Systems Biology of a genetically engineered Pseudomonas fluorescens with inducible exo-polysaccharide production: analysis of the dynamics a

The biosynthesis of the industrially important exopolysaccharide alginate will be used as a model system for global analysis of the functioning of metabolic networks in genetically engineered strains of the unicellular bacterium Pseudomonas fluorescens. T he level of synthesis of the polymer can be controlled at a wide range by addition of an externally added inducer. The network responses following from various levels of induction will be studied by omics techniques in cells growing in continuous cultures , both at steady-state and kinetically as a function of time after induction. In combination with data collected from randomly induced and targeted mutants an in silico model of the metabolic network will be constructed. The model will be used for simulat ions and refined through further studies of new targeted strain constructions. WP 1 Strain constructions and cultivations A major goal of WP1 is to grow cells in continuous cultures enabling analyses of the effects on the global metabolic networks result ing from a carefully controlled induction of alginate biosynthesis. Analysis will be performed at steady states and time-course perturbation experiments after induction of alginate synthesis. A second goal is to identify mutants affected in alginate biosy nthesis (up or down) by carrying out a global screen of a transposon insertion library in a genetically engineered strain with inducible alginate synthesis. A spin-off of the identification of mutants with blocked or reduced levels of alginate synthesis i s that the corresponding proteins represent potential new targets for treatment of P. aeruginosa infections in cystic fibrosis patients. A third goal of WP1 is to construct new mutants based on targeted gene interruptions and mutants expressing modified l evels of key enzymes. This activity will be ongoing throughout the entire project. NB! There are four WPs in this project, but this form dores not contain enough space to cover all four WPs. See Form 1.