Name: C. difficle p-cresol production
Keywords: Hpdbca decarboxylase

OmicsDI

Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

C. difficle p-cresol production

ABSTRACT: To identify the cellular localisation of the HpdBCA decarboxylase complex, a plasmid based HpdB-SNAP-tag translational fusion was constructed using a C. difficile compatible plasmid (pMTL84151) under control of the hpdBCA promoter. The hpdB coding sequence omitting the stop codon, was fused via a linker to a SNAP-tag (Table 1). Confirmation of HpdB linked to the SNAP-tag was carried out via western blot, and mass spectrometry, in which we identified six peptides, four with 100% identity and two with 99% identity unique to HpdB. Localisation of HpdB was visualised by confocal microscopy in 630Δerm and the p-cresol deficient mutant (hpdC::CT) (carrying the HpdB-SNAP-tag fusion (PhpdB-CDS-SNAP), in the presence and absence of p-HPA) to induce HpdBCA production.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Clostridioides Difficile Atcc 43255

TISSUE(S): Cell Culture

SUBMITTER: Steven Lynham

LAB HEAD: Dr Lisa Dawson

PROVIDER: PXD028948 | Pride | 2021-11-02

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

Project description:We apply a massively parallel reporter assay (MPRA) that relies on mRNA and plasmid tag sequencing (Tag-Seq) to compare the regulatory activities of more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. The resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution and can be used the to train quantitative sequence-activity models (QSAMs). Reporter Tag-Seq from HEK293 cells transfected with each of six MPRA plasmid pools, with and without stimulation (forskolin or Sendai virus). The reporter mRNAs contain unique 10 nucleotide tags that facilitates quantitation of their abundances. The same tags were also sequenced from each ransfected plasmid pool to facilitate normalization to plasmid copy numbers. The reporter constructs were designed according to two different mutagenesis strategies: 'single-hit scanning' and 'multi-hit sampling'. The specific variants are included in the processed data files.

2012-02-26 | E-GEOD-31982 | biostudies-arrayexpress

De novo deposition of the histone H3 variants H3.1 and H3.3 during DNA replication

Project description:We developed a new sequencing assay to track the de novo deposition of the histone H3 variants H3.1 and H3.3 during S phase. We use cells stably expressing H3.1-SNAP or H3.3-SNAP, and synchronize them in G1/S by double-thymidine block. The SNAP-tag enables to discriminate newly synthesized histones from preexisting ones, via a quench-chase-capture strategy. We applied this strategy to isolate new H3.1 and H3.3 after releasing cells into S phase, and probed their distribution by MNase digestion and sequencing. We could thus characterize H3.1 and H3.3 dynamics from early to mid S phase at genome-wide resolution. We further applied our method to investigate the consequences of perturbations upon deletion of the H3.3 chaperone HIRA. We used HIRA knockout and control cells, and compared H3.1 and H3.3 distribution to early replication patterns by EdU labeling and sequencing of nascent DNA.

2022-03-31 | E-MTAB-10619 | biostudies-arrayexpress

BIGenomics HPV zipcode array plasmid hybridizations

Project description:Hybridization of HPV genotype recognizing ligation probes to a zipcode (tag) microarray. The purpose of the experiment is to establish functionality and target specificity of the ligation probes by using a single template at a time with all of the probes. HPV plasmid DNA PCR products are used as templates for ligation probes. Each plasmid contains the full genome of a given HPV type. One type of plasmid is used per experiment to test the signals of all of the probes agains that template. 19 different plasmids are tested this way (i.e. 19 samples in the experiment).

2011-06-01 | E-MEXP-3152 | biostudies-arrayexpress

Novel genes involved in the pathophysiology of gonadotropin-dependent adrenocortical tumorigenesis in mice.

Project description:The inh-alpha/Tag mouse is a murine adrenocortical tumor model that combines gonadotropin-dependent tumorigenesis with SV40Tag expression (which inhibits p53). These mice have been extensively used to study adrenocortical and gonadal tumorigenesis, as they initially present with gonadal tumors. To induce adrenocortical tumorigenesis, the inh-alpha/Tag mice need to be prebubertally gonadectomized. Generally, females develop adrenal tumors faster and larger in size than males, with a very high individual variations of tumor volume and histological appearance. In this study, we used the inh-alpha/Tag mouse model to analyze the molecular basis of the LH-dependent adrenal tumorigenesis. Experimental inh-alpha/Tag and control littermates (C57Bl/6N) mice of both sexes were used as the animal model. inh-alpha/Tag and WT mice were gonadectomized at 21-24 days of age. Mice were sacrificed at the age of 30 weeks (+/-1 week) and adrenals snap-frozen in liquid nitrogen for microarray analysis. Additionally, a group of 27 week-old inh-alpha/Tag mice (+/-1 week) were treated for 3 weeks with GnRH antagonist cetrorelix acetate (#C5249, Sigma-Aldrich) (3mg/kg/48h). Mice were sacrificed at the age of 30 weeks (+/-1 week) and adrenals snap-frozen in liquid nitrogen for microarray analysis.

2017-02-01 | E-MTAB-5310 | biostudies-arrayexpress

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.

2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress

CapStarr-seq: a high-throughput method for quantitative assessment of enhancer activity in mammals

Project description:Here we developed CapStarr-Seq, a novel high-throughput strategy to quantitatively assess enhancer activity in mammals. This approach couples capture of regions of interest to previously developed Starr-seq technique. Extensive assessment of CapStarr-seq demonstrated accurate quantification of enhancer activity. Furthermore, we found that enhancer strength correlates with binding complexity of tissue-specific transcription factors and super-enhancers, while additive enhancer activity isolates key genes involved in cell identity and function. CapStarr-seq analysis in P5424 cell line (2 replicates), 3T3 cell line and in the plasmid library before (Input) and after transfection

2015-04-09 | E-GEOD-60029 | biostudies-arrayexpress

Global protein responses of multi-drug resistant plasmid containing Escherichia coli to beta-lactams and ciprofloxacin.

Project description:Our study aimed to analyse the responses of the clonal ST131 strain, containing the ESBL and multi-drug resistance plasmid under different beta-lactam and ciprofloxacin antibiotic stress in comparison with no antibiotic stresses in order to identify the specific response of a MDR plasmid to the specific antibiotic and the potential interaction between the plasmid and the host bacteria under different stresses. The aim was to understand how the plasmid and bacterial host proteins are influenced by the different antibiotic stresses. By analysing these factors systematically we aimed to identify the pathways and core or antibiotic specific responses of the bacteria. Using proteomics we provide an unbiased protein map of a pathogen with a MDR plasmid under antibiotic stress and compare it with the same bacteria in the absence of the stress.

2022-10-12 | PXD027164 | Pride

Genome-wide localisation map of ScRad9-9Myc overexpressed in pYX142 grown in SC (synthetic complete medium) along with BCS and BPS copper/iron chelators

Project description:This experiment was performed in order to assess the specificity of Rad9 binding to S. cerevisiae genome. In another ChIP-chip experiment in SC BCS BPS growth conditions we have found Rad9 present to a significant number of genomic loci and wanted to examine how specific this localisation is. For that we inserted Rad9 gene in the high copy plasmid pYX142 and transformed it in BJ5457 rad9delta yeast cells. Cells were grown under the same conditions and ChIP-chip was performed. By this experiment we aimed to examine quantitavely and qualitatively the Rad9-targets when Rad9 is overexpressed and to compare the findings with previous experiments of ours.

2014-11-01 | E-MTAB-2200 | biostudies-arrayexpress

Expression data from in vitro decidualized human uterine fibroblast cells (HuF cells) with or without NOTCH1 silencing

Project description:Decidualization is a critical process for embryo implatation during which uterine stromal fibroblasts are transformed into large, epithelioid-like decidual cell. NOTCH1 is recepotor of Notch signaling that plays important roles for cell-cell communication, which involves gene regulatory mechanisms that control multiple cellular differentiation processes during embryonic and adult life. Here we silenced NOTCH1 by shRNA in HuF cells and studied how it affects decidualization HuF cells were cultured, transfected with NOTCH1 shRNA via a lentivirus system, following by a in vitro induced decidualization by EPC cocktail (36 nM estradiol-17Î² and 1 Î¼M medroxyprogesterone acetate, and 0.1 mM dbcAMP) for 6 days. RNAs were extracted then hybridized on Affymetrix microarrays. There are four experimental conditions: 1) transfected with empty plasmid; 2) transfected with hNOTCH1 lentiviral-shRNA plasmid; 3) transfected with empty plasmid and treated with EPC cocktail; and 4) hNOTCH1 lentiviral-shRNA plasmid with subsequent treatment of EPC cocktail. HuF cells from two different patients were used in each group (HuF86P4 & HuF88P4).

2014-09-30 | E-GEOD-61827 | biostudies-arrayexpress

Coordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention

Project description:The N371 site on PKCe was mutated to TAG in an expression plasmid. This was co-transfected in a 1:1 ratio with a plasmid that encodes both an aaRS optimised to recognise AbK, and the tRNAPyl which recognises the UAG codon.

2021-11-03 | PXD025959 | Pride

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