"

From 2011.igem.org

  Hypothesis: 1) Increase in growth rate due to Proteorhodopsin proton efflux in minimal carbon media 2) Proton efflux generated by Proteorhodopsin increases ATP production Model Design Reconstruction and Mathematical Modeling of E.coli K12-MG1655 pathway with Proteorhodopsin Literature data: 1) Genome scale metabolic model thermodynamic data for genome scale E.coli K-12 MG1655 was derived. This was done by alignment with genomic annotation and the metabolic content of EcoCyc, characterization and quantification of biomass components and maintenance requirements of cell required for growth of the cell and thermodynamic data for reactions [1]. 2) Reconstruction of the pathway was carried out to suit our project, hence involving the effects due to Proteorhodpsin pumping activity. Data for pH gradient [2], the delta [H+] [3] was taken from literature and hence flux was calculated to formulate a comprehensive model. Conversion to Readable format for metabolic modelling tools: A Systems Biology Markup Language (SBML) file was created for the reconstructed model (model_PR) and Wildtype(model_WT). The flux balance studies were done by constraint based reconstruction and analysis FBA computations, which fall into the category of constraint-based reconstruction and analysis (COBRA) methods using the COBRA toolbox. The COBRA Toolbox is a freely available Matlab toolbox that can be used to perform a variety of COBRA methods, including many FBA-based methods. In Matlab, the models are structures with fields, such as 'rxns' (a list of all reaction names), 'mets' (a list of all metabolite names) and 'S' (the stoichiometric matrix). The function 'optimizeCbModel' is used to perform FBA. Also, gene deletion analysis and their effect on growth rates can also be modelled using COBRA toolbox. Procedure for setting up the model Simulation Design for Validation Glucose uptake flux (mMol/hr-dwt) Model Growth rate at complete inhibition of electron transport chain due to azide Growth rate at 70% electron transport chain inhibition by azide Growth rate in the absence of azide 2 Model_WT 0.0000 0.1576 0.1589 Model_PR 0.0000 0.1600 0.1609 3 Model_WT 0.0000 0.2449 0.2552 Model_PR 0.0035 0.2473 0.2572 4 Model_WT 0.0257 0.3322 0.3515 Model_PR 0.0306 0.3346 0.3535 5 Model_WT 0.0528 0.4195 0.4478 Model_PR 0.0577 0.4220 0.4498 6 Model_WT 0.0799 0.5068 0.5441 Model_PR 0.0848 0.5093 0.5461 8 Model_WT 0.1340 0.6785 0.7367 Model_PR 0.1389 0.6831 0.7387 10 Model_WT 0.1882 0.7609 0.8856 Model_PR 0.1931 0.7655 0.8901 12 Model_WT 0.2423 0.8433 0.9680 Model_PR 0.2472 0.8479 0.9725 1) 2) Experiement Design 3) 4) Results