Project description:The pha-4 locus encodes a forkhead box A (FoxA/HNF3) transcription factor homolog that specifies organ identity for Caenorhabditis elegans pharyngeal cells. We used microarrays to identify pharyngeal genes and analyzed those genes to determine which were direct PHA-4 targets. Our data suggest that PHA-4 directly activates most or all pharyngeal genes. Furthermore, the relative affinity of PHA-4 for different TRTTKRY (R = A/G, K = T/G, Y = T/C) elements modulates the onset of gene expression, providing a mechanism to activate pharyngeal genes at different developmental stages. We suggest that direct transcriptional regulation of entire gene networks may be a common feature of organ identity genes. A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's. Keywords: genotyping_design
Project description:Pseudomonas putida KT2440 is a well-known model organism for the medium chain length (mcl) PHA accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the -oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC are annotated in P. putida KT2440. To investigate the relationship of fatty acids - PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in WT and mutants under different growth conditions were analysed. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7 % of the cell dry weight (CDW). To identify other potential PHA monomer suppliers by analysing the proteome of the phaJ1maoCphaJ4. The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogeanses PedE and PedH as potential monomer suppliers, but when these were deleted PHA level remained at 2.2 – 14.8 % CDW depending on the fatty acid used, and whether nitrogen limitation was applied. To identify the other non-specific dehydrogenases supply monomers for PHA synthesis, we analysed the proteome of the sextuple mutant under nitrogen limiting and non-limiting conditions.
Project description:Pseudomonas putida KT2440 is a well-known model organism for the medium chain length (mcl) PHA accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the -oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC are annotated in P. putida KT2440. To investigate the relationship of fatty acids - PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in WT and mutants under different growth conditions were analysed. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7 % of the cell dry weight (CDW). To identify other potential PHA monomer suppliers by analysing the proteome of the phaJ1maoCphaJ4. The proteomes of the WT, phaJ1phaJ4 and phaJ1maoCphaJ4 strains in MSM medium with octanoate under nitrogen limited condition were detected. In addition, we found that the deletion of PhaJ1 in P. putida KT2440 has a negative impact on the PHA accumulation in cells cultivated on glucose with nitrogen limitation conditions. It seems PhaJ1 also mediates the synthesis of PHA when glucose was used as the carbon and energy source. To investigate the role of PhaJ1 in PHA accumulation with glucose, the proteomes of P. putida KT2440 wild type, phaJ1, phaJ1phaJ4 and phaJ1maoCphaJ4 mutant growing on glucose were detected and compared.
Project description:Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures. To maximize PHA production, mixed microbial cultures may be enriched for PHA-producing bacteria with a high storage capacity through the imposition of cyclic, aerobic feast-famine conditions in a sequencing batch reactor (SBR). Though enrichment SBRs have been extensively investigated a bulk solutions-level, little evidence at the proteome level is available to describe the observed SBR behavior to guide future SBR optimization strategies. As such, the purpose of this investigation was to characterize proteome dynamics of a mixed microbial culture in an SBR operated under aerobic feast-famine conditions using fermented dairy manure as the feedstock for PHA production. At the beginning of the SBR cycle, excess PHA precursors were provided to the mixed microbial culture (i.e., feast), after which followed a long duration devoid of exogenous substrate (i.e., famine). Two-dimensional electrophoresis was used to separate protein mixtures during a complete SBR cycle, and proteins of interest were identified.
Project description:modENCODE_submission_3158 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP37 (official name : OP37 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. made_by : R. Waterston and S. Kim ); Developmental Stage: L2; Genotype: unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L2; Target gene pha-4; Strain OP37 (official name : OP37 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. made_by : R. Waterston and S. Kim ); temp (temperature) 20 degree celsius
Project description:modENCODE_submission_2598 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP37(made_by : R. Waterston and S. Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] official name : OP37 ); Developmental Stage: late embryo; Genotype: unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage late embryo; Target gene pha-4; Strain OP37(made_by : R. Waterston and S. Kim description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] official name : OP37 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq
Project description:modENCODE_submission_3161 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP37 (official name : OP37 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. made_by : R. Waterston and S. Kim ); Developmental Stage: Young Adult; Genotype: unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage Young Adult; Target gene pha-4; Strain OP37 (official name : OP37 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. made_by : R. Waterston and S. Kim ); temp (temperature) 20 degree celsius
Project description:modENCODE_submission_3215 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP37(official name : OP37 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. made_by : R. Waterston and S. Kim ); Developmental Stage: L3; Genotype: unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L3; Target gene pha-4; Strain OP37(official name : OP37 genotype : unc-119(ed3) III; wgIs37 [unc-119(+) pha-4::TY1::EGFP::3xFLAG] outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The PHA-4::EGFP fusion protein is expressed in the correct pha-4 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the PHA-4 transcription factor. made_by : R. Waterston and S. Kim ); temp (temperature) 20 degree celsius