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Manipulating the 3D organization of the largest synthetic yeast chromosome [RNAseq]

ABSTRACT: Whether synthetic genomescan power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we reportde novosynthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bpSaccharomyces cerevisiaechromosome resulting from extensive genome streamlining and modification. During the construction ofsynIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE239534 | GEO | 2023/08/04

REPOSITORIES: GEO

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Manipulating the 3D organization of the largest synthetic yeast chromosome [WGS]

Project description:Whether synthetic genomescan power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we reportde novosynthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bpSaccharomyces cerevisiaechromosome resulting from extensive genome streamlining and modification. During the construction ofsynIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.

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RNA sequence (RNA-seq) profiling of SynXI strain

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Deep functional analysis of synII, a 770 kb synthetic yeast chromosome

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Deep functional analysis of synII, a 770 kb synthetic yeast chromosome

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MetaboLights MTBLS296 - GNPS Deep functional analysis of synII, a 770 kb synthetic yeast chromosome

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2018-08-07 | MSV000082770 | GNPS

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Project description:In recent years there has been considerable and growing interest in the 3-dimensional organization of genomes. In yeast this has included detailed FISH studies that show a surprising degree of spatial clustering of the tRNA genes, the localization of telomeres, centromere positioning, and global studies of chromosomal interactions.M-BM- In this study we performed an integrated biophysical M-bM-^@M-^S molecular study to produce the first high-resolution 3-dimensional reconstruction of a eukaryotic genome from first principles. Our conformations were produced using a using a molecular dynamics simulator to model the 16 yeast chromosomes as individual polymers. Critically, the compaction, folding and spatial organization of these chromosomes was informed by empirical data determined using proximity-based ligation. Our models allow us to investigate the structural organization and functional spatial clustering of the yeast nucleus at a resolution that has not previously been possible. We clearly show that yeast chromosomes have preferred yet non-exclusive positions. Surprisingly, while there are general trends relating chromosome position along the spindle pole body M-bM-^@M-^S nucleolus axis and chromosome size, some chromosomes have territories that contain two linked domains. Similarly, there is clear evidence for functional clustering of tRNAs and Gal4 protein binding sites. Intriguingly, these functional clusters are dynamic and the identity of the component elements alters in different model solutions. Genome Conformation Capture has been performed on exponentially growing Saccharomyces cerevisiae in media containing Glucose

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Design and experimental evaluation of a minimal, innocuous watermarking strategy to distinguish near-identical DNA and RNA sequences

Project description:The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavours, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The thirteen genes encoding proteins involved in the major pathway for sugar utilization (i.e glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not alter transcript abundance, protein activity and yeast physiology. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.

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