Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Relative expression profiling of ycdC knockout strain secreting HlyA protein

ABSTRACT: The most important approach to the development of platform organisms for recombinant protein production relies on random mutagenesis and phenotypic selection. Complex phenotypes, including those associated with significant elevated expression and secretion of heterologous proteins, are the result of multiple genomic mutations. Using next generation sequencing, a parent and derivative hypersecreter strain (B41) of Escherichia coli were sequenced with an average coverage of 52.8X and 55X, respectively. A new base-pair calling program, revealed a single nucleotide polymorphism in the B41 genome at position 1,074,787, resulting in translation termination near the N-terminus of a transcriptional regulator protein, RutR, coded by the ycdC gene. We verified the hypersecretion phenotype in a ycdC::Tn5 mutant and observed a 3.4-fold increase in active hemolysin secretion, consistent with the increase observed in B41. mRNA expression profiling showed decreased expression of tRNA-synthetases and some amino acid transporters in the ycdC::Tn5 mutant. This study demonstrates that power of next generation sequencing to characterize mutants leading to successful metabolic engineering strategies for strain improvement.

ORGANISM(S): Escherichia coli K-12 Escherichia coli

PROVIDER: GSE29266 | GEO | 2011/05/25

SECONDARY ACCESSION(S): PRJNA140275

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Relative expression profiling of ycdC knockout strain secreting HlyA protein

Project description:The most important approach to the development of platform organisms for recombinant protein production relies on random mutagenesis and phenotypic selection. Complex phenotypes, including those associated with significant elevated expression and secretion of heterologous proteins, are the result of multiple genomic mutations. Using next generation sequencing, a parent and derivative hypersecreter strain (B41) of Escherichia coli were sequenced with an average coverage of 52.8X and 55X, respectively. A new base-pair calling program, revealed a single nucleotide polymorphism in the B41 genome at position 1,074,787, resulting in translation termination near the N-terminus of a transcriptional regulator protein, RutR, coded by the ycdC gene. We verified the hypersecretion phenotype in a ycdC::Tn5 mutant and observed a 3.4-fold increase in active hemolysin secretion, consistent with the increase observed in B41. mRNA expression profiling showed decreased expression of tRNA-synthetases and some amino acid transporters in the ycdC::Tn5 mutant. This study demonstrates that power of next generation sequencing to characterize mutants leading to successful metabolic engineering strategies for strain improvement. 6 samples were analyzed with three biological replicates for each strain. Hly-ycdC+ is the control strain.

2011-05-25 | E-GEOD-29266 | biostudies-arrayexpress

Proteomic study in sucrose secreting mutant of cyanobacterium Synechococcus elongatus PCC 7942.

Project description:We conducted proteomic analysis of a strain engineered for inducible secretion of sucrose.

2022-03-21 | PXD027430 | Pride

Human peripheral IL-10-secreting vs non- IL-10-secreting B cells

Project description:Transcriptional profiling of human peripheral B cell subsets sorted by flow cytometry based on the secretion of IL-10 in response to CpG2006 stimulation. Goal was to identify molecular markers to distinguish IL-10-producing B cells form non-IL-10-producing B cells. Two-condition experiment, non-IL-10-secreting B cells vs. IL-10-secreting B cells. Healthy donors. Biological replicates: 6

2013-03-04 | E-GEOD-35002 | biostudies-arrayexpress

Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol [polyA]

Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries form little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.

2017-11-13 | GSE101519 | GEO

Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol [i5i7]

Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries from little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.

2017-11-13 | GSE101518 | GEO

Tn5 transposase-mediated library preparation of single-cell cDNAs for RNA-seq

Project description:Deep sequencing of single cell-derived genomic DNA and/or cDNAs brings novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-Seq requires multiple steps, including shearing the target DNA/RNA and following sequential enzymatic reactions, which result in consequent sample loss and stochastic variation at each step. Such variation may significantly affect the output from sequencing. We have found that a new technique of library preparation using hyperactive Tn5 transposase for the next-generation sequencer of Illumina's platform provided high-quality libraries from 100ng of short-length (average 700~800 bp) single-cell level cDNA. This new method reduced the number of steps in the protocol, which resulted in improved reproducibility and reduced variation among the specimens. Two methods of library preparation (sonication, tagmentation with hyperactive Tn5 transposase) were compared in the case of RNA-Seq for single-cell level cDNA. Technical triplicates were used.

2012-05-25 | E-GEOD-38198 | biostudies-arrayexpress

Next Generation Sequencing to analyse regulators of constitutive VWF secretion

Project description:Next Generation Sequencing to analyse regulators of constitutive VWF secretion

| PRJNA637494 | ENA

ChIPmentation: fast, cheap, low-input ChIP-seq for histones and transcription factors

Project description:Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is widely used to map histone marks and transcription factor binding throughout the genome. Here we present ChIPmentation, a method that combines ChIP with sequencing library preparation by Tn5 transposase (“tagmentation”). ChIPmentation introduces sequencing-compatible adapters in a single-step reaction directly on bead-bound chromatin, which reduces time, cost, and input requirements, making ChIPmentation a convenient and high-throughput alternative to existing ChIP-seq protocols.

2015-08-17 | GSE70482 | GEO

Human peripheral IL-10-secreting vs non- IL-10-secreting B cells

2013-03-04 | GSE35002 | GEO

N4- cytosine DNA methylation epigenetically regulates gene expression and pathogenesis in Helicobacter pylori

Project description:Next-generation sequencing (NGS) can reveal putative target which can be regulated by m4C modification. Comparison of wild-type strain with the strain harboring deleted M2.hpyAII mtase gene was done to identify the differentially expressed gene.

2017-02-01 | GSE94268 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data