Comprehensive representation

• To access pathway information, click on any [subsystem] in the [Pathways table].
• Click on a [Pathway] such as [TCA].
• The corresponding [Reaction table] is then displayed.

• We provide the option of displaying additional annotations for each reaction in the reaction table.
• By Clicking on [more information button], one can toggle on/off the columns of 'Official name', 'EC-Number' and the 'ORF-ID'.
• The [EC-Number] and [ORF ID] provides URL links to external [KEGG resources] upon clicking.

• KEGG resource page continued from previous slide.

• Upon clicking on the [Compound list table], the list of pathway compounds and their corresponding list of [Contributing reactions] is displayed.
• The reaction equation of each reaction can be displayed by clicking on the individual [reaction].

• The reactions in the pathway are displayed together in a graph diagram under clicking on [network visualization tab].
• A blue square represents a reaction.
• A red circle is a compound while a black circle is a cofactor.
• A cofactor can appear multiple times in a visualization.
• An arrow coming out of a cofactor into a reaction square means it is consumed during the reaction, while an arrow coming out of a reaction square into a cofactor means that the cofactor is produced. The lack of arrow head in a line implies a reversible reaction.
• The graph image helps to provide an intuitive global view of the pathway.

Dynamic visualization

• The WEbcoli graph editor is a web-client application for visually editing pathway diagrams
  
• Note: Java Run-Time Environment (JRE) version 1.5 or above required
• Movie clip: Click here to watch pathway assembly demo

• Click on [User defined pathways list] and follow by clicking on [Run Editor button] to open WEbcoli pathway editor.
• With the editor, one can stitch together existing pathways, edit reaction stoichiometry and organize graphical layout of the pathways.

• To start session after loading the graph editor, click on [Login], and enter the [User ID] and [Password] for WEbcoli.

• To start editing pathway diagram, first choose a strain from [Strain Pull-Down List].
• From the [Pathway List], click on any pathway follow by [Open]. To assemble multiple pathways, press down the keyboard-Ctrl while selecting the pathways required, and then click on [Assemble]. By pressing down on keyboard-shift when selecting any two pathways, will also select all pathways in-between.
• A [Reaction List], [Compound List] and [Pathway Diagram] become ready for editing. By Clicking on any individual reaction, the associated [Substrate List] and [Product List] become available for editing.
• The [Stoichiometry], [Co-factor Status] and [Node Degree] of reaction participants can be edited.

• Click on [Picking] to select individual node (colored orange). The selected node can then be moved without moving other nodes.
• By clicking on [Re Draw], the default layout of the diagram is restored.
• By clicking on [‘+’] and [‘-’], the pathway diagram can be zoomed-in and zoomed-out respectively.
• By clicking on [Moving], the entire pathway can be translated.
• Clicking on [Show cofactor] toggles the display of cofactor.
• Diagram resolution can be adjusted using the [Resolution Pull-Down List].

• By clicking on [Layout], many default layouts for pathway graph can be implemented.
• After editing, the new pathway can be saved by clicking on [Model], followed by [Save as] and then inputting [Pathway Name]. The new pathway can then be browsed in WEbcoli


• Note that before saving any pathways, the cofactors of the pathways must be shown. To do this, click on [Show Cofactor] before saving

Virtual design and modeling

• Genetic modification of wild-type strain by deleting or adding associated metabolic reactions.
• A wide range of queries for searching reactions available in other organisms from in-house KEGG database.

• To create a new strain, user has to register a new account and login.
• To view on list of available strains, scroll down [Strain pull-down list], or click on [All strains button].
• After clicking on [All strains button], one can load a strain by clicking on its [Selection button] and follow by clicking on [Open button].
• To delete a model and its results, click on its [Selection button] and follow by clicking on its [Del button].
• To create a new strain, click on [New strain button] button. Alternatively, after clicking on [All strains], click on [Create strain button].
• Note that a [Model summary] of selected strain is available upon clicking on its [Selection button].

• The next step in creating a new strain is to enter the [Strain Name] and its [description]. Click on [Save] to save the new model.
• The new model uses the Wild Type (Reed et al., 2003) as its template.

• User can create a new pathway by clicking on [User Defined category], and follow by clicking on [Create button].
• Entering a [Name] for the new pathway. Click on [Add button] to save the new pathway.
• Note that reaction can only be added to User Defined category after creating a new pathway.
• To add a new reaction to the newly created pathway, see the next slide.
• To delete a pathway, click on the [Pathway] to be deleted and follow by clicking on the [Delete button].

• To add a new reaction to the new strain, click on [Add Reaction] and a query table will be displayed.
• To query by form, select [by Form]. User have to decide on the [Category] and [Pathway] to assign the new reaction to. In construction a reaction, user has to give it a [Name], and specify the [Substrate] and [Product] as a minimum requirement.
• A compound suggestion engine gives candidate substrates and products in real-time when user enters a key word. To select a candidate compound, [tick] on it. If keyboard-[enter] is pressed upon entering key word, the key word is entered as a new compound if does not exist in database.

• To delete a compound in a reaction being composed, click on [x].
• The [Reversibility list] allows one to choose reaction reversibility. The [Stoichiometry] of participating compounds can also be specified in the field provided. Click on [Save] to save a ready reaction. User can create multiple reactions before clicking on [Close].

• To add a reaction by script, select 'By script' in the [Reaction submission mode pull-down list]. Choose subsystem by using [Category pull-down list] and the pathway by using the [Pathway pull-down list] to add to.
• Fill in the reaction in the [Text edit field] to comply with WEbcoli's reaction submission rules. Click on the [Reaction submission rules link] for an explanation of the submission rules.
• Click on [Save button] to save reaction in the strain.
• If Format Invalid' is displayed in the [Feedback box], user should further edit the reaction representation. 'N' and 'Y' means that the compound before it is *not present* in database, and *present* in database respectively.

• To add reaction from KEGG database, select 'From KEGG' from [Add reaction method pull-down list].
• Choose the subsystem from [Subsystem pull-down list] and the corresponding pathway from [Pathway pull-down list], the new reaction will be in.
• Choose the organism to search from the [Organism List]. One can specify if a compound, enzyme or gene is to be searched from the [Entity search option pull-down list].
• Enter keyword to search in the [Keyword field] and click on the [Search button]. [Candidate reactions] are then listed.
• Select a candidate reaction by clicking on its [Selection button], and follow by clicking on [Save-into-text-field button].

• Upon clicking on [Save-into-text-field button], the candidate reaction appears in the [Text edit field].
The user can edit the reaction to comply with WEbcoli's reaction representation format ( Click here Reaction submission rules) .
• Click on [Save button] to save reaction in the strain. If 'Format Invalid' is displayed in the [Feedback box], user should further edit the reaction representation.
• Click on [Close button].

• To add new compound to database, click on [Compounds added table] and follow by clicking on [Add compound button] button.
• Enter the [Compound ID field] and follow by the [Compound name field]. Specify the which cell compartment the compound is in, by using the [Compartment pull-down list].
• Also, specify the electric charge of the compound by filling in the [Electrical charge field].
• Click on [Save button] to save the new compound entry.
• To delete added compound, click on [Del button] of the corresponding compound.

• Clicking on the [Reactions deleted] table, one can see a list of reactions currently deleted.
• To undo deletion of reaction, click on corresponding [Del button].
• Additional reaction can be deleted by clicking on the [Knock out button]. The Gene knockout form will appear as depicted in the next slide.

• Enter search keyword in the [Search field].
• One can search gene/ORF-ID, enzyme name and reaction-ID in any and/or combination by ticking the corresponding [Search checkbox].
• Click on [Search button] to start search.
• A candidate list appears. Click on corresponding [Reaction checkbox] of selected reactions.
• Click on [Close button].

• Note that reactions can also be deleted or added while click-browsing through [Pathways table].
• Click on any [Pathway] in [Pathways table] and it's [Reactions], [Compounds] and [Visualization] tables appear.
• Reactions can be deleted by clicking on [Reactions] table followed by ticking [Knockout button] option.
• Reactions can be added by clicking on [Add reaction button]. The [Add-reaction function table] have been described in earlier slides.

Flux balance analysis

• Quantification of metabolic fluxes using flux balance analysis
• Comparative analysis of flux distributions under environmentally and/or genetically changed conditions
• Flux distribution diagrams for providing an intuitive understanding of metabolic behavior
• Movie clip: Click here for comparative analysis

• To do flux balance analysis, click on a [Condition] in the list of conditions.
• A condition description table describing [Constraints], [Objective Functions] and [Result] is generated.
• There are three [Constraints] available- Environmental, Reactions and Exchange.
• 'Environmental' constraint option provides a list of metabolites in the environment which can be in flux exchange with the cell.
• A positive (flux) value means that there is a net flow of metabolite into the environment, after taking contributions from all reactions which can deplete/add metabolite in the environment.
• The flux can be constrained to a single value by putting a value in the [Measure] field.
• Otherwise, the same value can be input for the [Lower] and [Upper] bound fields.
• By ‘mouse-ing over’ the [Balance] icon, the list of reactions contributing to the metabolite flux can be viewed.

• 'Exchange' constraint option allows flux constraints to individual reactions removing or adding metabolites to the environment.
• A positive flux value means that there is a net forward reaction and vice versa.
• Constraints can be similarly implemented by setting a fixed value in the [Measure] flux field or by setting the [Lower] and [Upper] bound field for flux.
• The [Upper] and [Lower] bound can be toggled by clicking on [Uptake toggle] or [Secretion toggle]. [Uptake toggle] gives the most relaxed uptake constraint of (-inf,0]. By definition, uptake of extracellular metabolites means that its flux values are constrained to negative values.
• Similarly, clicking on [Secretion toggle] gives the most relaxed constraint for a metabolite being secreted, i.e., [0,inf). By definition, the flux value of a secreted metabolite is positive.
• Constraint can be deleted by clicking on [Delete button].

• New constraint for any of the constraint options can be added by clicking on [Add Constraint] button. A [Search Table] appears. Use the [Search Table] to search for reactions to be constrained, by typing keyword in the [Search] field. Select reactions of interest by ticking the [ Tick Box]. Click [Close] to end.

• Clicking on the [Objective Functions] table give a list of objective functions. The objective functions can be maximized, minimized or deleted by clicking on [Maximize], [Minimize] or [Delete] buttons. More reactions can be specified as candidate objective function by clicking on [Add Objective function]. The functions of the [Search Table] have been detailed in the previous slide.

• After specifying flux constraints and objective functions, Click on [Solve] to get flux solutions.
• The [Objective Function Value] is reported beside the [Solve] button. If there's no solution or no unique solution, 'infeasible' and 'unbounded' will be reported respectively.
• Click on [Result] table to see the flux results. One can choose between reaction flux results or metabolite flux results by using [Result mode pull-down list] .

• The [Subsysystem pull-down list] and [Pathway pull-down list] can be used to display specific pathway results.
• Click on [Result Name] field, enter a name and click on [Save]. This save the current analysis result in the list of results under [Comparative Analysis].
• By clicking on [Show-overall-reaction button], one gets a stoichiometric overall reaction for the cell.
• By clicking on [Visualization button], flux results are presented in the form of a network diagram. Note that user must specify pathways for visualization. Visualization of all pathways are not allowed

• Flux balance analysis result can exported by clicking on [Export result button]. See section on ‘File export’ for more details.
• Click on [Result name field] field, enter a name and click on [Save button].
• This save the current analysis result in the list of results under [Comparative analysis table].

• Click on [Comparative analysis button], and select the results of interest for comparative analysis by clicking on the respective [Comparative analysis list selection button].
• Select 'Compound' in the [Flux result display mode pull-down list]. Notice the [Flux comparison result] compares the metabolite fluxes side-by-side for the three conditions solved earlier.
• The 'results' order can be changed by clicking on a result ( which is highlighted in pink after clicking), followed by clicking the [Result display order button].
• Click on [Delete button] to remove a result from list.

• Select 'Reaction' in the [Flux result display mode pull-down list].
• To display networks properly, it is important to specify the pathway using the [Subsystem selection pull-down list], followed by the [Pathway selection pull-down list]. Note that the display of all subsystems and pathways is not allowed. Here, we compare the reactions in the Glycolysis/Gluconeogenesis pathway.
• Notice the [Flux comparison result] compares the reaction fluxes side-by-side for the three conditions solved earlier.
• Click on the [Visualization button]. A graphical network comparison of reaction fluxes in the Glycolysis/Gluconeogenesis pathway is displayed as depicted in the next diagram.
• The [Flux intensity bar] in a reaction node gives visually the relative amount of flux under the three conditions. Refer to the next slide for the [Flux intensity bar].
• The coloring of the [Flux intensity bar] can be adjusted by filling in the [Range value field].

• This is a comparison of reaction fluxes in the Glycolysis/Gluconeogenesis pathway in the graphical network mode after clicking on the [Visualization button] in the previous slide.

File export

• While browsing through the pathways as described in section 1: ‘Comprehensive representation’, user can click on [Export] in the [Reactions] table to export reaction list in the pathway to the local computer.

• A SBML model pops up and can be saved using the web-browser 'File>save as' function.

• Flux balance analysis result can exported by clicking on [Export Result button]. An ‘export Result’ page will pop up. By clicking on [Save as.. button], the result can be saved as a text file.

Pathway assembly

• Pathway assembly is not available for wild-type template.
• Select the relevant strain to start pathway assembly
• Hold down CTRL-KEY and MOUSE-LEFT-CLICK to select multiple pathways during pathway assembly.
• Hold down SHIFT-KEY and MOUSE-LEFT-CLICK on any two pathways to include pathways between them.
• Click on 'Show Cofactor' button before saving assembled pathways.

Comparative analysis