Revision as of 16:23, 31 July 2017

Metabolic Modeling

Recent advances in computational biology include whole-genome metabolic networks. They describe the flow of metabolites within a particular organism. Using so called constraint-based method allows to make quantitative predictions about the phenotype while eliminating many of the complex parameters. Of particular interest to our project is flux balance analysis (FBA), which allows to predict the optimal steady-state biomass flux, which is often correlated with cell growth rate^[1] and is the most likely observed phenotype. One major advantage of FBA is that it does not require knowledge of enzymatic parameters. The mathematics of FBA is elucidated in this figure^[2]

One of the primary questions for the project is the following: will the synthetic P. denitrificans strain grow better than the unmodified one in presence of ammonia? To answer this question, we performed comparative analysis of the two Paracoccus strains using FBA on a whole-genome metabolic model. The analysis pipeline involved a slew of open-source computational tools, which we will describe below.

First, we reconstructed a metabolic model of Paracoccus denitrificans strain DSM 413 on a complete medium using ModelSEED^[3]. A complete medium is such that any nutrient, including ammonia, is available for uptake. Thus, the set of reactions included in the model is the biggest of all possible sets. Although the largest, this set is incomplete. In the next step, the model was gapfilled with all the reactions necessary for measurable cell growth.

The nature of our project dictates that we must be able to manually include several reactions, metabolites and genes (e.g. oxygenation of ammonia by the AMO enzyme complex) into the model. Such functionality is not available in ModelSEED. To implement this, we turned to COBRApy: Constraint-Based Reconstruction and Analysis^[4] package written in Python. COBRApy does not natively work with ModelSEED models. To overcome this, we used Mackinac package^[5] to convert the ModelSEED model into COBRApy. Using COBRApy, we can then add the new ingredients into the model and perform FBA to compare the biomass fluxes, and hence the growth rates, of the two Paracoccus strains. The script is available here.

First, the FBA was run on the gapfilled unmodified model, which initially contained 1550 reactions and 1556 metabolites. The optimal biomass flux was found to be \( 224.3248 \frac{\text{mmol metabolite}}{\text{g dry weight}\cdot\text{h}} \). Next, we add the reactions. Below is the list of all reactions added to the model. \(\ce{Q}\) and \(\ce{QH_2}\) represent ubiquinone and ubiquinol, respectively. \( \text{UqO} \) is the ubiquinone oxidoreductase enzyme which catalyzes the last reaction. \[ \ce{NH_3 + QH_2 + O_2 ->[\text{AMO}] H_2O + Q + NH_2OH} \] \[ \ce{NH_3 + NAD + H_2O ->[\text{AMO}] 2H^+ + NADH + NH_2OH} \] \[ \ce{NH_2OH + O_2 ->[\text{HAO?}] NO_2^- + H^+ + H_2O} \] \[ \ce{NH_2OH + 2Q + H_2O ->[\text{HAO}] NO_2^- + 2QH_2} \] \[ \ce{NO_2^- + NAD + H_2O ->[\text{nir}] H^+ + NO_3^- + NADH} \] \[ \ce{QH_2 ->[\text{UqO}] 2H^+ + Q} \] The optimal biomass flux of the modified model is \( 228.6980 \frac{\text{mmol metabolite}}{\text{g dry weight}\cdot\text{h}}\), with

ModelSEED

1

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Inspiration

Here are a few examples from previous teams:

References

[1] Feist, Adam M., and Bernhard O. Palsson. “The Biomass Objective Function.” Current opinion in microbiology 13.3 (2010): 344–349. PMC. Web. 28 July 2017.
[2] Cuevas, Daniel A. et al. “From DNA to FBA: How to Build Your Own Genome-Scale Metabolic Model.” Frontiers in Microbiology 7 (2016): 907. PMC. Web. 27 July 2017.
[3] Henry, C.S., DeJongh, M., Best, A.B., Frybarger, P.M., Linsay, B., and R.L. Stevens. High-throughput Generation and Optimization of Genome-scale Metabolic Models. Nature Biotechnology, (2010).
[4] COBRApy: COnstraints-Based Reconstruction and Analysis for Python.
[5] Mackinac: A bridge between ModelSEED and COBRApy to generate and analyze genome-scale metabolic models.

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-<p0> First, we reconstructed a metabolic model of <i>Paracoccus denitrificans strain DSM 413</i> on a complete medium using ModelSEED. A complete medium is such that any nutrient, including ammonia, is available for uptake. Thus, the set of reactions included in the model is the biggest of all possible sets. Although the largest, this set is incomplete. In the next step, the model was gapfilled with all the reactions necessary for measurable cell growth. </p0>
+<p0> First, we reconstructed a metabolic model of <i>Paracoccus denitrificans strain DSM 413</i> on a complete medium using ModelSEED<sup>[3]</sup>. A complete medium is such that any nutrient, including ammonia, is available for uptake. Thus, the set of reactions included in the model is the biggest of all possible sets. Although the largest, this set is incomplete. In the next step, the model was gapfilled with all the reactions necessary for measurable cell growth. </p0>
 <br></br>
-<p0> The nature of our project dictates that we must be able to manually include several reactions, metabolites and genes (e.g. oxygenation of ammonia by the AMO enzyme complex) into the model. Such functionality is not available in ModelSEED. To implement this, we turned to COBRApy: Constraint-Based Reconstruction and Analysis package written in Python. COBRApy does not natively work with ModelSEED models. To overcome this, we used Mackinac package to convert the ModelSEED model into COBRApy. Using COBRApy, we can then add the new ingredients into the model and perform FBA to compare the biomass fluxes, and hence the growth rates, of the two <i>Paracoccus</i> strains. The script is available <a href="https://2017.igem.org/Team:Virginia/Model/Files/CobraPD-LB.py">here</a>.</p0>
+<p0> The nature of our project dictates that we must be able to manually include several reactions, metabolites and genes (e.g. oxygenation of ammonia by the AMO enzyme complex) into the model. Such functionality is not available in ModelSEED. To implement this, we turned to COBRApy: Constraint-Based Reconstruction and Analysis<sup>[4]</sup> package written in Python. COBRApy does not natively work with ModelSEED models. To overcome this, we used Mackinac package<sup>[5]</sup> to convert the ModelSEED model into COBRApy. Using COBRApy, we can then add the new ingredients into the model and perform FBA to compare the biomass fluxes, and hence the growth rates, of the two <i>Paracoccus</i> strains. The script is available <a href="https://2017.igem.org/Team:Virginia/Model/Files/CobraPD-LB.py">here</a>.</p0>
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@@ Line 85: / Line 85: @@
 [1] <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912156/?report=classic">Feist, Adam M., and Bernhard O. Palsson. “The Biomass Objective Function.” Current opinion in microbiology 13.3 (2010): 344–349. PMC. Web. 28 July 2017.</a><br>
 [2] <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911401/">Cuevas, Daniel A. et al. “From DNA to FBA: How to Build Your Own Genome-Scale Metabolic Model.” Frontiers in Microbiology 7 (2016): 907. PMC. Web. 27 July 2017.</a><br>
+[3] <a href="https://www.ncbi.nlm.nih.gov/pubmed/20802497">Henry, C.S., DeJongh, M., Best, A.B., Frybarger, P.M., Linsay, B., and R.L. Stevens. High-throughput Generation and Optimization of Genome-scale Metabolic Models. Nature Biotechnology, (2010).</a><br>
+[4] <a href="https://www.ncbi.nlm.nih.gov/pubmed/23927696">COBRApy: COnstraints-Based Reconstruction and Analysis for Python.</a><br>
+[5] <a href="https://www.ncbi.nlm.nih.gov/pubmed/28379466">Mackinac: A bridge between ModelSEED and COBRApy to generate and analyze genome-scale metabolic models.</a><br>
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