Praveen Kumar Sappa

The aim of the study is to assess the global function of RNase Y in RNA processing and degradation in Bacillus subtilis. To this end we constructed a strain allowing controlled depletion of RNase Y and used microarrays to analyze the transcriptome in response to the expression level of RNase Y.

The objective of this project is an integrated understanding the metabolic, proteomic and genetic network that controls the transition from growth to glucose starvation. This transition is a fundamental ecophysiological response that serves as a scientific model for environmental signal integration and is pivotal for industrial fermentations of Bacillus that occur predominantly under nutrient starvation.

Keywords:
Glucose starvation, Transcriptomics, Proteomics, Metabolomics,Bacillus subtilis,

Bacillus subtilis was subjected to various stress conditions like high temperature(57°C), low temperature(16°C), high osmalarity(1.2M NaCl). The above mentioned stress conditions are again split into two different types as 'continuous stress condition' and 'sudden shock'. All the conditions were then done in biological triplicates.
Transcriptome for these samples was then analysed with Nimblegen Tiling array.

The aim of the study is to assess the global function of RNase Y in RNA processing and degradation in Bacillus subtilis. To this end we constructed a strain allowing controlled depletion of RNase Y and used microarrays to analyze the transcriptome in response to the expression level of RNase Y.

The main aim of this experiment is to actively grow B.subtilis in presense of glucose until high optical density in an aerobic fermentor and then, at a definite point of the growth, the glucose supply is shut down which leads to complete glucose exhaustion in the media. Simultaneusly samples for transcriptomics, intra and extracellular metabolomics, intra and extracellular proteomics are harvested through out the experiment.

The main aim of this experiment is to actively grow B.subtilis in presense of glucose until high optical density in an aerobic fermentor and then, at a definite point of the growth, the glucose supply is shut down which leads to complete glucose exhaustion in the media. Simultaneusly samples for transcriptomics, intra and extracellular metabolomics, intra and extracellular proteomics are harvested through out the experiment.

Growth of B.subtilis in shakeflask at 57°C; 16°C, 37°C(Control) and 1,2M NaCl in SMM.

BMM EtOH, 16, 57
SMM NaCl

Transcriptome analysis for the samples harvested from Chemostat cultivated samples.

Absolute quantification of proteins using heavy labeled QconCAT as an internal standard and quantifying the native proteins in the complex sample via scheduled Multiple Reaction Monitoring(MRM) .

B. subtilis was grown in SMM media with glucose as carbon source and the samples for RNA were harvested OD578nm- 1.0). The stress conditions that were applied over here are growthat 57°C, 16°C, 1.2M Nacl and 37°C(control).
All the samples were analysed for transcriptome as biological triplicates.

B. subtilis was grown in M9 media with glucose as carbon source and the samples were harvested during exponential phase (OD600nm- 0.4), early stationary phase(OD600nm- 1.3), late stationary phase(OD600nm- 1.0).
All the samples were analysed for transcriptome as biological triplicates.

Array hybridisation was done at Roche Nimblegen Inc. It cannot be displayed over here.

Recent studies revealed the unsuspected complexity of the bacterial transcriptome but its systematic analysis across many diverse conditions remains a challenge. Here we report the condition-dependent transcriptome of the prototype strain B. subtilis 168 across 104 conditions reflecting the bacterium's life-styles. This data set composed of 269 tiling array hybridizations allowed to observe ~85% of the annotated CDSs expressed in the higher 30% in at least one hybridization and thus provide an
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B. subtilis was grown in M9 media with glucose as carbon source and the samples for RNA were harvested at OD600nm- 0.4 , 1.3 and 1.0 ). Culture was done at 37°C and samples at OD600nm- 0.4(Exponential), OD600nm- 1.3 (Early stationary), OD600nm-1.0(Late Stationary). All the samples were analysed for transcriptome as biological triplicates.

Isolation of total RNA from Bacillus Subtilis using phenol-chloroform extraction method by maintaining cryogenec conditions initailly to prevent RNA degradation. Quality of the obtained RNA is then tested with Agilent Bioanalyser before proceeding for gene expression analysis.

SOP for ß-Galactosidase assay.

SOP for shake flask cultivation of B.Subtilis in Bacell-Sysmo

The reverse transcriptase synthesizes DNA, which complements the mRNA template
(complementary DNA, cDNA). Cy3/Cy5-dCTP are incorporated into cDNA during Reverse transcription. The obtained Cy3/Cy5 cDNA are then competitively hybridised onto Agilent microarray slide and subsequently scanned.

Cells were harvested from culture keeping the cells cold to quench the physiological condition of RNA and the cells were mechanically disrupted. RNA was isolated from the cells by conventional acid-phenol method and the quality was checked by Agilent bioanalyser.

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