Project description:We generated a collection of 13 plasmids, with each plasmid containing a variant of a CRISPR protospacer targeted by spacer 8 of the E. coli CRISPR-I array. We transformed the plasmids as a pool into delta cas3 E. coli cells expressing all other cas genes constitutively, with FLAG-tagged casA. We then used ChIP to enrich for CasA-bound protospacers. DNA surrounding the protospacers was PCR-amplified from input (pre-immunocrecipitation) and ChIP (post-immunoprecipitation) samples and sequenced.
Project description:The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome-wide screens in Escherichia coli and Salmonella. Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial âcoreâ small RNA of unknown function. Here, we report that ArcZ acts as a post-transcriptional regulator in Salmonella, repressing the mRNAs of the widely distributed sdaCB (serine uptake) and tpx (oxidative stress) genes, and of STM3216, a horizontally acquired methyl-accepting chemotaxis protein (MCP). Both sdaCB and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the tpx mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an arcZ deletion strain further argued for the existence of a distinct set of Salmonella loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady-state levels of >16% (â¥750) of all Salmonella mRNAs, and rendered the bacteria non-motile. Deep sequencing detected a dramatically changed profile of Hfq-bound sRNAs and mRNAs suggesting that the unprecedented degree of pleiotropic regulation might in part be caused by titration of Hfq binding by ArcZ. This study used three different approaches to identify target genes and biological role of the small RNA ArcZ in Salmonella Typhimurium. Transcriptomic analysis of ArcZ overexpression: Strain JVS-0082 (ÎarcZ) was transformed with plasmids pJV300 (control) and pKP48-1 (parcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptomic analysis of arcZ mutant strain: Strains JVS-007 (WT) and JVS-0082 (ÎarcZ) were transformed with plasmid pJV300 (control) and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptional effects of ArcZ pulse expression: Strain SL1344 was transformed with plasmids pKP8-35 (pBAD-control) and pKP4-13 (pBAD-ArcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture to an OD600 of 1.5. Expression of the insert was induced with L-arabinose (0.2% final concentrations) for 10 min. Three biological replicates were performed for each strain/condition.
Project description:To identify YehT-regulated genes, the transcriptome profiles of E. coli cells overproducing either the response regulator (RR) YehT or the RR KdpE (control) were comparatively analyzed. The expression level of 32 genes varied more than 8-fold.
Project description:To identify YpdB-regulated genes, the transcriptome profiles of E. coli cells overproducing either the response regulator (RR) YpdB or the RR YehS (control) were comparatively analyzed. The expression level of 15 genes varied more than 1.9-fold.
Project description:We created a mutator protein, Evolugene with very fast in vivo mutation rate and gene specificity in E. coli. To comprehensively analyze the specificity and mutagenicity, we sequenced ~3.3 kb DNA around the target gene from cells taken at cycle 27 by Illumina sequencing.
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. Rbf1 encodes a zinc-finger transcription factor that is expressed upon formation of an active bE/bW heterodimer. To analyse the dependency of b and rbf1, changes in gene expression were monitored in strain AB31 (Brachmann et al., 2001, Mol Microbiol 42, 1047-1063) and in the derivative AB31Δrbf1 in which rbf1 was deleted. AB31 harbours the active combination bE1 and bW2 under the control of the arabinose-inducible crg1 promoter. Samples were taken 3h, 5h and 12h after induction of bE/bW gene expression. Strain AB32, which harbors the incompatible bE2 and bW2 combination, was used as control. Strains were grown to an OD600 of 0.4-0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter-sterilized). For induction of the bE and bW genes, cells were inoculated in liquid array medium containing 1% (w/v) arabinose instead of glucose as a carbon source. For induction, cells were washed once with inducing medium; time point 0 h of the time course experiments corresponds to the first contact with inducing medium. Experiments were performed in two biological replicates for each time point.
Project description:We created a mutator protein, eMutaT7[transition] with targeted in vivo hypermutation in E. coli. To investigate the gene-specific mutagenesis, we sequenced ~3.3 kb DNA around the target gene from cells taken at cycle 0 (n = 1) and cycle 20 (n = 3) by Illumina sequencing.
Project description:Adr1 of Ustilago maydis is a protein kinase that is activated after separation from the regulatory subunit mediated by high cAMP levels. A copy of the gene under the control of the arabinose-inducible crg1 promotor was introduced into the Cbx locus of the wild type strain FB1 creating strain HE140. To monitor the transcriptional changes upon adr1 induction by arabinose, we conducted a time course experiment comparing the wild type transcriptional response before and 75 or 180 min after medium shift to complete medium containing arabinose, as well as the response of the mutant HE140 before and 75 or 180 min after induction of the additional copy of adr1 by arabinose. Keywords: gene induction Strains FB1 (control) and HE140 (a1b1 ipr[pcrg1:adr1]ips) were grown in CM medium containing glucose and were shifted to CM medium containing arabinose and grown for 75 or 180 min. Samples were taken before and after arabinose induction from two (glucose) or three (arabinose) biological replicates each.
Project description:AraC is an Escherichia coli transcription factor that regulates genes in response to the presence/absence of arabinose. We used transcription profiling to determine RNA levels in Escherichia coli K-12 strain MG1655 and MG1655 delta araC grown either in the absence or the presence of arabinose. Thus, we identified known and novel AraC-regulated genes. Data presented here are raw .CEL files as well as fully analysed data using GeneSpring