Unknown,Transcriptomics,Genomics,Proteomics

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Effect of LacI (lac repressor) on transcription in Salmonella

ABSTRACT: Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonellaâs close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique. Overall design The total RNA was isolated from Salmonella using âRNeasy Mini Kitâ (QIAGEN). Organism used: Salmonella enterica serovar Typhimurium LT2 * Slides: Agilentâs arrays (8x15k) AMADID: NO: 17809 * Starting material: Salmonella RNA in nuclease-free water * RNA Samples used: Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac) * Labeling kit: MessageAmpâ¢ II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790 * Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA * Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 * Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 * RNA quality was checked using Bioanalyzer. The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

SUBMITTER: DrSandeepa M E

PROVIDER: E-GEOD-15950 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. Here, we report the dynamic interaction of the DNA intercalator and potential anticancer, plant alkaloid, sanguinarine (SGR) with chromatin. Association of SGR with different levels of chromatin structure was found to be enthalpy driven. Apart from DNA, it binds with comparable affinity to histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR mediated repression of epigenetic marks and the alteration of chromatin geography also result in modulation of global gene expression. These data conclusively show that the anticancer DNA-binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context. The total RNA was isolated from control and treated cells using the TRIzol (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer), was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturer’s instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by the PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Five arrays were used with at least two biological treatments of cells, and dye-swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the downregulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all five replicates.

Project description:DNA binding anticancer agents cause alteration in chromatin structure and dynamics. Here, we report the dynamic interaction of the DNA intercalator and potential anticancer, plant alkaloid, Sanguinarine (SGR) with chromatin. Association of SGR with different levels of chromatin structure was found to be enthalpydriven. Apart from DNA it binds with comparable affinity to histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although, it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation dependent chromatin transcription. SGR mediated repression of epigenetic marks and the alteration of chromatin geography, also result in modulation of global gene expression. These data conclusively, show the anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context. The total RNA was isolated from control and treated cells using TRIZOL (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer) was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturerâs instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with at least one biological treatment of cells and dye swap experiment were included in the final analysis. . The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

Project description:Objective of the study is to find out the differentially regulated genes of Mycobacterium BCG strain in extended stationary phase comparison to log phase growth and resuscitation phase. Mycobacterium BCG culture was grown in Sauton medium at 37o C without shaking. Cells at A600 0.6-0.8 were harvested as log phase culture. The cells harvested after 5 months incubation at 37oC without shaking became non culturable and were harvested as extended stationary phase cells. The extended stationary phase cells were treated with resuscitation promoting factor (Rpf) from Micrococcus luteus and harvested after 8 days. Cells of this stage were treated as resuscitation phase cells. Gene expression profiling was carried out using Agilent microarray platform. Keywords: Extended stationary phase, Log phase growth and Resuscitation phase. â¢ Organism: Mycobacterium tuberculosis BCG â¢ Slides: Agilentâs Mycobacterium tuberculosis custom array 4x44k (G2514F) AMADID No: 016013 â¢ Labeling kit: Ambion Message Amp II Bacteria a RNA Amplification Kit Cat# 1790 â¢ Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA â¢ Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 â¢ Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 â¢ RNA quality was checked using Bioanalyzer. Hybridization protocol 825ng each of labeled control cRNA was mixed with 825ng of treated labelled cRNA, mixed with appropriate amount of blocking buffer and hybridization buffer (Agilent Technologies). The hybridization mix was applied on to the backings and hybridized to custom designed 60mer Mycobacterium tuberculosis microarray using sure hyb chambers at 65degC for 17 hours. Slides were washed with gene expression wash buffer 1 and 2 (Agilent Technologies) followed by Acetonitrile. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Slides were scanned at 5 micron resolution using Agilent scanner.Automated feature extraction was done using Agilentâs Feature Extraction Software. Data processing Analysis of feature extracted data was done using Agilentâs GeneSpring GX V 7.3.1 software. Pre-processing of the replicate experiment data was done to flag the outliers. Normalization of the data was done using per spot per chip intensity dependant lowess normalization and dye swap experiment was data transformed. Genes with log2 ratio of 1.29 and above in replicate experiments were considered as up regulated and 0.81 and below was considered down regulated. p-value was calculated by GeneSpring GX for each gene on the basis of replicate probes to indicate statistical significance. Biological significance of differentials were analyzed using Genotypics Biointerpreter - Web based tool for biological interpretation of given genelist.

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Effect of LacI (lac repressor) on transcription in Salmonella

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