Ontology highlight
ABSTRACT:
PROVIDER: PRJNA923142 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23059932_1.fastq.gz | Fastqsanger.gz | |||
| SRR23059932_2.fastq.gz | Fastqsanger.gz | |||
| SRR23059933_1.fastq.gz | Fastqsanger.gz | |||
| SRR23059933_2.fastq.gz | Fastqsanger.gz | |||
| SRR23059934_1.fastq.gz | Fastqsanger.gz |
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Project description:Synthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of 7.0x10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region of SPC110 as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.9x10-9. Combining SPC110-ERdd with a second ERdd tag on either DIS3 or RRP46 resulted in escape frequencies below the detection limit of the used assay (<2x10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms.
2024-02-05 | PXD042326 | Pride
Project description:A robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid
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Project description:Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here we show that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3) /MLL4 complex display impaired histone methylation (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus leading to defective immunoglobulin class-switching. We also show that PTIP accumulation at DSBs contributes to class-switch recombination (CSR) and genome stability independently from Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR, and suggest a non-redundant role for the MLL3/MLL4 complex in altering antibody effector function. Genome-wide analysis of histone modifications, PTIP, and Pol II in PTIP-WT and PTIP-KO mouse activated B cells.
2010-08-25 | E-GEOD-20852 | biostudies-arrayexpress
Project description:Robust yeast biocontainment system dependent on unnatural amino acid
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Project description:Class-switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B cell genome. Transcription at the immunoglobulin heavy-chain (Igh) locus targets CSRassociated DNA damage and is promoted by the BRCT domain-containing PTIP protein. Although PTIP is a unique component of the MLL3/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissect the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We find that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identify a tandem BRCT domain of PTIP that is sufficient for CSR and identify PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multi-functional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities. Genome-wide analysis of histone modifications in PA1-WT and -KO mouse activated B cells
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Project description:project abstract : H3K4 methylation is a well-conserved histone modification from yeast to human. Because H3K4 methylase Set1 and its complex, COMPASS (Complex of proteins associated with Set1), are conserved from yeast to humans, budding yeast has been studied as an acceptable model organism. Since COMPASS components affect Set1 protein stability and H3K4 methylation activity variously, it is important to study how Set1 is regulated by complex components. However, deletion mutant of Swd2 component of COMPASS is not viable, although overexpression of Sen1 fragment enables the construction of Swd2 deletion mutant. This study found that positioning epitope tag to the N-terminal of Swd2 did not decrease interaction between Swd2 and Set1, but reduced the stability of both proteins, Swd2 and Set1, and global H3K4 methylation. Also, we observed that overexpression of N-terminal tagged Swd2 caused increased Set1 protein level and bulk H3K4 methylation. Therefore, Set1 protein can maintain its protein level only when enough Swd2 exist to cover the protein amount of Set1. Also, by comparing RNA sequencing analysis of N-terminal tagged Swd2 and Swd2 deletion mutant with Sen1 fragment overexpression, we isolated genes regulated by Swd2. In conclusion, we suggest that the abundance of Swd2 is important to regulate the protein stability of Set1 and the regulation of gene expression.
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Project description:The yeast Saccharomyces cerevisiae is a highly powerful model for systems genetics. While the advent of ordered deletion libraries has considerably facilitated yeast screening, it has also narrowed the diversity of screenable variants and precluded the exploration of the non-coding genome. Here, we present a versatile, time- and work-efficient method to functionally explore the yeast genome at an unprecedented throughput and resolution, using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows the one-step mapping of all genetic loci in which a transposon can be inserted without disrupting a function necessary for cell growth. SATAY is especially suited to discover loci important for growth in various conditions. Here, we demonstrate that SATAY can be used to (2) reveal genetic interactions in single and multiple mutant strains, (1) reveal drug-resistant and -sensitive mutants, (3) detect not only essential genes, but also essential functional protein domains, and (4) generate not only null alleles, but also other informative genetic variants. Thus SATAY allows to easily explore the yeast genome at an unprecedented resolution and throughput.
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