The transition from growing to non-growing Bacillus subtilis cells

BaCell-SysMO 2
Modelling carbon core metabolism in Bacillus subtilis – Exploring the contribution of protein complexes in core carbon and nitrogen metabolism.

Bacillus subtilis is a prime model organism for systems biology approaches because it is one of the most advanced models for functional genomics. Furthermore, comprehensive information on cell and molecular biology, physiology and genetics is available and the European Bacillus community (BACELL) has a well-established reputation for applying
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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.

Experiments using shake flask cultures to measure dynamics associated with sigB response.

In this kind of studies sigmaB stress response is induced by the addition of artificial inducers of sigmaB. For example simgaB is downstream of a Pspac promoter and induced by the addition of IPTG.
A ctc::lacZ reporter gene is used to monitor sigmaB activity.

The aim of the study is to test the interplay of the effects of different transcriptional regulators on gene expression during different environmental responses in B. subtilis.
For a selected set of transcriptional regulators native proteins are fusion-labelled. The intensity of expression of each reporter protein is measured in the absence of several transcriptional regulators that compete for the RNA polymerase.

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.

Theoretical analysis of hypothetical sigma factor competition.
Based on the model 'transcription factor competition' possible dynamics of sigma factor competition are simulated and analysed using Lineweaver-Burk representations.

Measurement of intra- and extra-cellular metabolome.

We use BSA115 strain which lacks RsbU and RsbW proteins. Therefore, there is limited post-transcriptional regulation of sigmaB activity.
SigmaB itself is placed downstream of Pspac, inducible by IPTG. The lacZ reporter gene is downstream of Pctc promoter.
IPTG concentrations of 0.1, 0.2 and 1 mM are added in mid-exponential phase at an OD of appr. 0.3. The whole experiment runs for about eight hours.

The stressosome is an important sensor of environmental stresses in B. subtilis. It is formed by three protein types that form an icosahedral geometric protein complex. There are uncertanties how protein interactions take place, what the effects on the response behaviour of activation and inhibition of phosphorylation among proteins is, and what kind of proximal signal activates the stressosome in the first place.
To answer these questions a computational modelling approach was developed. This
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Theoretical analysis of hypothetical sigma factor competition.
Based on the model 'transcription factor competition' possible dynamics of sigma factor competition are simulated and analysed using Lineweaver-Burk representations.

batch fermatation - The transition from growing to non-growing Bacillus subtilis cells

batch fermatation - The transition from growing to non-growing Bacillus subtilis cells

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

Data sheet generated in Greifswald to measure the response of sigB activity in response to different media composition.

Strain BSA115 is grown until appr. OD 0.25 then expression of sigB is induced by the addition of IPTG. The extend of stress response is measured by the expression of lacZ via beta-Gal assay. The experiment lasts for appr. 400 min.

The stressosome is composed of three proteins that assemble in the form of an icosahedron. Icosahedra can be modelled in different ways with different abstraction levels regarding the original stressosome structure. The pdf-figure introduces geometric modelling of the stressosome using origami and particle dynamics simulations.

The pdf-file shows simulations of a hypothetical model of sigma factor competition. It simulates the dynamics that we can expect from the experiments and prepares for the analysis of the experimental data.
Analysis of sigma factor competition is based on a Lineweaver-Burk representation of RNApolymerase and competing sigma factors.

The figure contains information necessary to understand the mathematical model of experiments in BSA115. In these experiments sigB response is artificially initiated by the addition of IPTG while sigB is downstream of a Pspac promoter. The figure shows a flow-chart diagram that combines three hypotheses to explain experiments. It contains the ODEs and the fit of the respective models to the data.

only lacZ synthesis reduced by inhibitor in BSA115

This is a JWS model of the successful model for data representation. It realises regulation by a hypothetical sigB dependent protein that degrades beta-Gal.

The model represents a hypothetical situation in which an anti-sigmafactor reduces sigB efficacy.

The zip folder contains files that allow simulation of stressosome dynamics. The models are based on a cellular automaton approach. Each protein of RsbR and RsbS is located in the crystal structure of the stressosome. The proteins can be phosphorylated or not and these states determine the future of neighbouring proteins. To simulate the model open the file 'liebal_stressosome-model_12_workflow-matlab.m' in Matlab. It is written in the cell-model, put the cursor into a cell that you wish to
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The zip file contains model files and an experiment file. Unpack it in a directory and navigate with matlab to there. Use the 'matlab_execution_guide.m' for simulation and visualisation of the model. This file is written in matlab cell mode, so it is not a stand alone function.

Three models have been developed to test their capacity to reproduce the experimental data from Study: 'Controlled sigmaB induction in shake flask' with Assay: 'IPTG induction of sigmaB in BSA115'.
One model assumes a
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The zip-folder contains files for execution in matlab that allow for the simulation of stressosome dynamics and reproduction of published data on the stressosome. The important file for execution is 'liebal_stressosome-model_12_workflow-matlab.m'.

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.

Abstract (Expand)

Abstract (Expand)