developed an approach where modules are built using high-fidelity in vitro assembly strategies in a bacterial artificial chromosome (BAC) format. Then, the assembled modules are transferred in a simplified TAR step into a recipient YAC harboring the prokaryotic "stuffer" DNA that comprises a large portion of the final HAC construct. This approach is highly efficient with two-thirds of all screened yeast clones harboring the correct TAR product. Further, whole-genome Oxford Nanopore Technologies (ONT) sequencing/alignments, de novo assembly of the final YAC using a single ONT sequencing run, and close inspection of highly repetitive regions are all streamlined to rapidly validate clones that match the design. The fully sequenced, verified strain harboring a multi-module construct was then fused to human cells, where it efficiently formed functional HACs upon initial seeding with CENP-A-containing nucleosomes. We envision that the rapid assembly steps will be useful to quickly incorporate different functional modules, including diverse genetic cargoes, to engineer HACs with specific design features.

synthetic biology↗

bioRxiv2024

PEG Adjustment Enables Genome Transplantation Using Mycoplasma Mycoides Recipient

Pioneering advances in synthetic biology, initiated at the J. Craig Venter Institute, have enabled the creation of the first cell driven by synthetic genome and opened a new era of creating designer microbes. This technology offers far greater potential for genetic modification than traditional genome editing techniques and holds promise for the routine creation of synthetic cells as DNA synthesis costs decrease and genetic knowledge expands. Two essential technologies underpin this achievement: the ability to clone entire genomes in host organisms, such as Saccharomyces cerevisiae, and the successful transplant of these genomes into recipient cells to generate living organisms. In all previous work the recipient cell in genome transplantation experiments has been Mycoplasma capricolum. In this study, we explored the potential of using Mycoplasma mycoides strains as recipient cells for genome transplantation. By increasing polyethylene glycol (PEG) concentration from 5% to 10%, we successfully transplanted various M. mycoides strains using several M. mycoides recipient cells. Additionally, we demonstrated the ability to transplant M. capricolum genomes into M. mycoides recipient cells; although with lower efficiency compared to M. mycoides strains. These findings provide a modified transplantation protocol and likely get us closer to expanding genome transplantation to other bacterial species.

synthetic biology↗

bioRxiv2024

Robust and highly efficient transformation method for a minimal Mycoplasma cell

Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining of the genome of Mycoplasma mycoides. When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, CFU, and transformation efficiency, we found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early-exponential growth phase. Notably, we attained the transformation efficiency up to 4.4 x 10-2 transformants per cell per g plasmid DNA. We developed a method to obtain several hundred to several thousand transformants with less than 0.2 mL of cultured cells and 10 ng of plasmid DNA. Moreover, we established a transformation method using frozen stock of transformation-ready cells. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCEMycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand cellular process of life and for bioengineering because of their simple metabolism, small genomes and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtainable only 10 nanograms of plasmid DNA, which is around one-thousandth amount required for traditional mycoplasma transformations. Moreover, we established a convenient method using frozen stocks of transformation-ready cells. These improved methods play a crucial role in further studies using minimal cells.

microbiology↗

bioRxiv2023

Efficient Formation of Single-copy Human Artificial Chromosomes

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through [~]125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. Here, we describe an approach that efficiently forms single-copy HACs. It employs a [~]750 kb construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells. One-Sentence SummaryA quarter century after the first human artificial chromosomes, a solution to their uncontrolled multimerization is achieved.

molecular biology↗

bioRxiv2022

Minimal cell JCVI-syn3B as a chassis to reveal the mechanisms behind Mycoplasma host-pathogen interactions

Mycoplasmas are atypical bacteria that are obligate parasites of animals and plants. They are known for their capacity to contaminate and parasitize mammalian cell cultures, thereby offering fortuitous models to examine the host-microbe interaction. We used a near wildtype strain of the goat pathogen Mycoplasma mycoides (Mmc) and several minimized Mmc strains lacking genes not essential for growth in laboratory media to investigate host-mycoplasma interactions. Stains with near minimal genomes were incapable of surviving in co-culture with mammalian HEK-293T and HeLa cells. We identified a cluster of eight non-essential genes that when added back to the minimized strains enabled mycoplasma attachment to mammalian cells. Those genes did not restore the capacity of the minimal cell to grow when co-cultured with mammalian cells indicating processes of attaching to mammalian cells and parasitism involve different genes. Furthermore, minimized Mmc strains were not phagocytized by human myeloid cells unlike the near wildtype Mmc.

microbiology↗

bioRxiv2021

High content screening and computational prediction reveal viral genes that suppress innate immune response

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-{kappa}B and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCEInfectious diseases caused by viral pathogens exacerbate healthcare and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study we assembled a large viral gene library from diverse viral families and developed a high content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step towards early-stage evaluation of emerging and understudied viruses.

systems biology↗

bioRxiv2021

Metabolite Damage and Damage-Control in a Minimal Genome

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 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.

microbiology↗

bioRxiv2021

Evolution of a minimal cell

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.

evolutionary biology↗

bioRxiv2021

High-content screening of coronavirus genes for innate immune suppression revealsenhanced potency of SARS-CoV-2 proteins

Suppression of the host intracellular innate immune system is an essential aspect of viral replication. Here, we developed a suite of medium-throughput high-content cell-based assays to reveal the effect of individual coronavirus proteins on antiviral innate immune pathways. Using these assays, we screened the 196 protein products of seven coronaviruses (SARS-CoV-2, SARS-CoV-1, 229E, NL63, OC43, HKU1 and MERS). This includes a previously unidentified gene in SARS-CoV-2 encoded within the Spike gene. We observe immune-suppressing activity in both known host-suppressing genes (e.g., NSP1, Orf6, NSP3, and NSP5) as well as other coronavirus genes, including the newly identified SARS-CoV-2 protein. Moreover, the genes encoded by SARS-CoV-2 are generally more potent immune suppressors than their homologues from the other coronaviruses. This suite of pathway-based and mechanism-agnostic assays could serve as the basis for rapid in vitro prediction of the pathogenicity of novel viruses based on provision of sequence information alone.

systems biology↗

bioRxiv2020

Genetic requirements for cell division in a genomically minimal cell

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. While 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 resulting in irregular morphologies. The addition of 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents significantly less morphological variation than JCVI-syn3.0. We further identified seven of these 19 genes, including two known cell division genes ftsZ and sepF and five genes of unknown function, required together to restore cell morphology and division similar to JCVI-syn1.0. This surprising result emphasizes the polygenic nature of cell morphology, as well as the importance of a Z-ring and membrane properties in the physiology of genomically minimal cells.

synthetic biology↗