Leader : Uwe Sauer
Workpackage leader: Eidgenössische Technische Hochschule Zürich, Pr. Uwe Sauer
WP1.2 will develop quantitative and global technology:
A novel tiling DNA microarray based on the highly advanced Affymetrix technology will be designed for the specific needs of the project (ChIP-on-chip approach, unbiased inventory of RNAs);
High-throughput microscopy to study fluorescent reporter gene expression at the single-cell level;
Novel non gel-based strategies for high throughput protein separation to quantify proteins from cell extracts and to characterise post-translational modifications;
Quantitative determination of intracellular metabolite concentration (metabolomics);
Adaptation of intracellular 13 C-labelling flux methods to highly parallel analyses
In WP1.2 we will be constructing a tiling DNA microarray covering the entire chromosome of B. subtilis with very fine sampling. The array will be used in unbiased searches for novel transcription units and regulatory sequences, both in coding and in intergenic regions. Integral to the study is the development of tools for improving recombination in B. subtilis and the two selected pathogens. Major quantitative technologies that underpin systems biology are those of Proteomics, Metabolomics and Fluxomics and separation and analysis workflows will be developed in WP1.2. While the conceptual basis for these system-wide methods is laid out – and several of the partners in this consortium were key technology drivers in their areas – the focus here is to improve their applicability, resolution power, throughput, quantification and standardisation. In proteomics, for example, there will be a strong emphasis on the analysis of digested proteins in cell extracts, since this can provide quantitative data and the workflow is amenable to high throughput. Similarly mass spectrometry-based protocols will be developed for system-wide analysis of metabolites and fluxes.
In combination, WP1.1 and WP1.2 will enable the Biology Pillar to make full use of the ChIP-on-chip technology and to extend it, as well as the cell arrays technology, to pathogenic Gram-positive bacteria. The design of these tools will be specified through a close collaboration between the Biology and Computational Biology Pillars.