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A short tutorial

The bigg_textbook model is a very simplified metabolic network model of Escherichia coli K12. This toy model is almost identical to the one in Orth et al. (2010). Note that we can only recommend reading this excellent primer as a complement to our hands-on tutorial.

Warming up

Initialization
  • Reset your session
  • Pick the bigg_textbook E. coli model from the model and pathway repository
Question
  • What are the total number of reactions, chemical compounds, and compartments in the model?
Answer
  • This is automatically displayed in the sandbox: #reac=95, #chem=33 and #comp=3. Indeed a very small model.
Questions
  • What is the total number of external (or boundary) reactions?
  • What are the total numbers of reversible or irreversible reactions?
  • How many reactions occur in a single compartment only? What kind of reactions are the remaining reactions? What is their total number?
Answer
  • The answers can be found in the BC (Basic Classification) summary

Model boundary

Question
  • Restrict the display to external reactions in the reac_list view by typing BOUNDARY in the search field. What can you tell about the growth conditions, i.e. about the "growth medium" of E. coli K12 as modelled by bigg_textbook? What does it mean that some external reactions are unconstrained/unidirectional/bidirectional?
Some answers
  • D-glucopyranose is the sole carbon source that can be consumed with an absolute uptake rate of 10.0 (the negative sign indicates that this boundary reaction proceeds from right to left).
  • Hydrogenphosphate, ammonium, water, proton, oxygen, and carbon dioxide are freely available. They can be exchanged in both consumed and secreted as no flux constraints are placed on these reactions
  • The remaining organic compounds (e.g. acetate) occurring in the external compartment can only be exported and thus correspond to possibly secreted metabolites

Simplification

The bigg_textbook model is a simplified metabolic network.
  • What can you tell about its growth reaction? Is it biologically relevant?
  • What can you tell about its sulfur metabolism?
Answers
  • Identify the growth reaction in the reac_list view by, for example, typing BIOMASS in the search field... What a simplified growth reaction!! Only two amino acids are required for growth: glutamate and glutamine.
  • There are no sulfur compounds in the set of boundary reactions - the sulfur metabolism is completely ignored although sulfur atoms occur in some compounds, e.g. CoA!

Analyses

Run the GCR (groups of coupled reactions) and FBA (flux balance analysis) on bigg_textbook model.
  • Does the model grow?
  • Can you identify any reaction correlation groups? How do they relate to the flux distribution?
  • Can you identify any reaction correlation groups that cannot carry a flux in steady state? If so, why?

Model modification

The idea is to create a new model for the anaerobic growth of E. coli K12:
  • Determine the Reac ID of the boundary reaction of O2
  • Export the bigg_textbook model as an Excel spreadsheet (or in any other format, but Excel is rather convenient for performing a quick change)
  • Find the row with the correct Reac ID in the enzymes sheet and set the flux boundaries of O2 to [0;0]. What does this mean?
  • Save the model on the user side using a new filename.
  • Upload the new model using a new and informative model identifier, e.g. anaerobic_textbook
  • Inspect the original and modified models using the reac_list view. Can you identify any differences between the original and the modified model? If so, which ones?

More analysis

Run FBA on the anaerobic bigg_textbook model.

Questions
  • Can you identify any differences in the external flux distributions between the aerobic and anaerobic models?
  • Can you establish the overall growth stoichiometries for both models (hint: examine at the flux distribution of the boundary reactions). Are they compatible with E. coli K12 biology?
  • Can you identify any internal reaction(s) of the two models with altered directionality?
Answers
  • The different stoichiometries (aerobic versus anaerobic growth) are qualitatively correct from a microbial perspective:

    Aerobic respiratory metabolism10.0 D-glucopyranose + 17.75 dioxygen + 5.27 NH4(+) + 3.56 hydrogenphosphate-->18.87 CO(2) + 17.65 H(+) + 25.91 H2O + 0.97 BIOMASS
    Anaerobic fermentative metabolism 10.0 D-glucopyranose + 1.22 NH4(+) + 0.82 hydrogenphosphate + 0.40 CO2-->30.18 H(+) + 6.96 H2O + 0.22 BIOMASS + 17.68 formate + 8.42 acetate + 8.18 ethanol

    However, the absolute values of the fluxes are not really realistic, for example, the above stoichiometry implies that ~70% of the carbon of the D-glucopyranose is assimilated in aerobic conditions, which is certainly too high! Also, the (small) consumption of CO2 during anaerobic growth is suspect. These results can nevertheless be regarded as very encouraging given that (i) a stoichiometric model is a drastic simplification of a complete dynamic system and (ii) bigg_textbook is an excerpt of a larger E. coli genome-scale metabolic network, that is about 20 times larger.
  • Yes, the ATP synthase:

    4 H(+) extracellular + ADP + hydrogenphosphate <==> ATP + H2O + 3H(+)

    During aerobic growth, respiration generates a proton gradient, which is dissipated to generate ATP. During anaerobic growth, ATP consumption generates the proton gradient, which drives the various transport processes.

Reaction knockout

Run the reaction knockout analysis (RKO) on both the aerobic and the anaerobic model.
  • How many reactions are essential in each model (aerobic vs anaerobic growth)?
  • Are there reactions that are essential in only one model?
  • Are there reactions leading to a beneficial, i.e. producing more biomass, knockout? If so, why?
Answers
  • aerobicanaerobic
    improved01
    unaffected6454
    impaired1317
    lethal1823


  • The following cytoplasmic reactions are essential for anaerobic growth:

    1 ADP + 1 H(+) + 1 D-fructofuranose 1,6-bisphosphate(4-) <-- 1 ATP + 1 D-fructofuranose 6-phosphate
    1 phosphoenolpyruvate + 1 CO(2) + 1 H2O --> 1 Oxaloacetate + 1 H(+) + 1 hydrogenphosphate
    1 glyceraldehyde 3-phosphate(2-) + 1 glycerone phosphate<==>1 D-fructofuranose 1,6-bisphosphate(4-)
    1 D-glucopyranose 6-phosphate<==>1 D-fructofuranose 6-phosphate
    1 glyceraldehyde 3-phosphate(2-)<==>1 glycerone phosphate

    However, there is no reaction that is essential for aerobic growth!


A more realistic model

  • Add the model iAF1260 to your workspace and compare it to the bigg_textbook model with respect to the growth reaction and the boundary conditions.
  • Rerun the previous analyses and manipulations to get a feeling for a more realistic and complete model.