OptFlux3:RT

Example file

You can download it here -> IntegratedModelToy.zip

This tutorial uses an example based on work presented by 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 are characterized in the following way: 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.

• At 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.

Build a new project containing an integrated model

The user can build a project with an integrated model in two distinctive ways:

1. Through the "New Project Wizard Operation"

At the moment it is only possible to build an integrated model using the wizard operation from Flat Files, (new Formats will be available in a near future version).

• Step 1

• Step 2

The user have to choose the the toy.fluxes file as the Reactions File; the toy.matrix file as the Matrix File; the toy.metab file as the Metabolites File; the toy.gr file as the GPR Information and the toy.regNet file as the Regulatory Model File.

• Reader Configuration

In this step some definitions can be selected (depending from the type of the delimiters that are used on user Flat Files)

Continue to next panel Step 3 by pressing next. The user can select any of the options presented in the panel Step 3 or continue to next panel Step 4.

• Step 4

After selecting the biomass reaction, press the Finish button and a new project will be created. The model components will be displayed on Clipboard panel at left.

2. From an existing project

To create an integrated model project by this way it's required to have a metabolic model project already loaded (for more information click here). It's mandatory that the previous loaded metabolic model, have the GPR Rules information.

• The following dialog panel will be displayed

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

2. Choose Regulatory model reader type
You can select the type of reader for the regulatory network.

3. Choose Regulatory model file
You can select the regulatory model file. In our example the regulatory model file name is toy.regNet.

After pressing the Ok button the new project will be created, and the model components will be displayed on Clipboard.

How to perform a mutant simulation

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

• In both simulation types you can select the project to work, and set up your configuration.

Regulatory Model Simulation

1. Select genes to knockout
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).
Genes can be displayed by Name or ID.

2. Variables
Here you can define which variables (present in metabolic model or regulatory model) are to be considered in the simulation of the regulatory network.

3. Simulation Method
Methods to perform the regulatory network simulation. In future new methods will be added.

4. First Step Boolean Simulation
This parameter is related to BRNV simulation method (define how genes are knockout at beginning of regulatory simulation) , for example the user can assign a initial set of knockout genes instead of using the predefined OptFlux Strategy.

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

Integrated Model Simulation

1. Select genes to knockout
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:
SR-FBA and Integrated_BRN plus: FBA or pFBA or MOMA

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

Critical Genes

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

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

• The following dialog will be displayed.

1. Select Project
You can select a project to assign the set of critical regulatory genes.

2. Select File
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.

How to determining essential genes

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

1. Select Project
You can select a project to assign the set of critical regulatory genes.

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

3. 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.

4. Select Simulation Method
The critical genes operation can be performed using the same simulation methods, described in the Integrated Model Simulation section.

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

How to optimise the gene knockouts set

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

• The following dialog will be displayed.
  

1. Select Project
In the Project combo box select the project where you want to perform the optimization.

2. Select Simulation Method
OptFlux can use several simulation methods for knockout simulations, namely:
SR-FBA and Integrated_BRN plus: FBA or pFBA or MOMA

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

4. Select Critical Genes
If you have created or calculated a critical genes set, you can select them to be used in the simulation.

5. Select Objective Functions
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

6. Select Optimization Algorithm
Three optimization algorithms can be used:
Strengh Pareto Evolutionary Algorithm (MultiObjective algorithm);
Evolutionary Algorithm;
Simulated Annealing.

7. Maximum Number of knockouts
Is used to define the maximum of allowed gene knockouts, if Variable solution size is selected the solution will vary between 0 and maximum knockouts.

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

8. First Step Boolean Simulation
This parameter is related to BRNV simulation method (define how genes are knockout at beginning of regulatory simulation) , for example the user can assign a initial set of knockout genes instead of using the predefined OptFlux Strategy.

Analysing optimization solutions

When the optimization procedure completes, the solutions will be sent to the OptFlux clipboard:

Clicking it will launch the following view:

• The optimization view contains three distinct sections:
  • (TOP) The List of solutions and respective fitnesses.
  • (BOTTOM-LEFT) The decoded solution (you must select a solution either in the list or in the chart). This list will display the suggested gene deletions for the selected solution.
  • (BOTOM-RIGHT) The chart displaying the trade-of solutions (This chart is displayed as a pareto front when multiple objective functions were used, otherwise, the solutions will be ploted with the single objective function as the Y axis only)

• Pressing the << Add to simulation results button, will send the selected solutions to the list of simulations in the clipboard.