Mycoplasma pneumoniae MaxQuant Threshold
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Mycoplasma Pneumoniae (ncbitaxon:2104)
SUBMITTER: Jaume Pinyol
PROVIDER: MSV000079167 | MassIVE | Fri Jun 19 03:50:00 BST 2015
REPOSITORIES: MassIVE
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Project description:The screening of a cDNA derived expression library of Klebsiella pneumoniae MGH 78578 expressed in E.coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (ompA, mdh) and negative (pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from K. pneumoniae by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 14 novel immunogenic proteins could be identified.
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Project description:The screening of a cDNA derived expression library of Klebsiella pneumoniae MGH 78578 expressed in E.coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (ompA, mdh) and negative (pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from K. pneumoniae by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 14 novel immunogenic proteins could be identified. In total 1536 (4 x 384) different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: ompA1, ompA2 and mdh (3 x 40 replicates) as positive reference proteins, as they have been described as immunogenic before. gapA and pyrC (2 x 40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 376 different samples and 8 controls. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates). For identification rabbit polyclonal antibody to K. pneumoniae (Acris AP00792PU-N) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.
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Project description:Two main articles have used this data. The small bacterium Mycoplasma pneumoniae with its annotated 689 protein-coding genes and 44 RNAs constitutes an ideal system for global and conditional transcription analysis in bacteria. We have combined spotted arrays under more than 120 conditions with several strand-specific, high resolution tiling arrays to obtain an unprecedented level of detail of bacterial gene expression. We have found 68 new non-annotated transcripts, of which the vast majority are potential regulatory RNAs, 53 of them in antisense to known genes. Integration of all data confirmed a dynamic and complex view of bacterial transcription: Under reference conditions in a rich medium, 138 polycistronic and 212 monocistronic transcripts could be identified, with almost half of the polycistronic operons showing a ‘staircase’-like expression pattern, i.e. the expression level within each gene is constant, but succeeding genes have lower expression. Furthermore, under different conditions, operons can divide into smaller transcriptional units, possibly by utilization of internal promoters resulting in many alternative transcripts. More complex bacteria show similar responses to external stresses, although M. pneumoniae lacks the respective transcription regulators, indicating the existence of yet uncharacterized common response mechanisms. This is supported by the concerted expression of genes, some of which with common upstream DNA motifs, form distinct operons under different conditions indicating additional factors regulating their expression. Frequent antisense transcripts, alternative transcripts and multiple regulators per gene thus cannot longer be seen as indicators of eukaryote-specific regulatory complexity. Keywords: Stress, genetic modification, time series, drug treatment
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