Editing RegulatoryTool

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=Example file=

You can download it here ->


[[Image: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=
This brief tutorial will help you to begin working with RegulatoryTool4OptFlux.

*To start, you will need load a model in OptFlux(see how to [[HowTos| 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'''.

[[Image: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==
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
[[Image:PathForIntegratedModel.png]]


*The following dialog will be displayed.
[[Image:IntegrateRegulatoryModel.png‎]]

<b>1. <i>Select Project and Model</i></b>

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

<br>
<b>2. <i>File Options</i></b>

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.
[[Image:ClipBoardAfterIntegrateModel.png‎]]

==How to do a mutant simulation==
*You can access the '''Simulation''' option under the '''Plugins->Regulatory Tool->''' menu or right clicking on the Integrated Model icon on the clipboard.
[[Image:PathForSimAndOpt.png]]<br>
<br>
*In the Integrated Simulation you can select the model/project to work, and set up your configuration.<br>
[[Image:IntegratedSimulationOperaion.png]]
<br>
<b>1. <i>Gene Options</i></b><br>
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). <br>
<br>
<b>2. <i>Select Environmental Conditions</i></b><br>
If you have created [[Environmental conditions|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.<br>
<br>
<b>3. <i>Objective Function Definition </i></b><br>
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.<br>
<br>
<b>4. <i>Set Regulatory User Variables</i></b><br>
Here you can choose the value of all regulatory user variables, and see the values of the model variables.<br>
<br>
<b>5. <i>Select Simulation Method</i></b><br>
OptFlux can use several simulation methods for knockout simulations, namely:<br>
BRN+FBA, BRN+ROOM, BRN+MOMA and SR-FBA<br>
<br>
*And that's all!! You can press OK and the results will be loaded into the clipboard.<br>

[[Image:ClipboarIntegratedSi_mulation.png]]

==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 '''Plugins->Regulatory Tool->Critical Information''' menu in the Project Menu or right click on the Integrated Model icon in the clipboard.


[[Image:PathForCriticalGenes.png]]
*Choose the name of the project and the model to where you want to load the critical information and also the file that have the critical genes.
[[Image:LoadCriticalRegulatoryGenesOperation.png‎]]
*Now press OK and you can see a new element on the clipboard with the loaded critical genes.
[[Image:ClipboardAfterCriticalGenes.png‎]]
===How to determining essential genes===
*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.
[[Image:PathForCriticalGenes.png]]
*In the options you can select the model/project to work, and set up the process.
[[Image:ComputeCriticalGenesOperation.png‎]]

'''1. Set Regulatory User Variables'''<br>
Choose the value of all regulatory user variables.

'''2. Select Environmental Conditions'''<br>
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.
[[Image:ClipboardAfterCriticalGenes.png‎]]


==How to optimise the gene knockouts set==
[[Image:PathForSimAndOpt.png]]

[[Image:IntegrateOptimizationOperation.png]]

[[Image:ClipboardAfterIntegrateOptimization.png‎]]