Team:IIT Madras/Dry lab/Modelling

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<li>Reconstruction of the pathway was carried out to suit our project, hence involving the effects due to Proteorhodpsin pumping activity. Data for pH gradient <sup>[2]</sup>, the delta [H+] <sup>[3]</sup> was taken from literature and hence flux was calculated to formulate a comprehensive model.</li> <br/>
<li>Reconstruction of the pathway was carried out to suit our project, hence involving the effects due to Proteorhodpsin pumping activity. Data for pH gradient <sup>[2]</sup>, the delta [H+] <sup>[3]</sup> was taken from literature and hence flux was calculated to formulate a comprehensive model.</li> <br/>
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<div id="protocol1" style="cursor:pointer;"><h3><b><u>Model Construction</u></b></h3></div>
<div id="protocol1" style="cursor:pointer;"><h3><b><u>Model Construction</u></b></h3></div>
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Revision as of 03:11, 29 October 2011

bar iGEM 2011 - Home Page Indian Institute of Technology - Madras



In-Silico - Comparative Growth Analysis of Wild type vs PR Transformed cells

(Metabolic Modeling using Constraint Based Reconstruction and Analysis)

Abstract

    An extensive Reconstruction and Flux Balance Analysis study of metabolic pathways in E.coli at the genome scale, considering 1668 metabolites and 2383 reactions and their respective stoichiometry matrices was carried out using a Constraint Based approach. This model was validated with negative regulation of reactions by comparing with literature available for Oxidative Phosphorylation inhibitors. By including variations in substrate (glucose) concentrations under limiting conditions, we analyzed the global effects of Proteorhodopsin a.k.a. PR, (light-dependent proton pump) activity on the host system. Such a model which analyzes global effects on metabolic pathways is a novel addition to pre-existing kinetic models (at the protein level) of PR action.

Hypothesis

    Increase in cell growth rate due to the proton efflux generated by Proteorhodopsin in minimal carbon media

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.


Model Construction

Protocol for Metabolic Modeling


Click here to download SBML file for the genome scale e.coli (K-12 MG1665) model including Proteorhodopsin

Validation of Model


Simulations for Proof of Concept



References

  1. "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information" Adam M Feist, Christopher S Henry, Jennifer L Reed, Markus Krummenacker, Andrew R Joyce, Peter D Karp,Linda J Broadbelt, Vassily Hatzimanikatis and Bernhard Ø Palsson,Molecular Systems Biology-2007
  2. "Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host" A. Martinez, A. S. Bradley†, J. R. Waldbauer, R. E. Summons and E. F. DeLong,PNAS-2007
  3. "Light-powering Escherichia coli with proteorhodopsin" Jessica M. Walter, Derek Greenfield, Carlos Bustamante and Jan Liphardt,PNAS-2007