ChIP-sequencing of 3xFLAG-AntA from Streptomyces albus S4
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 3000
ORGANISM(S): Streptomyces albus
SUBMITTER: Ryan Seipke
PROVIDER: E-MTAB-7700 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:To evaluate the DNA binding of FscRI in Streptomyces albus S4 in vivo ChIP-Seq experiments were carried out on dusing anti-FLAG antibodies against 3xFLAG-FscRI protein using two biological replicates. The wild type strain was used as a control in experiments using the anti-FLAG antibodies as well as a DNA only control.
2016-11-14 | E-MTAB-5122 | biostudies-arrayexpress
Project description:To determine the influence of BldM and WhiI on the binding of each other to their respective binding sites in the genome of Streptomyces venezuelae, ChIP-Seq experiments were carried out using polyclonal antibodies against the wild type proteins as well as anti-FLAG antibodies against FLAG-tagged proteins. Null mutants of Streptomyces venezuelae lacking WhiI or BldM were used as controls. The wild tpe strain was used as control in experiments using the anti-FLAG antibodies.
2015-05-19 | E-MTAB-2698 | biostudies-arrayexpress
Project description:Understanding the liver stage invasion and development of Plasmodium falciparum parasites is difficult due to limitations in current methodologies, namely, a hepatocyte cell line that supports the invasion of sporozoites. In this work we seek to develop and characterize a subclone of the HC-04 cell line that supports higher invasion rates than the parental line. We make use of both RNA-seq and LC-MS/MS proteomics to characterize cells grown in two different mediums. Finally, we identify and validate a surface protein, glypican-3, which is deferentially expressed between parental and subclone cell lines, when compared to HepG2 cells, and demonstrate that antibodies against glypican-3 decrease parasite invasion.
2019-03-02 | E-MTAB-6919 | biostudies-arrayexpress
Project description:The whiH gene is required for the differentiation of aerial hyphae into spores in Streptomyces species. It is a predicted member of the GntR family of transcription factors and has been shown to bind specifically to a sequence in its own promoter. This ChIP-Seq experiment was carried out to determine all the binding sites whiH binds to in the genome of Streptomyces venezuelae. A whiH deletion strain was made and a FLAG tagged whiH protein was expressed in it from a genome-integrated plasmid. Then anti-FLAG antibodies were used for chromatin immunoprecipitation followed by high throughput sequencing. The wild type Streptomyces venezuelae strain (ATCC 10712) was used as a negative control. For both the FLAG-WhiH strain and the WT strain, non-immunoprecipitated (total) DNA was also sequenced to arrive at a background enrichment which could be subtracted from the enrichment in the immunoprecipated sample.
2018-12-31 | E-MTAB-6702 | biostudies-arrayexpress
Project description:Biosynthesis is an environmentally friendly and renewable way to synthesize a broad range of natural and some new-to-nature products; however, it lacks many of the reactions and flexibility of synthetic chemistry, an example being carbene mediated reactions. While it was recently shown that these reactions can be imported into the cell, carbene donors and unnatural cofactors needed to be added exogenously to the cells, precluding cost-effective scale-up of the reaction. Here we identified a biosynthetic gene cluster that when expressed in Streptomyces albus will produce the diazo compound azaserine, which we also demonstrated can serve as a carbene donor across a carbon-carbon double bond in another intracellularly produced molecule, styrene, thereby producing a cyclopropanated product. The reaction was catalyzed with P450 mutants harboring a native cofactor with excellent diastereoselectivity and moderate yield. This work establishes a platform for introducing carbene mediated reactions into microbes for bio-manufacture and contributes to sustainable green chemistry.
2023-05-03 | PXD037509 | Pride
Project description:The purpose of this experiment was to show that the iron-sulphur cluster ([4Fe-4S]) of whiB is essential for the DNA binding by WhiB and WhiA. The \\"serine version of whiB\\" mentioned below is a whiB in which the 4 cysteine residues which coordinate the [4Fe-4S] cluster were mutated to serines. ChIP-Seq using anti-FLAG anitbodies was performed in the following: 1. A whiB deletion strain expressing the FLAG-tagged serine version of WhiB (WhiBFLAG-Ser). 2. A whiB and whiA deletion strain expressing the FLAG-tagged serine version of WhiB (delAB_WhiBFLAG-Ser). 3. A whiB and whiA deletion strain expressing the serine version of WhiB and FLAG-tagged whiA (delAB_WhiAFLAG_WhiBcomp-Ser).
2021-08-16 | E-MTAB-10818 | biostudies-arrayexpress
Project description:To elucidate the effect of the rational ribosomal engineering on the changes in the expression of the endogenious biosynthetic gene clusters the transcriptome analysis was performed. The Streptomyces albus strains carrying mutations in rpsL gene (encode for ribosomal protein S12) and the deletion of the rsmG gene (16S rRNA methyltransferase G), as well as their combination were used for the experiment. The list of the strains with mutations is next: S. albus K88E, S. albus GI92, S. albus K88E-GI92, S. albus P91S, S. albus K88E-P91S, S. albus del rsmG, S. albus K88E-GI92 del rsmG, S. albus K88E-P91S del rsmG. Abovementioned strains along with S. albus native strain were grown in NL-19 production medium. Samples were harvested by centrifugation after 48 and 72 hours of cultivation. For total RNA isolation, S. albus cells were grown in SG medium (for ara expression) or NL19 medium (for indigenous BGC expression). Then, 2 ml of 2-day and 3-day cultures was spun down for 20 s at 14,000 × rpm, and the pellets were immediately frozen in liquid nitrogen and stored at ?80 °C. Total RNA extraction was performed using an RNeasy Kit (Qiagen, Hilden, Germany) as previously described (Huser et al. 2003). An RNase-Free DNase set (Qiagen) was used two times for on-column DNA digestion, and an additional DNase treatment was then performed with a DNase I kit (Roche Diagnostics, Mannheim, Germany) to ensure that all DNA was completely removed. To check the RNA samples for DNA contamination, PCR was performed using oligonucleotides designed to create two different products approximately 150 bp and 500 bp in size. Initially, RNA quality was checked with Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A Ribo-Zero rRNA Removal Kit for bacteria was obtained from Illumina (San Diego, CA, USA) and used to remove ribosomal RNA molecules from isolated total RNA. rRNA removal was checked using an Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries (Koepff et al. 2017). The resulting cDNAs were pair-end sequenced on an Illumina HiSeq 1500 system (San Diego, CA, USA) using a 70 bp read length. Short read alignments and differential gene expression were illustrated using ReadXplorer 2.2.0 (Hilker et al. 2014) and DEseq (Anders and Huber 2010), respectively.
2019-05-01 | E-MTAB-7715 | biostudies-arrayexpress
Project description:ChIP-seq was performed to map transcription factor (TF) binding in monocytes during in vitro differentiation. To map binding sites for TF lacking suitable antibodies we also performed mRNA transfections using synthetic mRNAs translating into 3xFLAG TF versions that were ChIPed using Anti-Flag antibody.
2021-02-03 | E-MTAB-9927 | biostudies-arrayexpress
Project description:WhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacterium Streptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA in Streptomyces development and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus, Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed that whiA is required for the initiation of sporulation septation and chromosome segregation in S. venezuelae, and several genes encoding key proteins of the Streptomyces cell division machinery, such as ftsZ, ftsW, and ftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor (WhiG) and the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, and filP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.
2016-05-20 | E-MTAB-4673 | biostudies-arrayexpress
Project description:We performed CUT&RUN assay with control Ig , anti-Flag and anti-ETO antibodies in Flag-tag AML1-ETO Kasumi-1 cells to determine binding occupancy of AML1-ETO following miR-130a KD. Kasumi-1 cells were transduced in 3 independent replicates for control and miR-130a KD samples with lentiviruses containing GFP reporter gene. GFP+ cells were sorted 3 days post-transduction and the cells were used in the CUT&RUN assay. Three antibodies were used for each biological sample.
| EGAS00001005867 | EGA