RegulatoryTool

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Example file[edit]

You can download it here -> IntegratedModelToy.zip


IntegratedModel.png

This tutorial uses this example based on an example from J. Kim and J. Reed.OptORF: Optimal metabolic and regulatory perturbations for metabolic engineering of microbial strains. BMC, 2010


  • The regulatory model is composed by a transcription factor (TF1) that is activated when metabolite S is present, activates the expression of two genes (G3, G5) and represses the expression of gene G1A.
  • The Gene Reactions rules said us that: R1 is catalyzed when the gene G1A and G1B are activated; R2 is catalyzed by the gene G2; R3 and R4 are catalyzed by the genes G3 and G4, respectively; finally, R5 can be catalyzed by either genes G5 or G6.
  • In the metabolic level, the substrate (S) is utilized to produce biomass (B) and by-products P1 and P2. The cellular objective is to maximize biomass production (B) and the engineering objective is the production of P1. Reaction R2 converts the internal metabolite I1 into product P1 and 0.08 biomass (B), whereas reaction R5 converts the internal metabolite I2 into product P2 and 0.12 Biomass. The stoichiometric coefficients of all other reactions reflect a one-to-one relationship between molecule quantities.


Brief tutorial[edit]

This brief tutorial will help you to begin working with RegulatoryTool4OptFlux.

  • To start, you will need load a model in OptFlux(see how to here). In the New Project Wizard you have to chose the the toy.fluxes file as the Fluxes File; the toy.matrix file as the Stoichiometric Matrix; the toy.metab file as the Metabolites File and the toy.gr file as the Load GPR Information.

LoadToyMetModel.png

  • On the wizard Step3, you have to chose indexing started at zero(0)
  • You must pay attention to select a correct biomass flux (R_Biomass).

How to integrate a regulatory model[edit]

To start using all features present in Regulatory plug-in, you have to integrate a regulatory model with a metabolic model previous loaded in OptFlux.

  • After loaded the metabolic model in OptFlux, accessing the Load Regulatory Net option under the Plugins->Regulatory Tool menu

PathForIntegratedModel.png


  • The following dialog will be displayed.

IntegrateRegulatoryModel.png

1. Select Project and Model
You can select the model/project to integrate the regulatory model.

2. File Options
You can select the regulatory model file. In our example the regulatory model file name is toy.regNet.


  • After execution, the Metabolic Model datatype will replaced by Integrated Model in the clipboard.

ClipBoardAfterIntegrateModel.png

How to do a mutant simulation[edit]

  • You can access the Simulation option under the Plugins->Regulatory Tool menu or right clicking on the Integrated Model icon on the clipboard.

PathForSimAndOpt.png

  • In the Integrated Simulation you can select the model/project to work, and set up your configuration.

IntegratedSimulationOperaion.png
1. Gene Options
Selecting in the Gene list you can add/remove (using the arrows buttons) genes to the knockout list (the list of genes to be knocked out, in the right).

2. Select Environmental Conditions
If you have created environmental conditions you can select them to be used as constraints in the simulation. These can be used to define the values of drain fluxes, i.e. the rates at which metabolites are consumed or produced.

3. Objective Function Definition
Here you can select the reaction to optimize (biomass, by default), and you can also define if you will be maximizing or minimizing that flux.

4. Set Regulatory Variables
Here you can define the value of all regulatory user variables, and see the values of the model variables.

5. Select Simulation Method
OptFlux can use several simulation methods for knockout simulations, namely:
BRN+FBA, BRN+ROOM, BRN+MOMA and SR-FBA

  • And that's all!! You can press OK and the results will be loaded into the clipboard.

ClipboarIntegratedSi mulation.png

Critical Genes[edit]

The regulatory plug-in allows the users to define which are the genes present on the integrated model that are essential for the strain to survive, i.e. to keep a value of growth (biomass reaction flux) different from zero. The essential genes can be calculated using BRN+FBA method. Also, the software allows the user to load these genes or reactions from a file (if they are available). The list of essential genes can be manually edited, allowing users to add or remove elements, given their knowledge or the purpose of their experiments.

How to do load critical genes from a file[edit]

  • To load the information from a file just access the Load Critical Regulatory Genes option under the Plugins->Regulatory Tool->Critical Information menu in the Project Menu or right click on the Integrated Model icon in the clipboard.


PathForCriticalGenes.png

  • The following dialog will be displayed.

LoadCriticalRegulatoryGenesOperation.png
1. Select Project and Model
You can select the model/project to integrate the regulatory model.

2. File Options
You can select the file that have the critical genes information.

  • Now press OK and you can see a new element on the clipboard with the loaded critical genes.

ClipboardAfterCriticalGenes.png

How to determining essential genes[edit]

  • You can access the Compute Critical Regulatory Genes option under the Plugins->Regulatory Tool->Critical Information menu or by right clicking on the Integrated Model icon on the clipboard.

PathForCriticalGenes.png

  • In the options you can select the model/project to work, and set up the process.

ComputeCriticalGenesOperation.png

1. Set Regulatory User Variables
Here you can define the value of all regulatory user variables, and see the values of the model variables.

2. Select Environmental Conditions
If you have created environmental conditions you can select them to be used as constraints in the critical genes/reactions calculation. This means that in this case you will calculate essential genes in a given environment.

  • And that's all !! Now you can press OK and check the results in the clipboard.

ClipboardAfterCriticalGenes.png

How to optimise the gene knockouts set[edit]

  • You can access the Optimization option under the Plugins->Regulatory Tool menu.

PathForSimAndOpt.png

  • The following dialog will be displayed.

IntegrateOptimizationOperation.png
1. Select Project and Model
In the Project combo box select the project where you want to perform the optimization, in the Model combo box select the model in the project that you want to use.

2. Select Simulation Method
OptFlux can use several simulation methods for knockout simulations, namely:
BRN+FBA, BRN+ROOM, BRN+MOMA and SR-FBA

3. Select Environmental Conditions
If you have created environmental conditions you can select them to be used as constraints in the simulation.

4. Select Objective Function
OptFlux can use two types of objective function:
BPCY - Biomass-Product Coupled Yield; YIELD - Product Yield with Minimum Biomass.
The first calculates the product of the biomass flux and the compound production flux; the second, returns the value of the target compound production flux divided by the substrate consumption flux, if the biomass is larger than a minimum value, defined by the user

5. Objective Function Configuration
Here you can set the configuration of the selected objective function. In both BPCY and YIELD objective functions you can select the flux to be optimize (the compound you wish to produce) and the flux that represents the biomass. On BPCY you can also select the substrate flux that is in use. On YIELD you have to set the percentage of the wild-type minimum value.

6. Select Optimization Algorithm
OptFlux allows you to perform optimizations using one of the following algorithms:
Cellular Genetic Algorithm, Evolutionary Algorithm, Simulated Annealing

7. Optimization Basic Setup
Here you can configure the maximum number of solution evaluations (simulations) and check the expected time to perform that number of evaluations.
You can also set up the maximum number of knockouts and if the set of knockouts should be static or have a variable size.

8. Essential information
If you loaded/created some essential genes you can define if it is possible to knockout some critical genes.

9. Set Regulatory Variables
Here you can define the value of all regulatory user variables, and see the values of the model variables.

ClipboardAfterIntegrateOptimization.png