ABSTRACT: The trans-acting mutant genetic selection was performed as described in Baniulyte et al, 2017. Chromosomal mutations were identified by whole genome sequencing.
INSTRUMENT(S): Illumina MiSeq
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
Project description:In order to identify cellular factors that influence the efficiency of AAV-HR mediated TI, we performed an unbiased genome-wide screening in a library of near haploid human cells (HAP1) mutagenized by retroviral insertions.
Project description:We have examined and compared the transcriptome of T. reesei growing on wheat straw and lactose as carbon sources under otherwise similar conditions. Gene expression on wheat straw exceeded that on lactose, and 1619 genes were found to be only induced on wheat straw but not on lactose. They comprised 30 % of the CAZome, but were also enriched in genes associated with phospholipid metabolism, DNA synthesis and repair and iron homeostatis. Two thirds of the CAZome was expressed both on wheat straw as well as on lactose, but 60 % of it at least >2-fold higher on the former. Major wheat straw specific genes comprised xylanases, chitinases and M-CM-^_-mannosidases. Interestingly, the latter two CAZyme families were significantly higher expressed in a strain in which xyr1 encoding the major regulator of cellulase and hemicellulase biosynthesis is non-functional, demonstrating that XYR1 is a repressor of these genes. We used two biological replicas of four T. reesei strains growing on glucose, lactose, and on wheat straw
Project description:Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage (ICH), the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets. The objective of this study was to describe the number and protein content of blood-derived EVs following an ICH in an animal model in rats treated with allogenic or xenogenic (human) EVs. The protein content of the EVs from the treated animals and control group was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points.
Project description:Unbiased forward genetic screens to identify host factors for DENV1 and JEV in 293FT cells. Unbiased forward genetic screens to identify host factors for HCoV-229E in Huh7.5.1 cells.
Project description:To search for host factors regulating SARS-COV-2 infection, we performed a genome-wide loss-of-function CRISPR/Cas9 screen in haploid human ESCs. The regulators were identified by the quantification of enrichment of their mutant clones within a pooled loss-of-function library upon SARS-COV-2 infection.
Project description:West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen using CRISPR/Cas9. HEK 293FT cells were infected with lentivirus expressing sgRNAs and then transfected with a Cas9 expressing construct. WNV infection killed most cells during a 12d selection. Survivor cells were harvested, from which DNA was isolated. The sgRNAs integrated in genome of survivor cells were amplified with PCR. The PCR product was sequenced with Illumina MiSeq to profile the sgRNA population in the survivor cells. Three replicates were conducted. Similarly, a second round of screen was conducted. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J2, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Examination of sgRNA populations in survival 293FT cells
Project description:Food-borne illness arising for Shiga-toxigenic Escherichia coli is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. Attachment of the bacteria to host tissue is one of the first steps in the interaction, and, as with mammalian hosts, has shown to be mediated by a combination of non-specific and specific adhesin-mediated interactions. We took a high-throughput positive-selection approach to investigate adherence mechanisms for E. coli O157:H7 isolate Sakai by inoculating a BAC clone library onto spinach, which was quantified by microarray hybridisation and gene loci enrichment measured using a Bayesian hierarchical model. The screen involved four successive rounds of adherence to spinach roots, resulting in 115 CDS credible candidates, covered by seven contiguous genomic regions. Two candidates regions selected for functional assessment included a chaperone-usher fimbrial gene cluster (loc6) and the type two secretion system (T2SS). The TS22 was found to significantly enhance binding to spinach roots and leaves, demonstrated with a BAC-T2SS clone and by mutagenesis of the secretin protein, EtpD. Both etpD and the inner membrane anchor protein gene etpC were expressed at 18 degree celsius, and expression of etpD was demonstrated for STEC (Sakai) resident in the apoplastic spaces in spinach leaf tissue. Together, these data indicate a novel function for STEC T2SS in adherence to plant tissue. Experiment 2: full replicated control screen. Conditions as for Experiment 1 (E-MTAB-5923), but no spinach root present. Two BAC clones (BAC2B5 & BAC2B24) used for additional positive controls.
Project description:We analyzed the transcriptional profile of P.aeruginosa PA14 grown under 14 different environmental conditions. These included conditions of growth within biofilms, at various temperatures, osmolarities and phosphate concentrations, under anaerobic conditions, attached to a surface and conditions encountered within the eukaryotic host. We found that >30% of the PA14 genome was differentially regulated at least under one of the 14 environmental conditions (referred to as the adaptive transcriptome). Most of the genes were also differentially regulated upon sigma factor hyper expression and/or inactivation (GEO accession number GSE54999) and many of those belonged to primary alternative sigma factor regulons. The samples of P. aeruginosa PA14 wild type strain were cultivated under 14 different experimental conditions and were analyzed by RNA-seq. For each condition, at least two biological replicates were generated Please note that PA14 is our standard lab strain used for all generated data in this records. There is no mutation introduced for any of those experiments, and thus, the descriptions only highlight the growth conditions.
Project description:In recent years, due to the influence of climate change and rising sea temperature, the incidence of Vibrio alginolyticus infections is increasing, and becoming the second most common Vibrio species reported in human illness. Therefore, better understanding of the pathogenic mechanism of V. alginolyticus infection is urgently needed. Vvrr1 (Vibrio virulence regulatory RNA 1) is a new found ncRNA predicted to be closely related to the adhesion ability of V. alginolyticus through the previous RNA-seq. In this study, the target genes of Vvrr1 were fully screened and verified by constructing Vvrr1 over-expressed strains and proteome sequencing technology.