Project description:Short Description: Oleaginous microalgae are capable of producing large quantities of fatty acids and triacylglycerides. As such, they are promising feedstocks for the production of biofuels and bioproducts. Genetic strain-engineering strategies offer a means to accelerate the commercialization of algal biofuels by improving the rate and total accumulation of microalgal lipids. However, the industrial potential of these organisms remains to be met, largely due to the incomplete knowledgebase surrounding the mechanisms governing the induction of algal lipid biosynthesis. Such strategies require further elucidation of genes and gene products controlling algal lipid accumulation. In this study, we have set out to examine these mechanisms and identify novel strain-engineering targets in the oleaginous microalga, Chlorella vulgaris. Comparative shotgun proteomic analyses have identified a number of novel targets, including previously unidentified transcription factors and proteins involved in cell signaling and cell cycle regulation. These results lay the foundation for strain-improvement strategies and demonstrate the power of translational proteomic analysis. Experimental: Lysates were diluted 1:300 for quantitation in order to abate interference due to the chlorophyll content of the samples. Quantitation was performed by Qubit fluorometry (Invitrogen) and 20 ug of each sample was solubilized in LDS buffer, heated at 85 C for 5 min and separated on a 4-12% Bis-Tris Novex mini-gel (Invitrogen) using the MOPS buffer system. The gel was stained with coomassie and each lane was excised into 40 equally sized segments. The gel bands were processed using a robot (ProGest, DigiLab) with the following protocol: - Washed with 25mM ammonium bicarbonate followed by acetonitrile. - Reduced with 10mM dithiothreitol at 60 C followed by alkylation with 50mM iodoacetamide at RT. - Digested with trypsin (Promega) at 37 C for 4h. - Quenched with formic acid and the supernatant was analyzed directly without further processing. The gel digests were analyzed by nano LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Orbitrap Velos Pro. Peptides were loaded on a trapping column and eluted over a 75um analytical column at 350 nL/min; both columns were packed with Jupiter Proteo resin (Phenomenex).The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 FWHM resolution and MS/MS performed in the LTQ. The fifteen most abundant ions were selected for MS/MS. Mascot DAT files were parsed into the Scaffold software for validation, filtering and to create a non-redundant list per sample. Data were filtered using a minimum protein value of 99%, a minimum peptide value of 50% (Prophet scores) and requiring at least two unique peptides per protein.
Project description:This study investigates the proteome of the flagellum of the parasite Trypanosoma brucei. This parasite is the causative agent in African trypanosomiasis. This structure contains a 9 + 2 axoneme structure, highly conserved throughout eukaryotic evolution. The kinetoplastid flagellum additionally has a trypanasome-specific structure, the paraflagellar rod. Here, we have performed analyses based upon 1D- and 2D-gel separation followed by LC-MS/MS identification of peptides associated with flagellar proteins.
Project description:Differences in genome size and gene content are among the most important signatures of microbial adaptation and genome evolution. Here, we investigated the patterns of genome variation among strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Using the sequenced strain Rm1021 as a reference, the genome size and gene content variations were analyzed among ten diverse natural strains, through pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01Mbp, with the greatest variation arising from the pSymA replicon, followed by that of pSymB. No observable size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of ORFs on pSymA were variably absent/present, followed by 12.7% on pSymB, and 3.7% on the chromosome. However, the percentages of ORFs that were variably duplicated were more evenly distributed among the three replicons, 11.0%, 16.5% and 15.3% respectively for ORFs on pSymA, pSymB and the chromosome. Among the 10 strains, the percentages of absent ORFs ranged from 1.51% to 6.35% and those of duplicated ORFs ranged from 0.27% to 8.56%. Our analyses showed that host plants, geographic origins, multilocus enzyme electrophoretic types, and replicon sizes had little influence on the distribution patterns of absent or duplicated ORFs. The proportions of ORFs that were either variably absent/present or variably duplicated differed greatly among the functional categories, for each of the three replicons as well as for the whole genome. Interestingly, we observed positive correlations among the three replicons in their numbers of absent ORFs as well as the numbers of duplicated ORFs, consistent with coordinated gene gains/losses in this important bacterium in nature. microarray:Sm6kOligo
Project description:Biological replicates: 393-395 (crude extract): protein extraction 399-401 (40% AS): protein extraction+40% AS precipitation 405-407 (crude extract+MOAC): protein extraction+phosphoprotein enrichment by MOAC 408-410 (40% AS+MOAC): protein extraction+40% AS precipitation+phosphoprotein enrichment by MOAC -determination of protein concentration by 2D-Quant Kit -LC-MS with LTQ Orbitrap Velos: nanoLC, precursor scan Orbitrap, fragmentation CID, fragment scan LIT -Thermo Proteome Discoverer 1.3 with phosphoRS 1.0 -Mascot 2.3 -database TAIR 10 -Scaffold 4 Technical replicates two measurements each (e.g. 393, 393_2)
2014-01-28 | PXD000421 | Pride
Project description:RNA-seq of CAR-Ms and Gel-CAR-Ms
Project description:Differences in genome size and gene content are among the most important signatures of microbial adaptation and genome evolution. Here, we investigated the patterns of genome variation among strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Using the sequenced strain Rm1021 as a reference, the genome size and gene content variations were analyzed among ten diverse natural strains, through pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01Mbp, with the greatest variation arising from the pSymA replicon, followed by that of pSymB. No observable size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of ORFs on pSymA were variably absent/present, followed by 12.7% on pSymB, and 3.7% on the chromosome. However, the percentages of ORFs that were variably duplicated were more evenly distributed among the three replicons, 11.0%, 16.5% and 15.3% respectively for ORFs on pSymA, pSymB and the chromosome. Among the 10 strains, the percentages of absent ORFs ranged from 1.51% to 6.35% and those of duplicated ORFs ranged from 0.27% to 8.56%. Our analyses showed that host plants, geographic origins, multilocus enzyme electrophoretic types, and replicon sizes had little influence on the distribution patterns of absent or duplicated ORFs. The proportions of ORFs that were either variably absent/present or variably duplicated differed greatly among the functional categories, for each of the three replicons as well as for the whole genome. Interestingly, we observed positive correlations among the three replicons in their numbers of absent ORFs as well as the numbers of duplicated ORFs, consistent with coordinated gene gains/losses in this important bacterium in nature. microarray:Sm6kOligo A total of 12 strains were included for the microarray analyses in this study. The 12 strains included two reference strains and ten other natural strains. Reference strain Rm1021 was the strain with a completely sequenced genome, from which the whole genome microarray was based on (Krol et al. 2004). The second reference strain RmF909 was derived from strain Rm1021 but with a deletion of 575 ORFs located on the megaplasmid pSymB (genomic location 106724-735431, Charles et al. 1991). The two reference strains were used for positive and negative controls in our microarray hybridization experiments and for establishing threshold values for determining whether an ORF is present or absent among natural strains.