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USER MANUAL - Universidade do Minho

1. 1 1 210000000000000 nnn nennen 18 19 3 4 Identifying essential genes nennen nnne nena 20 3 5 Using Environmental 5 00 21 3b SIN IU PEN 22 3 6 1 Using the Simulation 23 3 6 2 Flux Variability Analysis 24 3 7 Optimization with Evolutionary 5 0 0 25 3 7 1 235 2 0 25 3 8 Optimization with Simulated 27 3 9 Optimization with Local Search 30 3 10 Using the Optimization 31 4 OPERATIONS COMPLETE 1 1 1 4 32 A A Fl E DEM 32 4 4 1 32 4 1 2 LOAG 34 ps uo 35 OptFlux Metabolic Engineering Workbench LER Eoque 36 37 42 3
2. GLUR 0 R ASPOS ADSLIr ADSL2r ARGSL R_SUCD1i 0 DAPE R 0 UAGAAT R UHGADA 0 1 x R_C1815N R 1415 0 ACODA UHGADA ACKr CYSS 0 ORPT OMPDC 0 D AKGDH 0 00215 x R_BiomassEcoli 2 0 x R PRAMPC R ASPOS 31xR BiomassEcoli R TYRTA 0 DDPA R TKT2 0 UAGDP R UAGAAT R UAGCVT R LIAGPT3 2 O FHL DHORD2 R_SUCD4 GCALDD NADH6 CYTBO32 0 MPD R GLUDy IGPS GLUPRT 0 25 x R BiomassEcoli R ASPTA V R_DRPA IGPS R TKT2 F6PA GAPD R 0 manne Amcie Reactions Steady State Equations OptFlux Metabolic Engineering Workbench 6 2 11 SIMULATION SOLUTION VIEWER Name Simulation Solution Viewer Viewer This viewer shows the complete information about a certain simulation procedure The complete list of knockouts and Description the values for all the fluxes in the simulated model are herein contained Components Maximized Flux The name and value for the flux selected for maximization in this simulation Simulation Method The name of the simulation method used in this simulation Knockouts The list of knocked out fluxes in this solution Th
3. 1 53 OptFlux Metabolic Engineering Workbench 6 2 2 Equivalent Variables 54 6 2 3 Environmental Conditions 55 6 2 4 Metabolites 56 6 2 5 Model Graph Viewer Graph Overlap essen eene 57 6 2 6 Model Properties VIBWET orbes ab EDU DUE DUE KE DR EX tea ED rus tea petas CODI CH 58 6 2 7 Optimization Solutions VIBWOGLT xo iar teen teur Na pete uo E Ope uk abra Ra 59 6 2 8 Reactions Viewer Flux 60 6 2 9 Reactions Viewer Stoichiometric 4 eene 61 6 2 10 Steady State Equations Viewer Stoichiometric 62 6 2 11 Simulation Solution 63 6 2 12 Variability Analysis 64 6 2 13 Zero Variables 2 65 7 5 9 OD 7 3 Flat Files for 5 66 74 1 Flux Bounds File ir tes Eee va ds 66 7 4 2 Sparse Matrix File NR T 66 7 13 Full 67 71 4 Metabolites o ERREUR GUI M 67
4. reactions sec reactions The integration of the SBML visualization graph and the metabolic model objects is done using the names of the reactions fluxes Therefore the visualization graph can be related to the whole set of fluxes or only to a subset 3 3 SIMPLIFYING MODELS OptFlux Metabolic Engineering Workbench In the Optimization menu Preprocessing sub menu the operation Model Simplification allows to choose among two types of simplification procedures or both e Remove variables reactions that are constrained by the metabolic model to have flux values of 0 e Identify groups of equivalent variables and replace these groups by a single variable This functions is used to simplify an initial model NOTE The Project s Simplified Data will be modified Some Simulation Results may be deleted as well Project 2003 Reed s E coli MI flux to maximize vgrowth zero fluxes V equivalent variables cancel These operations create a new metabolic model denoted as simplified model and also produce lists of the identified variables kept in data objects named Equivalent Variables and Zero Valued Variables It should be noticed that the identification of zero value variables is time demanding 3 4 IDENTIFYING ESSENTIAL GENES This operation also in the Preprocessing sub menu allows the identification of genes that when knocked out lead to a biomass reaction flux o
5. Search Search for the desired flux Fluxes The list of possible targets for deletion The user must select the flux to be deleted in the simulation Add selected flux to Pressing the button will add the flux selected in the above knockouts list list to the knockouts list on the right Data The Model to use in this simulation Algorithm The algorithm to use in the simulation Either FBA Flux 2 You Type Search OptFlux Metabolic Engineering Workbench Balance Analysis MOMA Minimization Of Metabolic Adjustment or ROOM Regulatory On Off Minimization of metabolic fluxes Flux to maximize The flux to maximize in the LP or MOMA problem use env conditions Environmental Conditions if any to use Knockouts Presents the user with the list of currently select fluxes to be knocked out in this simulation 4 2 4 FLUX VARIABILITY ANALYSIS Analyzes the maximum possible value of a selected flux for a range of values for the biomass Data The Model to use in this simulation Flux to Compare Flux to analyze against the biomass growth associated flux Interval Step 0 11 The step size for the biomass to use between simulations Must be a value between and 1 4 2 5 ENVIRONMENTAL CONDITIONS Interface to define the environmental conditions These will later be available in simulation and optimization tasks Data S
6. Use Conditions anaerobic E 8 Fat Q cancel OptFlux Metabolic Engineering Workbench 3 6 2 FLUX VARIABILITY ANALYSIS FVA An additional feature in OptFlux is the Flux Variability Analysis FVA that allows the user to examine the maximum possible value of a selected flux for a range of values for the biomass typically varying from 0 to 100 of its value in the wild type strain Optimization Visualizatia Simulation Wizard WildType Simulation Mutant Simulation Flux Variability Analysis Environmental Conditions Users can launch this operation by accessing the Simulation Menu as depicted in the picture above This will launch the operation panel Flux Variabilty Analysis Operation Please select a flux to compare against biomass and also the step interval Data Original Metabolic Model Flux Compare xyl D e Interval step 0 1 0 05 Cancel Run Simulation The selected interval step must be between 0 and 1 Users should take into account that the smaller the step size the longer this operation will take Results will be kept inside the Variability Analysis Results list in the Clipboard OptFlux Metabolic Engineering Workbench 3 7 OPTIMIZATION WITH EVOLUTIONARY ALGORITHMS Evolutionary Algorithms EAs are optimization algorithms based on an analogy with natural evolution In this application
7. anaerobic coli i parameters the number of generations the EA s population size The multiplication of the values of these two parameters gives an approximation of the number of solutions that will be evaluated during the search process The optimization procedure using EAs is typically time consuming In SBREAs an important feature is the possibility of evolving solutions with variable number of knockouts meaning that the user does not have to define a priori the number of genes to be deleted information that is usually not known Additionally it is possible to define an upper limit on the number of genes to be deleted since solutions with a large number of knockouts are difficult to implement in the lab The output of the EA is a set of processed solutions that result from the application of OptFlux Metabolic Engineering Workbench a simplifying process to the raw solutions obtained by the EA This process removes all the repeated solutions and removes from each solution the knockouts that are not strictly necessary to obtain a good fitness value The solutions resulting from this process can be kept in an object of the type Solution Set and further analysed This object is essentially a list of objects of the type Simulation Solution mentioned before ordered by the value of objective function Thus each solution in the list can be selected and analysed using the tools mentioned in the previo
8. Created From Model Properties OptFlux Metabolic Engineering Workbench 6 2 7 OPTIMIZATION SOLUTIONS VIEWER Name Optimization Solutions Viewer Type Viewer This Viewer provides a list of all the different solutions Description gathered during an optimization procedure Only different solutions are kept and they are sorted by the best fitness Components Solutions The list of solutions kept after the optimization procedure In this panel some details about the solution selected in the solutions list are shown Specifically the name and value Details of the maximized flux the desired flux the substrate flux the fitness function and the simulation method Knockouts The list of knocked out fluxes in the solution selected from the solutions list Launches Simulation Solution Viewer see 6 2 9 View Complete Info containing the complete information about the selected solution EN fitness desired flux maximized flux 0 0 0 53911 solutions details flux to maximize R_BiomassEcoli 0 53911 desired flux EX M succ 0 substrate EX M glc D e 6 fitness BPCY 0 simulation method knockouts view complete info Optimization Solutions J OptFlux Metabolic Engineering Workbench 6 2 8 REACTIONS VIEWER FLUX BOUNDS Name Reactions Viewer Tyne Viewer Editor This Viewer is also an Editor It all
9. Universidade do Minho IBB CEB INSTITUTE FOR BIOTECHNOLOGY AND BIOENGINEERING CENTRE OF BIOLOGICAL ENGINEERING CCTC COMPUTER SCIENCE AND TECHNOLOGY CENTER SCHOOL OF ENGINEERING UNIVERSITY OF MINHO UsER MANUAL Document revision based on OptFlux v1 35 OptFlux Metabolic Engineering Workbench OPTFLUX METABOLIC ENGINEERING WORKBENCH Universidade do Minho IBB CEB INSTITUTE FOR BIOTECHNOLOGY AND BIOENGINEERING CENTRE OF BIOLOGICAL ENGINEERING CCTC COMPUTER SCIENCE AND TECHNOLOGY CENTER SCHOOL OF ENGINEERING UNIVERSITY OF MINHO O IBB CEB CCTC rights reserved OptFlux Metabolic Engineering Workbench TABLE OF CONTENTS TABLE OF CONTENTS 3 LICENSES 6 1 INTRODUCTION 7 2 BASIC CONCEPTS AND INTERACTION 1 2 4 1 8 2 1 Datatypes and 5 8 2 2 Plug in Based 8 2 3 User Interaction and the MVC 9 3 OPTFLUX GETTING STARTED visssssssssnvedsensasacsnsccnecssccassssoncsontesscnnesonaueretes 11 3 1 Creating projects and loading data 11 3 1 1 Using the New Project cator RR EUR 12 3 2 Model graph
10. e STEP3 OPrioN1 Representation The desired representation of the individuals to be used by the Evolutionary Algorithm Either Set Based Representation or Binary Based Representation population size The number of individuals that the EA population should have generations The number of generations that the EA should evolve corresponding to the number of iterations in any classic algorithm Knockouts The number of knockouts that the algorithm must perform OptFlux Metabolic Engineering Workbench Corresponds to the size of the individuals themselves variable size The user must select this to use a variable sized genome The algorithm will try to select the optimum size for the individuals use essential genes If a list of essential genes a CriticalBox instance exists in the project the user can use it The genes contained in that list will never be knocked out Select the flux to maximize in the LP MOMA problem LP MOMA ROOM Usually the biomass flux desired flux Select the flux to maximize in the evolutionary algorithm process Usually it corresponds to the product that the user wants to maximize the production of substrate Select the flux to use as the substrate e g carbon source etc simulation method Select the simulation method used Either LP or MOMA objective function Select the objective function to use by the EA Currently only BPCY is avail
11. Optimization Wizard Please set the optimization and simulation parameters Optimization Wizard Please set the optimization and simulation parameters EA Parameters SA Parameters representation Set Based Representation initial delta tinai population size 10 number of trials generations 1000 number of function evaluations 100 knockouts knockouts variable sze variable sze use essential genes use essential genes Simulation Parameters Simulation Parameters flux to maximize in FBA MOMA ROOM varowth s desired suce __ flux to masamize in FBA MOMA ROOM vgrowth ls desired aabut e substrate gle le substrate 4abutle simulation method we simulation method ob objective function Ca E Step 3 Define simulation and Step 3 Define simulation and optimization parameters for optimization parameters for the EA algorithm Optimization Wizard the SA algorithm Define environmental external conditions if necessary search external fluxes addto erv conditions Step 4 Define environmental J Usa Conditions anaerobic E co conditions or use previously created ones if necessary OptFlux Metabolic Engineering Workbench 4 OPERATIONS COMPLETE REFERENCE In this chapter a complete reference the operations of OptFlux is presented The structure will follow the structure of the menus 4 1 FILE Simulation Optim
12. Upper 0 0 0 0 remove from environmental conditions notes anaerobic E coli cancel create When the data object EnvironmentalConditions has been created the field Use Environmental Conditions appears as selectable in simulation and optimization processes and the user can choose between the available instances and use them in the simulation optimization This allows the observation of the behaviour of the organism when the original conditions are replaced by the chosen ones while keeping the original stoichiometric matrix model safely unmodified OptFlux Metabolic Engineering Workbench 3 6 SIMULATION The simulation menu operations allow the user to test the behaviour of both wild type and mutant strains under different conditions The simulation of mutants is possible by selecting the reactions that should be eliminated from the metabolic model from the complete list of reactions The environmental conditions can be defined by appropriately setting the limits of the fluxes as described in the previous section The result of the simulation will consist on the numerical values for the complete set of the fluxes of the metabolic model In this version the simulation methods included are Flux Balance Analysis FBA Minimization of Metabolic Adjustment MoMA and the Regulatory On Off Minimization ROOM In FBA a linear programming problem is solved where one flux typically representing biomass production is
13. process Usually it corresponds to the product that the user wants to maximize the production of substrate Select the flux to use as the substrate e g carbon source etc Simulation method Select the simulation method used Either LP or MOMA objective function Select the objective function to use by the EA Currently only BPCY is available 4 4 OPTIONS 4 4 1 PROJECT e PREFERENCES Edition of some options of the Project The only available option at the moment is the edition of the user experience and the proxy information An advanced user will have access to more options inside some operations Project Select the Project whose preferences will be edited User Level Select the user level experience and proficiency for the selected Project Either BEGINNER INTERMEDIATE ADVANCED 4 5 HELP 4 5 1 ABOUT Launches an information panel about the coordination and development team of OptFlux as well as institutions associated with this project OptFlux Metabolic Engineering Workbench 5 DATATYPES COMPLETE REFERENCE This chapter presents a complete reference about the Datatypes used in OptFlux listed alphabetically 5 1 AIBENCH TYPES OF DATATYPES In AlBench several Datatypes have been defined There are three major types that give the programmer the ability to define any other Datatype This type of Datatype is the simplest one It work
14. there are two variants of EAs namely a Set based Representation Evolutionary Algorithm SBREA and traditional binary based Genetic Algorithms GA 3 7 1 SOLUTIONS ENCODING When using the traditional binary based representation the solutions will have the size of the complete genome i e if the model that the user is using has n fluxes the solutions length will be also n Binary Solution Encoding index 1 2 3 4 5 6 1 1 n 1 In value 1 1 1 a 1 P The above image shows how a solution is encoded in the binary representation The solution has n indexes the same as the number of fluxes in the model For any given index the corresponding value tells the GA if that gene is prone to be knocked out value 0 or not value 1 On the other hand the set based representation allows for the definition of shorter solutions The solution will have as many indexes as the number of knockouts Set Based Solution Encoding index 1 2 4 5 161 12 21 123 232 234 345 471 567 value The image above shows a typical instance of a set based encoded solution The values corresponding to the indexes are the indexes of the fluxes in the original model that should be knocked out It is obvious that a solution of this type will have much smaller solutions allowing the execution of the EA to be less time and memory consuming The aim is to obtain m
15. 7 2 Flat Files for Other Comiponefhits e DRE 68 7 2 1 Critical Reactions File ui re Y o toa teretes 68 Vid TGS A E 68 73 1 Pur e SBME File S eine EU nine 68 7 3 2 CellDesign r File AREE 68 OptFlux Metabolic Engineering Workbench LICENSES For this user manual This work is licensed under the Creative Commons Attribution Share Alike 3 0 Unported License To view a copy of this license visit http creativecommons org licenses by sa 3 0 or send a letter to Creative Commons 171 Second Street Suite 300 San Francisco California 94105 USA For the OptFlux software Copyright 2009 IBB CEB Institute for Biotechnology and Bioengineering Centre of Biological Engineering CCTC Computer Science and Technology Center University of Minho This is free software you can redistribute it and or modify it under the terms of the GNU Public License as published by the Free Software Foundation either version 3 of the License or at your option any later version This code is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU Public License for more details You should receive a copy of the GNU Public License along with the code If not see http www gnu org licenses Created inside the SysBio Research
16. Engineering Workbench e The original metabolic model object that contains information on the reactions fluxes and metabolites involved including the stoichiometric matrix e A simplified metabolic model OPTIONAL generated when a simplification of the original model is requested The information contained is the same as in the original metabolic model plus information on the equivalent variables and zero values discovered during the simplification procedures graph representation of the model loaded from a SBML file that can be used to visualize the model or a part of it A list of critical reactions i e reactions that cannot be knocked out to maintain biomass production e A list of results from model simulations using FBA MoMA ROOM of wild type or mutant strains e A list of results from optimization algorithms objects of type Solution Set that hold sets of solutions of the previous type e Alistof variability analysis results using Flux Variability Analysis Lists of different environmental conditions that can be used in the simulation of the model 3 1 1 USING THE NEW PROJECT WIZARD The New Project Wizard is accessible both through the File Menu and the Toolbar Simulation X Optimization New Project Wizard New Project Wizard This will launch the New Project Wizard step 1 panel In OptFlux a metabolic model can be loaded from 3 difference sources This choice is made
17. ROOM objective function Select the objective function to use by the EA Currently only BPCY is available STEP4 OpTION1 OPTION2 Search Search for the desired flux external fluxes The list of possible fluxes Typically only the external fluxes detected automatically by OptFlux will be listed This can be however overridden using Project Preferences with User Level ADVANCED see 4 6 1 edit In this panel the user can edit the value of the flux selected in the above list and add it to the list of modified fluxes modified fluxes The list of fluxes that the user has modified This list will define the Environmental Conditions themselves Use the remove from environmental conditions button to remove an undesired flux from this list Use conditions Users may choose to use a previously created environmental condition 4 3 2 PREPROCESSING MODEL SIMPLIFICATION Two different options to simplify the model thus reducing the complexity Project The Project that contains the model to simplify Zero Value Must be selected in order to perform the zero values simplification Equivalent Fluxes Must be selected in order to perform the equivalent fluxes simplification to max The name of the flux to be maximized FIND CRITICAL REACTIONS This operation tries to automatically find the critical reactions in the model i e those that are mathemat
18. early in the wizard and affects the rest of its behaviour FROM LOCAL FLAT FILES Step 1 In the first step the user must input a valid project name anything different from white spaces or tabs No proxy information is necessary in this step Step 2 In the second step the user must select three files e The first contains the reactions names and their flux limits e The second contains the stoichiometric coefficients e The third contains the metabolite names optional OptFlux Metabolic Engineering Workbench New Project General Project Options Project Name 2003 Reed s Ecoli None HTTP O SOCKS Model Source Flat Files O SBML O BioModels gt Next cancel New Project Please select the files for the following fields Fluxes File home pmaia dEReyFluxesEcoli txt Stoichiometric Matrix J home pmaia data SparseMatrixEcoli txt Sparse Full Metabolites File Jhome pmaia data MetabolitesEcoli txt 4 Back gt Next cancel Step 3 In step 3 the first option concerns the indexing used in the stoichiometric matrix if the SPARSE option was selected The rest of the options are relative to each of the three files selected previously For each one the user should select the appropriate separator Step 4 In the fourth step OptFlux automatically tries to find the biomass growth associated flux s
19. effective when the user press this button and accept them in the posterior dialog Essential Genes Gene Name 1205 1405 C1415N 1605 C1615N C1815N CHORM CHORS CLPNE coat co2 e Editing Box Gene Name add mm remove selected genes 7 accept changes Critical Reactions OptFlux Metabolic Engineering Workbench 6 2 2 EQUIVALENT VARIABLES VIEWER Name Equivalent Variables Viewer Tyne Viewer yy ep oe Components Search AYT Search Search for the desired flux in the list Equivalences The list of equivalent variables Equivalences Equivalent Variables ACHBS 242 lt gt DHAD2 281 THRD 12 lt gt KARAZI 149 2 38 lt gt DURIPP 240 5 5 128 lt gt 51 48 lt gt NNDPR 224 lt gt 53 lt gt QULNS 185 169 lt gt MTRK 44 lt gt SPMS 157 R_DDPA 33 lt gt 0 5 112 R_GMHEPAT 94 lt gt R GMHEPPA 172 lt gt AGMHE 212 lt gt 57 244 lt gt GMHEPK 250 R_RBFSb 293 lt gt R_FMNAT 208 lt gt R_RBFK 228 R_UAGDP 51 lt GIPACT 254 30 lt gt DPR 217 lt gt PPCDC 82 lt gt 23 lt gt PNTK 259 lt gt 6 1 lt gt DHDPRy 93 lt gt 5 261 lt gt DHDPS 287
20. maximized In MoMA wild types follow the same strategy as with FBA while for mutants a quadratic problem is defined where the purpose is to minimize the differences of the fluxes to the wild type solution The implementation of ROOM was based on a LP relaxation of the original MILP formulation proposed by the original authors The interface allows the user to define the flux to be maximized in the linear programming problem typically the flux over the reaction representing biomass formation The simulation operations 77 create data objects of type 1 1 1 ower Bound er Bound Simulation Solution as x EDT ED ALAR 10000 0 result These objects have w 100000 5 52 A 10000 0 two viewers one allows 100000 hecki the values A checkin s oco obtained for all the fluxes in gt Bum 10000 0 the model after the 1 10000 0 ALCD19 10000 0 simulation The other allows tca Eins 1502519 10000 0 these values to 2222 150100 superimposed over the 19000 100000 model graph using the gt add selected flux to knockouts list BioVisualizer options Both Original Metabolic Model E Remove viewers can be selected by Jus different tabs Flux to max min vgrowth J minimize se env conditions Cancel OptFlux Metabolic Engineering Workbench 3 6 1 USING THE SIMULAT
21. to OptFlux OptFlux Metabolic Engineering Workbench Currently the insertion of new plug ins has to be done manually in a simple process 1 Download the plug in from the OptFlux website check for versions 2 Copy the downloaded archive to SOPTFLUX_HOME plugins_bin 3 Restart OptFlux 2 3 USER INTERACTION AND THE MVC Since OptFlux is an AlBench based application the user interaction is thought to be as simple as possible The Model View Controller MVC architectural pattern has been used in every step of development of A Bench as well as of OptFlux resulting in a great deal of decoupling between the operational data and the views As mentioned before in OptFlux a View is related to a given Datatype If there is no View associated with a Datatype a Default View is launched a bean inspector The Views will by default be launched on the right side of the work area The original layout of the components can be observed in the diagram bellow An area denoted as other tools is also available It is used to launch several different tools usually not directly related with the problems solved in OptFlux By default that area is turned off resulting in an expansion of the working area but it can be easily turned back on MENU TOOLBAR CLIPBOARD VISUALIZATION AREA STATUS BAR File Simulation Optimization Options d Simulation Wizard 4 Optimization Wizard T A 6 New Project Wiz
22. user should manually select it ostane LJ case sensitive Back Finish cancel 3 2 MODEL GRAPH VISUALIZATION The application is integrated with a complementary tool that allows model visualization Metabolic models are represented as graphs with different kinds of nodes e g genes enzymes metabolites An SBML file can be loaded option File Load SBML for Visualization to construct the graph if the model has not been created directly from a SBML file This will create a data object of the type ModelGraph OptFlux Metabolic Engineering Workbench File Simulation Optimization Visualization Options Help New Project Wizard dk Simulation Wizard p Optimization Wizard T Type NAD sfcA Q NADH pri 1 1 1 28 D Lactate Pyruvate Coenzyme tdcE 2 3 1 54 Formate L hycG hycF hycE hycD fdhF Acetyl phosphate ia 2 51 Sl SI Sl B SI S SI SI SI SI Sl SI SI Bite 51 Sl 51 Sl SI Bs s100 s95 Show Genes Show Proteins V Show Simple Molecules Show Complex Molecules Show Secondary Reactions Model Graph AlBench simple molecules E proteins 8 fons genes
23. 7 4 2 1 Simulation Wizard 38 8 2 2 Wild Type S IAM Ga ned ub RAM nd 39 4 2 3 Mutant nennen snas sisse sense nera sans sensns nan 39 2 2 4 Flux Variability aeo ton uc de he ug a EE Re Se ER pd E 40 4 2 5 Environmental 5 enne 40 4 3 ODUIMIZATION 41 4 3 1 Optimization WIZANG ric tasso aperi ble stes eget est 41 4 3 2 2 nennen se sata 43 4 3 3 EA OptimizatlOTi eon ep vt iE Rea EE ad vL eoe 44 2 3 4 SA OptimizatiOTn EU EXC HAY RC EXE CON ER 45 4 3 5 7 1 45 4A mU Ui m H 46 PROJ OCU E 46 ERU 46 D aU oin amd RSV UR Sol me 46 5 DATATYPES COMPLETE 64 47 5 1 AlBench types of Dataty
24. A m 568 E de EL 4 561 563 Et 570 s 556 EH Sh 571 ADP ec 572 an ADP AMP 874 957 Acetyl phosphate Show Genes Show Proteins V Show Simple Molecules Show Complex Molecules V Show Secondary Reactions Simulation Solution Graph Overlap OptFlux Metabolic Engineering Workbench 6 2 6 MODEL PROPERTIES VIEWER Name Model Properties Viewer Type Viewer Editor Description This Viewer is also an Editor It allows for the insertion of notes relative to the metabolic model Components The date when the Model was created Equals the creation Date of Creation date of the Project Fluxes Count The number of fluxes found in the Model Metabolites Count The number of metabolites found in the Model Created From The paths of the files from which the Model was created Annotations Optional annotations that the user can add to better describe the Model Save Saves the modifications if any that the user made in the notes area origi Date of Creation Fluxes Count Metabolites Count Sun Feb 08 22 08 12 WET 2009 1218 fluxes counted 904 metabolites counted SBML home pmaia data ecoli_old Ec_iJR904_GIcMM xml
25. D Name Publication ID Last Modified BIOMDOOO00000030 BIOMDOO00000033 BIOMDOO00000032 BIOMDOO00000023 0000000024 BIOMD0000000031 Markevich2004_MAPK_orderedMM2kinases z Fetch Info BIOMD0000000031 2008 08 21111 49 29 00 00 New Project Please validate the following ra celular Compartment Step 3 Extra_organism gt Next cancel step 3 4 external metabolites MtrpLe M 15dap asnLe M fum e M acac e nad internal metabolites hco3 c M eig3p c M phaccoa c M lald L c a lt lt add lt lt add all M p hthr Back Next Q Cancel OptFlux Metabolic Engineering Workbench The third step is relative to the extra cellular environment OptFlux will automatically try to find the extra cellular compartment and the respective metabolites This heuristic can however fail In that event the user must manually select and validate this information Step 4 In the fourth step OptFlux New Project automatically tries to find Please select the biomass flux the biomass growth associated flux since this information is essential for Selected Biomass Flux vgrowth ID glyc e both simulation and imi optimization procedures ee If the heuristic fails to detect TE the correct flux the
26. DITIONSSET Type List Description This Datatype holds the list of Environmental Conditions created so far Viewers NONE Not Viewable Contains Name Description Viewers List Each Environmental Conditions Environmental EnvironmentalCondit define limits value modifications to a Conditions Viewer ions set of chosen fluxes 5 2 4 EQUIVALENT VARIABLES EQUIVALENCESBOX Simple Description This Datatype holds the Equivalent Variables Those variables that are constrained in the LP problem to have the same value OptFlux Metabolic Engineering Workbench Viewers Equivalent Variables Viewer 5 2 5 MODEL GRAPH Type Simple Description This Datatype holds the graph information correspondent to the metabolic pathway loaded from a SBML model Viewers Model Graph Viewer 5 2 6 OPTIMIZATIONRESULTS Type List Description This Datatype holds the list of optimization procedures done so far Viewers NONE Not Viewable Contains Name Description Viewers List Each SolutionSetBox contains a Optimization SolutionSetBox series of solutions for one Solutions Viewer optimization procedure performed 5 2 7 PROJECT Type Complex Description This Datatype basically holds everything All the other Datatypes listed in this chapter are in one way or another i
27. Group http sysbio di uminho pt OptFlux Metabolic Engineering Workbench 1 INTRODUCTION The OptFlux application includes a number of tools to support in silico metabolic engineering The application allows the user to load a genome scale stoichiometric model of a given organism This will serve as the basis to simulate the wild type and mutants original strain with a set of selected gene deletions The simulation of these strains will be conducted using a number of approaches e g Flux Balance Analysis FBA Minimization of Metabolic Adjustment MoMA or Regulatory On Off Minimization ROOM that allow the set of fluxes in the organism s metabolism to be determined given a set of environmental constraints The software also includes a number of optimization methods to reach the best set of gene deletions given an objective function typically related with a given industrial goal This application is complemented by an independent visualization tool named BioVisualizer which allows the visualization of graphs representing metabolic networks These graphs have a number of distinct node types e g metabolites enzymes reactions and connections One of the major features of this tool is the ability to associate numerical values to the different types of nodes and edges in the graph The integration of the two applications allows the visualization of the metabolic network superimposed by the values of the fluxes of a given simul
28. ION WIZARD A wizard for simulation is also available in the software The user can access it both through the Simulation Menu or the Toolbar The general steps of the process are the following Simulation Wizard Simulation Wizard step 2 5 Please select the type of simulation to perform Please select the model to use in the simulation 2003 Reed s E coli Wild Type Simulation Mutant Simulation Original Metabolic Model is Step 1 Select Project and Step 2 Wild type or Mutant Model original simplified Mutant selected simulation selection Simulation Wizard Simulation Wizard select the flux to be optimized and the type of optimization Design the mutant strain selecting the fluxes to knockout Knockouts Wild type selected Select to optimize varowth Lower Bound Upper Bound Maximization ES 10000 0 Choose an objective P add selected flux to knockouts list gt Minimization Algonthm ROOM Back Next cancel 41 Step 4 Select flux to optimize and optimization goal Step 3 Mutant strain design Select reactions to knock out Simulation Wizard Define environmental external conditions if necessary search external fluxes modified fluxes Name Lower upper Step 5 Define environmental conditions or use previously created ones if necessary
29. M dhap c M c M adp c M pi c M 4r5au c M nh4 c M ppi c M M met L c M pyr c M succ c M thf c M nadp M h2o c M skm c M glu M g3p c M pyr c M ppi c M 2 0xM c M amp M adp omi m o RA Reactions OptFlux Metabolic Engineering Workbench 6 2 10 STEADY STATE EQUATIONS VIEWER STOICHIOMETRIC MODEL Name Steady State Equations Viewer Type Viewer Description This view presents the stoichiometric model in the form of steady state equations for the balanced metabolites Components Search Search Search for the desired flux or metabolite in the Steady State The complete list of balanced metabolites and respective Equations steady state equations present in the model search Metabolite Steady State Equations Steady State Equation hco3 M c M trpLc M dctp c M asnLc M dhf c M glu D c M fum c M 26dap M c M u3aga c M tdeACP c M ac c M arot5p M nad c MtyrLc M 4 M uacgam c R_PRFGS ACCOACr 0 R_PTAr R_ACKr 0 R_UAAGDS UGMDDS 0 0 034 x BiomassEcoli TRPAS2 20 0 0254 x R BiomassEcoli RNTR3 0 0 229 x BiomassEcoli ASNS2 0 R_TMDS DHFR GCALDD 0 UAAGDS a MPD _ 2
30. METABOLITES VIEWER Name Metabolites Viewer Tyne Viewer Components Search AYT Search Search for the desired metabolites in the list Metabolites The list of the metabolites themselves Abbreviation Metabolites Complete Name Compartment Name Compartment Location M hco3 c M actp c M ugmd c M trp L c M dctp c M asnLc M dhf c D c M fum c M 26dap M c M u3aga c M tdeACP c M ac c M orotsp c M nad c M tyrL c M c M uacgam c M q8h2 c M glu L c M g3p c M Bicarbonate CHO3 Cytosol INTERNAL M Acetyl phosphate C2H30 Cytosol M UDP N acetylmuramoyl L Cytosol NTERNAL M L Tryptophan C11H12N202 Cytosol TERNAL M_dCTP_C9H12N3013P3 Cytosol TERNAL M_L_Asparagine_C4H8N203 Cytosol TERNAL M 7 8 Dihydrofolate 19 Cytosol TERNAL M D Glutamate C5H8NO4 Cytosol TERNAL M Fumarate C4H204 Cytosol TERNAL M 2 6 Diaminohepta Cytosol TERNAL M UDP 3 O 3 hydroxytetra Cytosol NTERNAL cis tetradec 7 enoyl Cytosol TERNAL M Acetate C2H302 Cytosol TERNAL M_Orotidine_5 phosphate Cytosol TERNAL M Nicotinamide adenine di Cytosol NTERNAL M_L Tyrosine C9H11N03 Cytosol ITERNAL M D Enythrose 4 phosphat Cytosol TERNAL M UDP N acetyl D Cytosol TERNAL M Ubiquinol 8 C49H7604 Cytosol NTERNAL M L Glutamate C5H8NO4 Cytosol TERNAL M Glyceraldehyde 3 phosp Cytosol TERNAL Meta
31. ONS Loads a list of critical reactions from file The loaded reaction must be contained in the model otherwise an Exception will be thrown Project The Project to where the list of essential genes will be loaded BIN or ASCII The type of file where the list is saved in Either BIN binary file or ASCII text file File The location of the file itself ASCII The file format for the critical reactions file can be found in 7 2 1 BIN This file does not have a human readable format It can be generated inside OptFlux using the Critical Reactions Viewer see 6 2 1 The user can afterwards save the list in a Binary format to ease the loading parsing times 4 1 3 SAVE SAVE PROJECT As Saves a Project to file The file will be saved in a binary format thus not human readable A XML representation is being studied for the next version Project The Project that to be saved File The file to where the project will be saved OptFlux Metabolic Engineering Workbench 4 1 4 EXPORT EXPORT FLUXES Export the list of fluxes to a text file FBAFluxes The FBAFluxes instance to be exported to text file File The file to where the fluxes will be saved EXPORT MATRIX Export the matrix to a text file The matrix will be saved in a SPARSE format either it was loaded as SPARSE or FULL matrix Matrix The FBASparseMatrix instance to export to text file F
32. R DKMPPD 74 lt gt R UNK3 B1 lt gt R MDRPD 265 89 lt gt ATPPRT 205 lt gt HISTP 238 lt gt PRATPP 142 lt gt IGPDH 186 gt 269 107 lt gt KDOCT2 30 lt gt KDOPS 104 280 gt R MCOATA 291 PGL 155 lt gt GND 268 138 lt gt ACLS 223 lt gt 1 282 96 lt gt ICDHyr 188 55 EC 173 lt gt TDSK 57 lt gt 2 20 lt gt 51 19 lt gt R USHD 69 lt gt 2 56 R_SHSL1 18 lt gt HSST 137 lt gt METS 190 lt gt R CYSTL 207 122 lt gt HYD1 218 279 lt gt 1 95 lt gt R IPMD 136 gt gt Equivalent Variables J OptFlux Metabolic Engineering Workbench 6 2 3 ENVIRONMENTAL CONDITIONS VIEWER Name Reactions Viewer Tyne Viewer Description ie bec bounds for a given Environmental Components Search AYT Search Search for the desired flux in the list Fluxes The list of the fluxes themselves search Environmental Conditions Flux Lower Bound Upper Bound Type EX 02 0 0 0 0 EXTERNAL Environmental Conditions J OptFlux Metabolic Engineering Workbench 6 2 4
33. The difference in energy that corresponds to an acceptance probability of 5096 of worse solutions at the beginning of the run AE final delta The difference in energy that corresponds to an acceptance OptFlux Metabolic Engineering Workbench probability of 50 of worse solutions at the end of the run e trials The number of iterations per temperature NFEs The number of function evaluations Using these parameters the initial temperature the final temperature and the scale parameter are computed by OptFlux using the following equations _ log 0 5 AE log0 5 log T log To trials a exp The advantage of using and AE is that it allows the user who knows the fitness landscape of the optimization problem to automatically define the temperatures by reasoning over the values of the objective function Supplying the number of function evaluations instead of the scale parameter allows the user to accurately define the number of function evaluations the optimization algorithm will use enabling a simpler comparison with other approaches OptFlux Metabolic Engineering Workbench 3 9 OPTIMIZATION WITH LOCAL SEARCH Local search algorithms are used OptFlux to improve a particular solution Therefore this operation can be applied to any existing solution wild type or mutant Basically the local search algorithm tries to add a gene deletio
34. able OPTION2 initial delta The initial state of energy for the annealing process see 3 8 final delta The final state of energy for the annealing process see 3 8 number of trials The number of trials to execute at each temperature see 3 8 number of function An approximate maximum number of function evaluations evaluations Allows an easier comparison with other methods see 3 8 knockouts The number of knockouts that the algorithm must perform Corresponds to the size of the individuals themselves variable size The user must select this to use a variable sized genome The algorithm will try to select the optimum size for the individuals use essential genes If a list of essential genes a CriticalBox instance exists in the project the user can use it The genes contained in that list will never be knocked out Select the flux to maximize in the LP MOMA problem LP MOMA ROOM Usually the biomass flux desired flux Select the flux to maximize in the evolutionary algorithm process Usually it corresponds to the product that the user wants to maximize the production of substrate Select the flux to use as the substrate e g carbon source etc If the variable size option is selected this value is only used as the initial size of the individuals OptFlux Metabolic Engineering Workbench simulation method Select the simulation method used FBA MOMA or
35. algorithm are the initial and final temperatures the SA Parameters number of iterations me initial delta performed at each final delta temperature and the total number of trials 50 number of function number of function evaluations 50000 evaluations knockouts 12 variable size L The choice of these use essential genes parameters is of paramount Simulation Parameters importance to the pe rformance of the flux to maximize in FBA MOMA ROOM vgrowth algorithm If the initial substrate glc D e temperature is too low or the cooling schedule is not slow enough the optimization process may become stuck in local optimum On the other hand if the initial temperature is too high the cooling is too slow or the number of iterations per temperature is too high the algorithm wastes a potentially large amount of computational time while searching for solutions simulation method objective function BPCY The cooling schedule used by OptFlux is among the most popular ones where the temperature decreases exponentially defined according to the following equation To ensure that the cooling schedule is sufficiently slow the parameter should be given values close to the unity As the choice of initial and final temperatures is problem dependent it was decided to use as configuration parameters the following AEo initial delta
36. ard f Clipboard enu Opt ux v Project 2003 Reed s coli Original Model Flux Bounds EH Metabolites E stoichiometric Model Y Simulation Results Wild Type FUM Mutant Optimization Results Environmental Conditions anaerobic E coli Variability Analysis Results V Pyruvate Metabolism Clipboard e NAD sfcA 111 28 Lactate 9 tdcE NADH i Pyruvate 2 3 1 54 Formate PEL E 6 2 1 1 e Coenzyme A Acetyl phosphate Acetate Name Type OptFlux Metabolic Engineering Workbench A screenshot of OptFlux is shown bellow to give an idea of the default layout of the system Looking closely it can be seen that once a data object is selected in the Clipboard the corresponding Viewer is launched on the right side Pyr SIM 0 NAD SM SIM H SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM SIM GENE GENE GENE erur s56 s64 s67 s98 s99 s76 s78 s101 s100 s95 ane sa60 sa62 sa63 5 64 sa65 sa67 sa69 sa68 sa73 5 71 5 72 5 77 5 78 5 75 5 76 5 79 5 87 5 82 5 84 5 85 5 59 5 57 5 58 70 74 sal sal 86 88 sal sa
37. ation a feature well integrated in the OptFlux application The values of the fluxes for the wild type and different mutants can therefore be adequately visualized in this way providing useful analysis tools for the researchers Additionally this software is compatible with several SBML Systems Biology Markup Language standards allowing the utilization of models stored in public databases e g like BioModels or built in other software tools e g like Cell Designer both for simulation and visualization The OptFlux application is being developed taking as a basis the A Bench framework This is an environment for the development of Data Mining Bioinformatics tools using the Java programming language The details of this project a collaboration between the universities of Minho Portugal and Vigo Spain as well as updated documentation can be found at the web site http www aibench org This document briefly explains the functionalities of the OptFlux application and the way it can presently be used This is however still a preliminary version of the documentation Therefore it should serve as the basis to understand the potential of the approach and be faced as a basis for future development OptFlux Metabolic Engineering Workbench 2 BASIC CONCEPTS AND INTERACTION 2 1 DATATYPES AND OPERATIONS Every application built based on A Bench is organized around the concepts of datatypes and operations define
38. bolites OptFlux Metabolic Engineering Workbench 6 2 5 MODEL GRAPH VIEWER GRAPH OVERLAP Name Model Graph Viewer Type Viewer This Viewer provides graphical visualization of the previously loaded model in the form of a graph Description The Graph Overlap Viewer is exactly the same as the Model Graph Viewer but it only appears in the visualization of a SimulationSolution It provides an overlap of the simulation values and the Model Graph Components I Zooms in MOUSE WHEEL DOWN has the same effect 1 1 Zooms out MOUSE WHEEL UP has the same effect Show Genes Enable Disable the visualization of genes in the graph Show Proteins Enable Disable the visualization of proteins in the graph Show Simple Enable Disable the visualization of simple molecules in the Molecules graph Show Complex Enable Disable the visualization of complex molecules in the Molecules graph Show Secondary Enable Disable the visualization of the secondary reactions Reactions in the graph Table A list of all the components shown in the pathway Sp Id con 305 558 58 560 Or 0 has 862 ane 563 5 n 565 pria PFL 556 sa 2 3 1 54 9 lioc us m NADH adhe s 866 2 3 569 S 12 1 10 Co
39. ction are all properly identified Components Search Search Search for the desired flux or metabolite in the Reactions The complete list of reactions present in the model a search Name Reactions Reactants Direct Products IMPD R_PGSA_EC R_ASPCT R GLUDy R PPND R DTMPK R DRPA UAMAS GALLI R DHAPT R PGMT R DHPPDA2 R_SHSL1 R_SHK3Dr IGPS ADKI ADNKI R ADK3 M imp c M c M nad c M gln L c M atp c M imp 2 0 02 x M cdpdag EC c M alyc3p c M asp L M cbp M glu L c M M 20 c M nad c M pphn c 2 0xM atp c M dtmp c M 2dr5p c M uamr c M ala L M atp c M mlthf c M nadp c M h2o Mutpc M glpe Mhe M pep c M c c c nadph 5 0x M 20 M succoa M cys L c M hom L c M 5m M M nadph M prpp c c M chor M amp c M adn c M atp c M amp c fprica c M glu L c M nadh c M gmp M ppi c 0 02xM EC cmpce Mhec M pi c M_cbasp_c Mhc M c 4 c M nadphce M nadh M co2 c M 34hpp M 2 0 c M g3p M acald c M pi c M uama M M lOfthf M nadph c Mhc M M ppi M pyr c
40. ctions Steady State Equations Zero Values Contains the list of variables that are Zero Variables constrained to be 0 by the LP Viewer ZeroBox problem i Contains the list of variables that are Equivalent tea irai constrained by the LP problem to Variables Viewer have the same value OptFlux Metabolic Engineering Workbench 5 2 9 SIMULATION RESULTS Type List Description This Datatype holds the list of simulations performed so far Viewers NONE Not Viewable Contains Name Description Viewers List Each SimulationSolution represents SimulationSolution SimulationSolution a the outcome of a solved LP Viewer problem 5 2 10 VARIABILITY ANALYSIS RESULTS Tyne List Description This Datatype holds the list of variability analysis performed so far Viewers NONE Not Viewable Contains Name Description Viewers List Each FVASimulationResult represents Variability Analysis FVASimulationResult the outcome of a variability analysis Viewer 5 2 11 ZERO FLUXES 7 Simple Description This Datatype contains the list of variables that are constrained to be 0 by the LP problem Viewers Zero Variables Viewer OptFlux Metabolic Engineering Workbench 6 Viewers EDITORS COMPLETE REFERENCE In this chapter the Viewers corresponding to the Dataty
41. d as follows DATATYPES define the types of data that are of interest to a given application For each data object one or more visualizers can be defined to show its content to the user in a given perspective Data objects can have a hierarchy where a given object A contains objects B and C these objects are called compound objects The set of data objects and their hierarchy in a given application are shown in a tree the clipboard typically appearing in the left side of the screen In this tree compound objects can be opened to show their contents a list of other data objects When a given data object is selected double clicked the available viewers if any are launched in the right area of the screen OPERATIONS each operation defines a function that takes zero or more data objects as its inputs and can create as an output zero or more data objects and or merely change the input data objects Operations can be accessed through the menu options being typically grouped in several menus and sub menus Operations can also be run from the clipboard by right clicking context menus a data object this will show the list of all available operations using that data object as an input 2 2 PLUG IN BASED FRAMEWORK AlBench uses Platonos http platonos sourceforge net a powerful plug in engine This gives AlBench s built applications such as OptFlux the power to be easily upgraded or extended e Apply new plug ins
42. ded External Metabolites internal Metabolites The list of detected or manually assigned The list of internal metabolites Users can add external metabolites This information is or remove metabolites to the external essential for simulation purposes Sink fluxes metabolites list if OptFlux heuristics failed to will be created if non existent correctly detect them STEP4 5 1 SOURCE2 AND SOURCE 3 Displays the currently selected flux that will be assumed by OptFlux as Selected Biomass flux the biomass growth associated flux The list of fluxes present in the model The biomass growth associated NAME flux should be selected by default by OptFlux if not the user must manually select it Field used to perform a quick search for the desired flux Must only be Search used if OptFlux heuristics fail to correctly detect the biomass growth associated flux 4 1 2 LOAD LOAD PROJECT Recovers a Project from a previously saved file File The location of the file where the project was saved to LOAD SBML FOR VISUALIZATION Loads a SBML model with a graph representation to be visualized OptFlux Metabolic Engineering Workbench Project The Project to where the SBML model will be loaded Model Type The type of SBML model that will be loaded Either SBML or CSBML File The file where the SBML model is located LOAD CRITICAL REACTI
43. duction of substrate Select the flux to use as the substrate e g carbon source etc simulation method Select the simulation method used Either FBA MOMA ROOM objective function Select the objective function to use by the EA Currently only BPCY is available use env conditions The Environmental Conditions if any to use 4 Tf the variable size option is selected this value is only used as the initial size of the individuals OptFlux Metabolic Engineering Workbench 4 3 4 SA OPTIMIZATION Optimization through Simulated Annealing Project Select the Project containing the desired Model initial delta The initial state of energy for the annealing process see 3 8 final delta The final state of energy for the annealing process see 3 8 number of trials The number of trials to execute at each temperature see 3 8 number of function approximate maximum number of function evaluations evaluations Allows an easier comparison with other methods see 3 8 knockouts The number of knockouts that the algorithm must perform Corresponds to the size of the individuals themselves variable size The user must select this to use a variable sized genome The algorithm will try to select the optimum size for the individuals use essential genes If a list of essential genes a CriticalBox instance exists in the project the user can use it The genes contain
44. e constrained by the linear problem have a value equal to zero are presented Components Search AYT Search Search for the desired flux in the list lero Valued Variables The list of variables constrained to have value equal to zero Flux Name Zero Valued Variables R SUCBZS 1 R_DCYTt2 R SUCBZL UACMAMO UDPGALM GPDDA2 R DHCINDO R_SOTA R IDOND2 GLUTRR R_HCINNMt2r R_EX_gly_e_ ICHORSi R PACCOAL R_XYLIL R PTRCabc CYTDt2r EX e R EDTXS4 R TAUDO D mew zero Variables OptFlux Metabolic Engineering Workbench 7 FILE FORMATS This chapter explains the format of the files supported by OptFlux 7 1 FLAT FILES FOR MODELS 7 1 1 FLUX BOUNDS FILE For n reactions R there must be n lines in the file one per each flux There are four fields in each line separated by commas tabs or any other separator that the user decides First comes the reaction name then the reaction reversibility either for Irreversible or R for Reversible OPTIONAL then the flux lower limit and finally the flux upper limit reversibility R1 jower_bound R1upper_bound R2 eversibility R2iower bound RNyeversibility Rhiower bound bound 7 1 2 SPARSE MATRIX FILE Each line defines the presence of given metabolite M in a reaction R and the correspondent
45. e type of SBML and the container file and the type of SBML therein contained either Pure SBML CellDesigner SBML SBML Normal CellDesigner org SBML files level 2 version 3 supported e CellDesigner SBML CellDesigner s generated files containing layout and visual information 2 Back Next cancel version 4 0 If this option is selected OptFlux will try to generate a visualization graph representing the model Large models genome scale are very hard to render and to prevent that OptFlux will not create the graph if a model with 200 metabolites is loaded SBMLFile data ecoli old Ec 18904 GlcMM xml Step 3 The third step is relative to the extra cellular environment OptFlux will automatically try to find the extra cellular compartment and the respective metabolites This heuristic can however fail In that event the user must manually select and validate this information Step 4 In the fourth step OptFlux automatically tries to find the biomass growth associated flux since this information is essential for both simulation and optimization procedures If the heuristic fails to detect the correct flux the user should manually select it OptFlux Metabolic Engineering Workbench New Project Please validate the following ra celular compartment Extra organism internal metabolites A M hco3 c lt lt add act
46. e values for all the fluxes of the model after the Flux Values simulation procedure Search AYT Search Search for the desired flux in the list Export Values Exports the above list to a text file E maximized flux R_BiomassEcoli 0 53911 Flux Values lt m Flux EX M 12ppd 5 EX M 15dap EX M 26dap M e EX M 2ddglcn EX M 3hcinnm e EX M 3hpppn EX M 4abut EX M EX M acac e EX M acald EX M acgam EX M acmana EX M acnam EX M ade EX M adn e EX M akg e EX M ala D e simulation method FBA knockouts search show only non zero 0 values export all values Simulation Solution Graph Overlap OptFlux Metabolic Engineering Workbench 6 2 12 VARIABILITY ANALYSIS VIEWER Name Variability Analysis Viewer Type Viewer This viewer shows a chart that represents the variability Description analysis process usually of a given flux in function of the biomass growth associated flux Components Export to JPEG Allows writing the chart to an external JPEG image Flux Variability Analysis 0 5 0 6 vgrowth Concentration Export to JPEG Chart 6 2 13 ZERO VARIABLES VIEWER OptFlux Metabolic Engineering Workbench Name Zero Variables Viewer Tyne Viewer Description In this Viewer a list of fluxes that ar
47. ed in that list will never be knocked out flux to maximize Select the flux to maximize the LP MOMA problem FBA MOMA ROOM Usually the biomass flux desired flux Select the flux to maximize in the evolutionary algorithm process Usually it corresponds to the product that the user wants to maximize the production of substrate Select the flux to use as the substrate e g carbon source etc simulation method Select the simulation method use FBA MOMA or ROOM objective function Select the objective function to use by the EA Currently only BPCY is available 4 3 5 LOCAL OPTIMIZATION Local optimization of a previously achieved solution BestNeighbour and HillClimbing techniques are available Solution Select the solution to improve 5 If the variable size option is selected this value is only used as the initial size of the individuals OptFlux Metabolic Engineering Workbench algorithm Select the algorithm to use in the improvement process Either BestNeighbour or HillClimbing see 3 9 use essential genes If a list of critical reactions a CriticalBox instance exists in the project the user can use it The genes contained in that list will never be knocked out to maximize Select the flux to maximize in the LP MOMA problem LP MOMA ROOM Usually the biomass flux desired flux Select the flux to maximize in the evolutionary algorithm
48. elect the Project that contains the desired Model Search AYT Search Search for the desired flux external fluxes The list of possible fluxes Typically only the external fluxes detected automatically by OptFlux will be listed This can be however overridden using Project Preferences with User Level gt ADVANCED see 4 6 1 etit In this panel the user can edit the value of the flux selected in the above list and add it to the list of modified fluxes modified fluxes The list of fluxes that the user has modified This list will define the Environmental Conditions themselves Use the remove from environmental conditions button to remove an undesired flux from this list notes Additional and fully optional comments that the user wishes to add to this Env Conditions e g Aerobic Anaerobic etc OptFlux Metabolic Engineering Workbench 4 3 OPTIMIZATION visualization Of Optimization Wizard Preprocessing EA Optimization SA Optimization Local Optimization 4 3 1 OPTIMIZATION WIZARD STEP1 Project Select the Project where the model to be user is contained Model Select the model to use in the simulation usually the original or the simplified metabolic model STEP2 Choose optimization Select the Optimization Algorithm to use either Evolutionary algorithm Algorithm Option1 or Simulated Annealing Option2
49. ess the SBML specifications for SBML L2V3 please refer to http www sbml org specifications sbml level 2 version 3 release 2 sbml level 2 version 3 rel 2 pdf 7 3 2 CELLDESIGNER SBML FiLES CellDesigner SBML specifications are not available to the public The users must contact the authors in order to access them www celldesigner org
50. f near Therefore the corresponding reactions cannot be removed from the metabolism in order to keep the organism biologically viable Project 2003 Reed s E coli biomass flux vgrowth cancel The list of genes that is created in this option can be used in the optimization OptFlux Metabolic Engineering Workbench algorithms in order to limit the search space This list can be manually edited using the visualization of the corresponding data object Essential Genes 3 5 USING ENVIRONMENTAL CONDITIONS The Environmental Conditions are a mechanism developed to ensure that a researcher has the possibility to change some conditions of a problem leaving the stoichiometric matrix of the original model untouched This means that the user can change the limits of certain fluxes creating a data object EnvironmentalConditions that can later be used either in simulations or optimizations These fluxes are by default limited to the external fluxes the ones that represent the environmental conditions themselves availability of carbon source and oxygen for example This restriction can be however overcome to grant the possibility to change the limits of internal fluxes as well see 4 10 data 2003 Reed s coli search 2 external fluxes Name lac D e r edit v to env conditions modified fluxes lower upper o2 e 0 0 0 0 Lower
51. ically prone to be found OptFlux Metabolic Engineering Workbench Project The Project that contains the data to max The name of the flux to be maximized 4 3 3 EA OPTIMIZATION Optimization through Evolutionary Algorithms Project Select the Project containing the desired Model representation The desired representation of the individuals to be used by the Evolutionary Algorithm Either Set Based Representation or Binary Based Representation population size The number of individuals that the EA population should have generations The number of generations that the EA should evolve corresponding to the number of iterations in any classic algorithm Knockouts The number of knockouts that the algorithm must perform Corresponds to the size of the individuals themselves variable size The user must select this to use a variable sized genome The algorithm will try to select the optimum size for the individuals use essential genes If a list of essential genes a CriticalBox instance exists in the project the user can use it The genes contained in that list will never be knocked out to maximize Select the flux to maximize the FBA MOMA ROOM FBA MOMA ROOM problem Usually the biomass flux desired flux Select the flux to maximize in the evolutionary algorithm process Usually it corresponds to the product that the user wants to maximize the pro
52. ile The file to where the matrix will be saved EXPORT METABOLITES Export the list of metabolites to a text file Metabolites The FBAMetabolites instance to be exported to text file File The file to where the metabolites list will be saved EXPORT ENVIRONMENTAL CONDITIONS Export the environmental conditions to a text file Env Conditions EnvironmentalConditions instance to be exported to text file File The file to where the Environmental Conditions will be saved EXPORT COMPLETE MODEL Exports the complete model to text files A prefix for the files must be provided as well as a directory to contain them OptFlux Metabolic Engineering Workbench Model The DataBox instance that to be saved to text files NOTE three separate files will be created concerning the fluxes the matrix and the metabolites Path The location where the files should be created Note A folder must be selected not a file Prefix The prefix that the files will have The complete names will be PREFIXFluxes txt PREFIXMatrix txt PREFIXMetabolites txt EXPORT CRITICAL REACTIONS Export the list of critical reactions to a text file CriticalBox The CriticalBox instance to be exported to text file File The file to where the critical reactions list will be saved 4 1 5 QUIT Quits OptFlux asking for confirmation and saving Project Select the Project s tha
53. ince this information is essential for both simulation and optimization procedures If the heuristic fails to detect the correct flux the user should manually select it OptFlux Metabolic Engineering Workbench New Project Select the file options please Indexing starts at zero 0 V one 1 Fluxes File Separator tab w space _ user defined Stoichiometric Matrix File Separator comma tab w space _ user defined Metabolites File Separator comma tab w space user defined 4 Back gt Next cancel New Project Please select the biomass flux Selected Biomass Flux vgrowth glyc e lac D e lacte mal4te 2 pyrte succ e Search vgrowth _ case sensitive 9 Back gt Finish o Cancel OptFlux Metabolic Engineering Workbench FROM A LOCAL SBML MODEL Step 1 In the first step the user must input a valid project name anything different from white spaces or tabs No proxy information is EE necessary in this step The user must select the SBML option in the bottom New Project General Project Options Project Name 2003 Reed s Ecoli HTTP 2 SOCKS Model Source Flat Files SBML 2 BioModels gt Next cancel Step 2 In the second step the user New Project must select the file to load Please select th
54. ization LL New Project Wizard 4 1 1 NEw PROJECT WIZARD Provides a step by step wizard to create a new Project in the Clipboard This is always the first operation to be executed OptFlux otherwise no other will work e STEP1 Project Name The name of the project Name validation will be performed The type of proxy to bypass if necessary Either NONE HTTP or SOCKS Proxy Host proxy host if HTTP or SOCKS is selected proxy port if HTTP or SOCKS is selected Model Source The source of the model to be loaded Either Flat Files Source1 SBML Source2 or BioModels Source3 This option will affect the behavior of the wizard for the following steps and therefore the following steps will be described with Source1 Source2 Source3 depending on the context they apply to OptFlux Metabolic Engineering Workbench e STEP2 SouRcE1 The file containing the flux bounds for the reactions For supported Fluxes File formats refer to 7 1 1 The file containing the matrix with the stoichiometric coefficients SPARSE Select this option if the loaded matrix is in sparse format Stoichiometric Matrix Refer to 7 1 2 for description of the sparse format FULL Select this option if the loaded matrix is in its full format Refer to 7 1 3 for description of the full format Metabolites File The file containing the metabolite
55. l sal aal V Show Secondary Reactions Status Bar 4 Ic G mes ation Area OptFlux Metabolic Engineering Workbench 3 GETTING STARTED This chapter will focus on the main tasks that can be performed with OptFlux The main techniques that OptFlux uses will also be briefly explained A full list of operations datatypes viewers and other details will be presented in chapters 4 5 and 6 3 1 CREATING PROJECTS AND LOADING DATA The Project is the main data object in OptFlux It is a compound object used to hold other objects such as a metabolic model its visualization graph and the results of the pre processing simulation and optimization operations conducted over this model In this version the full content of a project in this case the Escherichia coli project is the following ey Project col 2003 Reed s E coli 45 original Model Simulation Optimization gt File WildType Simulation Mutant Simulation Flux Variability Analysis FS Flux Bound rename element Simulation Results Optimization Results i Environmental Conditions Variability Analysis Results Pyruvate Metabolism E OptFlux Metabolic
56. n to an existing solution trying to improve the objective function All possible genes are attempted and the best solution is returned data amp method solution Wild Type iv algorithm Complete HillClimbing use essential genes simulation parameters flux to maximize in FBA MoMA ROOM growth desired flux substrate glc DCe simulation method ROOM objective function BPCY cancel This cycle be executed once Best Neighbour repeated while it provides an improved solution over the starting point HillClimbing If an improved solution is found the best solution is added to the clipboard OptFlux Metabolic Engineering Workbench 3 10 USING THE OPTIMIZATION WIZARD A wizard for optimization is also available in the software The user can access it both through the Simulation Menu or the Toolbar The general steps of the process are the following Optimization Wizard Optimization Wizard Please select the model to use in the optimization Please select the optimization algorithm to use Project 2003 Reed s E Evolutionary Algorithm U Simulated Annealing Simplified Metabolic Model O canca Step 1 Select Project and Model original simplified Step 2 Select EA s or SA s as optimization algorithm SA s selected
57. nside a Project This is the root Datatype Viewers NONE Not Viewable Contains Name Description Viewers Metabolic Model Holds the metabolic model itself Metabolic Model DataBox Viewer OptFlux Metabolic Engineering Workbench Simplified Metabolic The copy of the original metabolic Metabolic Model Model model after some simplifications Viewer SimplifiedDataBox Simulation Results The list of all the simulations NONE SimulationResults performed so far Optimization Results The list of all the optimization NONE OptimizationResults procedures performed so far Environmental The list of environmental conditions NONE Conditions List created so far EnvironmentalCondit ionsSet This Datatype holds the graph Model Graph Model Graph information correspondent to the Viewer ModelBox metabolic pathway loaded from a SBML model 5 2 8 SIMPLIFIED METABOLIC MODEL SiMPLIFIEDDATABOX Type Complex Description This Datatype holds the Metabolic Model itself Viewers Metabolic Model Viewer Contains Name Description Viewers Reactions Contains the flux names and limits Reactions Viewer FBAFluxes Metabolites Contains the metabolites Metabolites Viewer FBAMetabolites information abbreviation name compartment name and compartment location Stoichiometric Model FBASparseMatrix Contains all the stoichiometric coefficients Rea
58. odified microbial strains with better production capabilities that OptFlux Metabolic Engineering Workbench are evaluated by their fitness function For each potential solution a given mutant strain this function is calculated by performing a simulation using the FBA MOMA or ROOM approaches as described above In this version the BPCY Biomass Product Coupled Yield is used as a fitness function BPCY is an objective function that allows simultaneous maximization of both product and biomass and that can be associated with the productivity of a given bioprocess Project The interface allows the user to define the fluxes to be maximized both by the FBA MOMA or ROOM typically the flux leading to 2003 Reed s E coli EA Parameters representation Set Based Representation biomass and the age Fo population size 100 optimization process the flux mo leading to the target product used E R the calculation of the BPCY e g succinate or ethanol as well as use essential genes the substrate uptake flux corresponding to the main carbon source whose value is Simulation Parameters flux to maximize in FBA MOMA ROOM vgrowth desired flux succte i needed for the calculation of the substrate glc D e fitness function BPCY FBA objective function BPCY The user can also define a few EA use env conditions
59. other separator The first position contains the metabolite abbreviation if the file exists this is the only one that is necessary the second one contains the complete name of the metabolite The third and fourth positions contain the compartment name and location respectively abbreviation 1 1 comptocation 2 2 2 2 abbreviation M3name compName M3 comptocation MNabbreviation MN OptFlux Metabolic Engineering Workbench 7 2 FLAT FILES FOR OTHER COMPONENTS 7 2 1 CRITICAL REACTIONS FILE OptFlux allows users to load the critical reactions information from a previously saved or manually created file The format of this file is the following each line in the file must have the name one reaction that is critical that is for m reactions there will be m lines in the file When manually editing these files special attention must be taken since all the names in this file must be present in the flux bound files otherwise OptFlux will ignore them 1 R7 name 7 3 SBML FILES 7 3 1 PURE SBML FILES OptFlux uses libSBML and therefore can support any SBML file that libSBML supports In the current version SBML level 2 version 3 is the latest supported though it handles any prior version To acc
60. ows the selection of the Description type of the flux INTERNAL gt EXTERNAL EXTERNAL gt INTERNAL Components Search AYT Search Search for the desired flux in the list Fluxes The list of the fluxes themselves Fluxes Reaction Name Lower Bound Upper Bound Type R_IMPC 999999 0 999999 0 INTERNAL R IMPD 0 0 999995 0 INTERMAL R_PGSA_EC 999999 0 999999 0 INTERNAL R_ASPCT 0 0 999999 0 INTERNAL R GLUDy 999999 0 999999 0 INTERNAL R_PPND 0 0 999999 0 INTERNAL 999999 0 999999 0 INTERNAL R_DRPA 0 0 999999 0 INTERNAL R_UAMAS 0 0 999995 0 INTERMAL 999999 0 999999 0 INTERNAL R 0 0 999999 0 INTERNAL R DHAPT 0 0 999999 0 INTERNAL R_PGMT 999999 0 999999 0 INTERNAL 999999 0 999999 0 INTERNAL R_DHPPDA2 0 0 999999 0 INTERNAL R SHSL1 0 0 999999 0 INTERNAL R_SHK3Dr 599999 0 999999 0 INTERNAL IGPS 0 0 999999 0 INTERNAL 999999 0 999999 0 INTERNAL R_ADNK1 0 0 999999 0 INTERNAL R_ADK3 999999 0 999999 0 INTERNAL 7A INTERNAL Reactions En OptFlux Metabolic Engineering Workbench 6 2 9 REACTIONS VIEWER STOICHIOMETRIC MODEL Name Reactions Viewer Type Viewer In here a full view of the Stoichiometric Matrix is presented The Metabolites and its coefficients Description presented in the form of equations for each different flux The Reactants Products and the direction of the rea
61. p M eig3p c lt lt add all M phaccoa c M lald L c rem gt gt c M_2ahbut_c M phthr c mat 1 4 Back ED Next o Cancel external metabolites MtrpLe M 15dap MasnLe M fum e M acac e M 26dap M e MtyrLe New Project Please select the biomass flux Selected Biomass Flux vgrowth ID glyc e lac D e lacte mal4te o2 e pyrte succ e Search vgrowth case sensitive 4 Back Finish cancel FROM THE REMOTE BIOMODELS REPOSITORY Step 1 In the first step the user must input a valid project name anything different from white spaces or tabs When the user computer is behind a proxy that information must be provided to OptFlux in order to grant remote access Project Name Model Source OptFlux Metabolic Engineering Workbench New Project General Project Options Something from Biomodels _ None Host proxy domain com HTTP m Port 3128 O SOCKS O Flat Files 2 SBML BioModels Step 2 In the second step the user must select the file which model to load The above list Next o Cancel New Project Please choose the desired model from the list bellow BIOMDOOQ00000031 shows only the model ID but when the Fetch Info button is pressed a more complete description of the selected model is provided I
62. pes cccccccccccccsesssseeececeeeesseeeaeseeeceeeeeessaeaaesnenes 47 5 2 OptFlux Datatypes ri pud 47 5 2 1 Critical Reactions 11 nennen enne enm nnne nennen 47 5 2 2 Original Metabolic Model 55 atre denk npe bewk ea 48 5 2 3 Environmental Conditions 48 5 2 4 Equivalent Variables EquivalencesBox cccccssscccecsssseccessececeesessececseseeeecesssaeeeeeesesees 48 5 2 5 Graph 49 54245 OPE MZ SEO MRSS UES NE DU EK 49 Eds PEERS 49 5 2 8 Simplified Metabolic Model 50 5 2 9 Simulation Results 00000 0 51 5 2 10 Variability Analysis 51 5 2 11 Zero aaa e badai 51 6 VIEWERS EDITORS COMPLETE REFERENCE 52 6 1 Viewers or taie Funes Ruin on UE Fe up UM wwe 52 6 2 OptFlux Viewers and 53 6 2 1 Critical Reactions
63. pes presented in the previous chapter will be described and explained also listed alphabetically 6 1 VIEWERS OR EDITORS Since OptFlux follow the MVC software architectural pattern the models in this case the data and the viewers are structurally detached Nevertheless it is possible with some viewers to edit data associated with the models and these changes can be passed to the corresponding data This fact claims for a separate classification for those Viewers that have editing capabilities and those that do not We will call them Viewers or Viewers Editors depending on their editing abilities The viewers will always be launched in the visualization area emphasized in the picture bellow VISUALIZATION AREA OptFlux Metabolic Engineering Workbench 6 2 OPTFLUX VIEWERS AND EDITORS 6 2 1 CRITICAL REACTIONS VIEWER Name Critical Reactions Viewer Tyne Viewer Editor Description 2 Editor It allows for the edition of the Components Search AYT Search Search for the desired flux in the list Essential Genes The list of essential genes Gene Name The list of genes that aren t in the list yet add Adds the flux selected in the combo to the above list remove selected Removes the genes select in the list The removed genes genes will be added to the end of the Gene Name combo box The edition of the essential genes list is done in memory accent changes The changes are only
64. rently select fluxes to be knocked out in this simulation e STEPA OPrION1 OPTION2 Select a flux to Optimize The desired flux to optimize in the simulation usually the biomass growth associated flux The optimization objective either Maximization Minimization OptFlux Metabolic Engineering Workbench e STEP5 OpTION1 OPTION2 Search Search for the desired flux external fluxes The list of possible fluxes Typically only the external fluxes detected automatically by OptFlux will be listed This can be however overridden using Project Preferences with User Level gt ADVANCED see 4 6 1 edit In this panel the user can edit the value of the flux selected in the above list and add it to the list of modified fluxes modified fluxes The list of fluxes that the user has modified This list will define the Environmental Conditions themselves Use the remove from environmental conditions button to remove an undesired flux from this list Use conditions Users may choose to use a previously created environmental condition 4 2 2 WILD TYPE SIMULATION Simulation of the Wild Type of the organism Data The Model to use in this simulation biomass flux The flux to be maximized in the LP problem use env conditions The Environmental Conditions if any to use 4 2 3 MUTANT SIMULATION Simulation of mutant strains Search
65. s as a Simple wrapper for any type of data that the user wishes but can only contain one instance of a given type of data This Datatype was created do hold Lists of other Datatypes list Since the objective is for it to hold instances of Lists it can hold as many objects as the List capacity itself This is the most elaborate Datatype It was created to act as the Complex glue between all the other Datatypes This Datatype can hold Simple List and other Complex Datatypes in it 5 2 OPTFLUX DATATYPES 5 2 1 CRITICAL REACTIONS CRITICALBOX Tyne Simple Description This Datatype holds the list of essential genes The essential genes are those genes needed to maintain the organism s biomass production Viewers CriticalBox Viewer OptFlux Metabolic Engineering Workbench 5 2 2 ORIGINAL METABOLIC MODEL DATABOX Type Complex Description This Datatype holds the Metabolic Model itself Viewers Metabolic Model Viewer Contains Name Description Viewers Reactions C FBAFluxes Contains the flux names and limits Reactions Viewer Contains the metabolites Metabolites information abbreviation name FBAMetabolites compartment name and Metabolites Viewer compartment location Stoichiometric Model Contains all the stoichiometric ee FBASparseMatrix coefficients Equations 5 2 3 ENVIRONMENTAL CONDITIONS ENVIRONMENTALCON
66. s information For the format of this file please refer to 7 1 4 SouRcE2 Select this option if the loaded model is in Pure Pure SBML SBML format refer to 7 3 1 Select this option if the loaded model 15 CellDesigner SBML CellDesigner s SBML format refer to 7 3 2 SBML File The SBML file containing the model to be loaded SouRcE3 The list of ID s provided by the BioModels database One must be selected to be loaded By pressing this button OptFlux will fetch complementary information Fetch Info for the Model ID selected in the list above The information will be placed in the fields to the left of this button e STEP3 SouRcE1 number used to identify the first index used in sparse matrices Indexing starts at only either 0 or 1 Fluxes File Separator The separator used to discriminate elements in this file Either Comma Tab White Space or a User Defined Separator Stoichiometric Matrix The separator used to discriminate elements in this file Either File Separator Comma Tab White Space or a User Defined Separator OptFlux Metabolic Engineering Workbench Metabolites File The separator used to discriminate elements in this file Either Separator Comma Tab White Space or a User Defined Separator SOURCE2 AND SOURCE3 The compartment representing the extra cellular environment Extra cellular Compartment in the SBML model loa
67. stoichiometric coefficient Coeff Each line is composed of three elements separated by any separator the user wishes First comes the metabolite index in the metabolites file then the reaction index in the flux bounds file and finally the stoichiometric coefficient MOindex RO MO RO Coeff MO index R10index MO R10 Coeff R33index MO R33 Coeff Mhinge RXindex Mn Rx Coeff OptFlux Metabolic Engineering Workbench If a metabolite appears in five reactions there will be five lines for that metabolite one for each reaction Note that some users find more intuitive the indexation beginning in 1 instead of 0 OptFlux supports that option also 7 1 3 FULL MATRIX FILE Each line represents a metabolite M and each column represents a reaction R A given position M R represents the M th metabolite in the metabolites file if one exists and R the R th reaction in the fluxes file The value for that position is the correspondent stoichiometric coefficient The fields are separated by any separator the user wishes M1 R1 Coeff M1 R2 Coeff 1 Rm Coeff M2 R1 Coeff M2 R2 Coeff M2 Rm Coeff M3 R1 Coeff M3 R2 Coeff Coeff Mn R1 Mn R2 Coeff Mn Rm Coeff 7 1 4 METABOLITES FILE The file format for the metabolites file is very simple For each metabolite there will be a line in this file Each line can have up to 4 components Each component is separated by tabs M or any
68. t should be saved before quitting and add them to the list bellow list below When the user adds a Project with the above button they will be added to this list This list represents the Projects that should be saved prior to quit 4 2 SIMULATION Swen Optimization visualizatir Simulation Wizard WildType Simulation Mutant Simulation Flux Variability Analysis Environmental Conditions OptFlux Metabolic Engineering Workbench 4 2 1 SIMULATION WIZARD STEP1 Project Select the Project where the model to be user is contained Model Select the model to use in the simulation usually the original or the simplified metabolic model STEP2 Select the type of simulation to perform either Wild Type Tyne of Simutation Option1 simulation or a Mutant Strain Option2 simulation STEP3 OPrioN2 ONLY Search for the desired flux The list of possible targets for deletion The user must select the flux to be deleted in the simulation Add selected flux 10 Pressing the button will add the flux selected the above list to knockouts list the knockouts list on the right Algorithm The algorithm to use in the simulation Either FBA Flux Balance Analysis MOMA Minimization Of Metabolic Adjustment or ROOM Regulatory On Off Minimization of metabolic fluxes Knockouts Presents the user with the list of cur
69. us section 3 8 OPTIMIZATION WITH SIMULATED ANNEALING Simulated Annealing SA is an optimization algorithm inspired in the annealing process used in metallurgy where a melt initially at high temperature is slowly cooled so that the system at any time is approximately in thermodynamic equilibrium As the cooling proceeds the system becomes more ordered approaching a minimal energy state when the temperature reaches zero If the initial temperature of the system is too low or the cooling process is not sufficiently slow the system may become trapped local minimum energy state In the original Metropolis scheme an initial state of a thermodynamic system is chosen at energy and holding temperature constant The initial configuration is perturbed and the change in energy AE is computed A better configuration is always accepted while a worse configuration is only accepted with a probability given by the Boltzmann factor _AE p accept e The Interface allows the user to define several different parameters Similarly to the EA interface it allows the user to define the fluxes to be maximized both by the FBA or MoMA and by the SA optimization process as well as the substrate uptake flux OptFlux Metabolic Engineering Workbench Furthermore the interface allows the definition of some of the parameters of the SA itself The configuration _ Project parameters for the T 2003 Reed s E coli

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