MetaFluxNett

An Operating Manual

Ver.1.8.6.2

2005.04. MBEL@KAIST

CONTENTS
The MetaFluxNet Software............................................................................................ 4
1.1. Introduction ........................................................................................................ 4 1.2. System Requirements PC................................................................................ 5 1.2.1. Hardware Requirements ....................................................................... 5 1.2.2. Software Requirements......................................................................... 5

Installation....................................................................................................................... 6
2.1. Installation.......................................................................................................... 6 2.1.1. Installation of prerequisites ................................................................. 6 2.1.2. Installation in Progress and Completion ........................................... 7 2.2. Uninstalling MetaFluxNet................................................................................ 8

First Steps ........................................................................................................................ 9

3.1. Starting the Main Program .............................................................................. 9 3.2. Menu and Tool Bar .......................................................................................... 10 3.2.1. File Menu................................................................................................. 10 3.2.2. Model Menu ............................................................................................ 10 3.2.3. Flux Analysis Menu............................................................................... 10 3.2.4. Visualization Menu ............................................................................... 11 3.2.5. About Menu............................................................................................. 11 3.2.6. Tool Bar ................................................................................................... 11

Building the Model ....................................................................................................... 12

4.1. Creating a project file .................................................................................... 12 4.2. Constructing a new model ............................................................................ 13 4.2.1. Add Metabolites .................................................................................... 13 4.2.2. Add Reactions........................................................................................ 14

Metabolic Flux Analysis ............................................................................................... 15

5.1. Starting the analysis ...................................................................................... 15 5.2. System Analysis .............................................................................................. 16 5.3. Solve the system ............................................................................................. 17 5.4. Flux Balance Analysis.................................................................................... 18 5.5. Comparative MFA ............................................................................................ 19 5.6. Model Export .................................................................................................... 20 5.7. Miscellaneous .................................................................................................. 21

User Registration .......................................................................................................... 23 Contact Information ..................................................................................................... 25

Chapter I
The MetaFluxNet Software
1.1. Introduction
MetaFluxNet is a program for managing information on the metabolic reaction system and for quantitatively analyzing metabolic fluxes in an interactive and customized way, which allows users to interpret and examine metabolic behavior in response to genetic and/or environmental modifications. As a result, quantitative in silico simulations of metabolic pathways can be carried out for understanding the metabolic status and for designing the metabolic engineering strategies Main features are 1. 2. 3. 4. 5. CONSTRUCTION OF REACTION MODEL METABOLIC FLUX ANALYSIS VISUALIZATION OF FLUX DISTRIBUTION COMPARATIVE ANALYSIS & FLUX PROFILING EXPORTING MFN TO SBML & MATLAB

1.2. System Requirements PC

1.2.1. Hardware Requirements Minimum hardware configuration for MetaFluxNet: Intel P-II (or compatible) CPU, 133 MHz 512 KB cache memory 128 MB of main memory 40 MB hard disk Recommended hardware configuration for MetaFluxNet Intel P-III (or compatible) CPU, 500 MHz 512 KB cache memory 384 MB of main memory 100 MB hard disk 1.2.2. Software Requirements Supported Operating Systems: Windows 98, ME, 2000, NT4.0, XP Microsoft Windows NT 4.0 requires Service Pack 6a. Note: Make sure you have the latest service pack and critical updates for the version of Windows that you are running. To find recent security updates, visit Windows Update Microsoft Internet Explorer 6.0 or higher and Internet Explorer 6.0 Service Pack 1. Microsoft .NET Framework Version 1.1 Redistributable Package or higher Java 2 Platform, Standard Edition, v 1.4.2 (J2SE) or higher

Chapter 2
Installation
Open a web browser and visit the following URL: http://mbel.kaist.ac.kr/mfn. Fill in the user registration form in the Download page to get the License Agreement Form. Submit the License
Agreement form to leesy@kaist.ac.kr. When we receive the signed form, we will send you an email with password to access and download MetaFluxNet.

2.1. Installation
Click . Then, proceed as follows. 2.1.1. Installation of prerequisites The Setup Wizard will automatically check for all the prerequisites for the installation and install those that are missing. 1. Automatic Detection of the Absence of Microsoft.NET and Its Installation

2. Automatic Detection of the Absence of Java 2 Platform and Its Installation

2.1.2. Installation in Progress and Completion

After prerequisites installation, the Setup Wizard will begin installing MetaFluxNet.

2.2. Uninstalling MetaFluxNet

To uninstall MetaFluxNet, go to Start Menu -> Programs -> KAIST MBEL. Then, click Uninstall. This will lead to automatic deletion of all the files related to MetaFluxNet. If you need the MetaFluxNet data, make sure to save it as backup on another directory before uninstalling.

Chapter 3
First Steps
3.1. Starting the Main Program
To start MetaFluxNet, double-click the shortcut icon (on your desktop) or press the Start button on the taskbar and select KAIST MBEL -> MetaFluxNet under Programs menu.

3.2. Menu and Tool Bar

3.2.1. File Menu

1. New Project: Create a new model 2. Open Project: Open an existing mfn file 3. Save Project: Save a model to mfn file 4. Save As: Save a model to a different mfn file 5. Import: Import a model from an existing SBML or MFAML model 6. Export: Export a model to SBML or MFAML model 7. Exit: Close MetaFluxNet

3.2.2. Model Menu

1. Metabolite: Show a metabolite list 2. Model Information: Show the description of the model

3.2.3. Flux Analysis Menu

Perform the flux analysis

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3.2.4. Visualization Menu

Show a model and flux distribution as a network 3.2.5. About Menu

1. 2.

Registration: Register Procedure for commercial users. About MetaFluxNet: Show the information of MetaFluxNet

3.2.6. Tool Bar

New project: Create a new model Open project: Open an existing mfn file Save project: Save a model to mfn file Model information: Show the description of the model Metabolite list: Show the metabolite list Flux analysis: Perform the flux analysis Visualization: Show the model as a network About : Show user registration procedure and information of MetaFluxNet

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Chapter 4
Building the Model
4.1. Creating a project file
1. To create a new project, click the New Project button on the tool bar or click File and New Project from the menu. It opens the New Project window. 2. Type in the Title and Description of the project. This will help you recognize the model later. 3. Click the Save Project button in tool bar to save the project as filename in the specified directory

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4.2. Constructing a new model

4.2.1. Add Metabolites

1. Click Metabolite List on the tool bar or click Model->Metabolite from the menu. 2. Once Metabolite List window appears, click the Add button to add a new metabolite to the list. 3. Determine Metabolite Name, Metabolite Type (whether it is intracellular or extracellular) and type in Synonyms if necessary.

Note: Definition of metabolite type

Uptake: extracellular metabolite which

enters the system boundary.

Secretion: extracellular metabolite which

leaves the system boundary.

Unknown: extracellular metabolite which

enters or leaves the system boundary.

Intermediate: intracellular metabolite.

4. Press the Save button. 5. Detail button displays the same window as the one from the Add button. It shows the saved data of a metabolite. 6. Delete button erases a metabolite from the list. 7. List view button displays a new window, which shows a list of both reactions and metabolites. It can be saved as a text format.

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4.2.2. Add Reactions

1. Click the Add button on the main window. It opens Reaction Information window. 2. Define Reaction Name. 3. Define Substrate Information and Product Information. Click the Add & Modify button. Once AddReactant window appears, select Substrates from the Metabolites list on the left-hand side. Click the >> button to save it. Determine Stoichiometry and Primary of the corresponding Substrate (or Metabolite). Primary determines whether to show the metabolite on the dynamic graph or not. If checked, it appears on the graph. Repeat this procedure for Products. 4. Save the information. 5. Check reversibility of Reaction (Direction) 6. Define Synonym, Gene, EC number if desired. 7. Repeat this procedure for the remaining reactions. 8. Detail button leads the user to the same window as the one from the Add button. It shows the saved data of a reaction. 9. Delete button erases a reaction from the list. 10. List view button displays a new window, which shows a list of both reactions and metabolites. It can be saved as a text format.

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Chapter 5
Metabolic Flux Analysis
5.1. Starting the analysis
1. To perform system analysis and flux balance analysis, click the Flux Analysis button on the tool bar or click the Flux Analysis from the menu. It opens Metabolic Flux Analysis window. 2. Uncheck a reaction in Reaction List Tab if you wish to exclude it from the analysis. This is equivalent to gene deletion or gene inactivation under specific circumstance. 3. Specify the lower and upper bounds or input the measurement of reaction if available. 4. Specify the boundary condition of

extracelluar metabolite in Metabolite List Tab if available.

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5.2. System Analysis

1. To model investigate system, the click determinacy, the System

redundancy and consistency of the Analysis button. 2. System Analysis windows shows the following results Determinacy: classify the system as overdetermined, determined or underdetermined system Redundancy: classify the systems as redundant or non-redundant system Balanceability: balanceable fluxes can be detected Consistency: check if the system satisfies the mass balance for intracellular metabolite. Calculability: calculable fluxes can be uniquely determined 3. Click the Apply button if you wish to apply system analysis result to the model.

Note: Definition of Status

C: Calculable flux B: Balanceable flux

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5.3. Solve the system

1. To solve the system, click the Solve button. 2. Solution method window shows the determinacy and redundancy, 3. It also suggests a relevant method.

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5.4. Flux Balance Analysis

1. Select an objective function candidate by clicking the corresponding reaction from the Reaction List Tab or a metabolite from Metabolite List Tab, and press Select the objective function button. 2. Choose the objective function in the candidate list 3. Click the FBA button to calculate the steady state intracellular flux. 4. Optimal Solution and flux distribution is calculated. 5. Once FBA result is obtained, the Add result to Comparative MFA button becomes active. This transfers the FBA result to Comparative MFA. 6. Click the Graph button in order to view the intracellular flux distribution graphically.

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5.5. Comparative MFA

1. Once analysis result is obtained, the Add result to Comparative MFA button becomes active. This transfers the result to Comparative MFA. 2. Type the result name and press OK button. 3. Analysis result is added to comparative analysis list. 4. If you want to delete any result column, choose one by clicking column header and click the Delete button. 5. It enables the user to conveniently compare the flux distribution results obtained from each specific case study 6. Check the flux (click on the column header to check all) and then click the New plot page button to graphically view the flux profile of a set of specific reactions. This subsequently displays Flux Profile View window. 7. Determine Title, X and Y axis, and display methods. Then click the Draw button. 8. The Multiple Display Method provides different Flux Profiles on the same graph.

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5.6. Model Export

As the number of software package for systems biology is increasing, it has become crucial for the users to exchange data between other programs for deeper investigations. Therefore, MetaFluxNet supports Systems Biology Markup Language (SBML), and Metabolic Flux Analysis Markup Language (MFAML). Furthermore, it can also transfer the data to many other mathematical applications including Comparative, LP SOLVE, CPLEX LP, MPS, GAMS, AMPL, LINDO and MATLAB. This exporting data function can be performed by clicking the Export button in Metabolic Flux Analysis window.

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5.7. Miscellaneous

Metabolite List tab shows mass balance equations of all the metabolites stored in the file. This is particularly useful for validating the consistency of analysis results. This tab shows intermediate and extracellular metabolites separately. If analysis is already performed, various properties of metabolites can be obtained. For intermediate metabolites, flux sum for each metabolite is calculated and dead ends can be identified. For extracellular metabolites, secretion or uptake rate of each metabolite (solution) is calculated.

In the Metabolite List Tab in Metabolic Flux Analysis window, it is also possible to view the overall reaction of the biochemical model system.

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Users can perform Flux Balance Analysis with a different mathematical setup, which is available in Objective Function Tab in Metabolic Flux Analysis window. By setting up specific solver parameters, user may get more accurate Flux Balance Analysis results, which otherwise might not be obtainable. Following is a brief explanation of primal/dual algorithm parameters.

Dantiz: In this option, all non-basic variables are checked (full pricing) and the one which violates its optimality condition the most is selected. This rule is quite expensive (dot products with all non-basic variables). Steep: Steepest edge is the most powerful normalized pricing algorithm but it is computationally the most expensive. It is a full pricing and does not adapt to the multiple pricing scheme. It can dramatically reduce the total number of iterations but the work per iteration can be so large that there can be no computational gain. Devex: Introduced by Paula Harris, this was the first effective normalized pricing; today we see it as an approximation to steepest edge. It requires less work per iteration but still can reduce the number of iterations quite considerably. Partial: To alleviate the computational burden, only a part of the non-basic variables is scanned and the best candidate from this part is selected. In the next step, the next part is scanned, and so on.

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Chapter 6
User Registration
MetaFluxNet is limited to 300 reactions for academic users. If you want the version of MetaFluxNet which supports unlimited model analysis, you must obtain the user license.

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1. To register, click the About -> Registration from the menu. It opens the License Agreement Form window. 2. Fill the Name and Affiliation. Then click the User Key Creation button. Then save the user key file in the specific directory 3. Send 4. Receive the the user key file file to from leesy@kaist.ac.kr via email. license leesy@kaist.ac.kr via email 5. Restart MetaFluxNet 6. Save the license file in the specific directory 7. Open the License Agreement Form window again. 8. Click the Register License Key button. 9. Choose the license file and click OK button

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Chapter 7
Contact Information
1. Prof. Sang Yup Lee (leesy@kaist.ac.kr) for general questions 2. Hongseok Yun (hsyun@pse.kaist.ac.kr) for any bugs and technical problems

Department of Chemical &Biomolecular Engineering Korea Advanced Institute of Science and Technology 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea Tel: 82-42-869-3930, Fax: 82-42-869-3910

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