Minnesota/21 October 2008

Synthetic Biology Software Suite (SynBioSS)

Mathematical modeling plays an important role in capturing the behavior of synthetic biological constructs and in guiding the design of new synthetic systems. Although there is no shortage of outstanding models in synthetic biology, the field has suffered from the absence of a common modeling approach. For example, Gardner and coworkers captured bistability in a brilliant model (Gardner et al. 2000) and Elowitz and Leibler captured oscillations with an ingenious model (2000), but Gardner’s model cannot be used for Elowitz’s system. Thus we propose to develop a process for generating synthetic gene network models that is applicable to any synthetic construct and is suitable for automation. SynBioSS is a complete suite of software for the modeling and simulation of arbitrary synthetic genetic constructs. There are three components in SynBioSS: Designer, Wiki and Simulator. SynBioSS Designer is a web application for the automatic generation of sets of biomolecular reactions. Tutorials and examples are available on synbioss.sourceforge.net for various synthetic biological constructs (oscillators, switches, AND gates). Users can enter the molecular parts of a synthetic biological system involved in gene expression and regulation (e.g., promoters, transcription factors, ribosomes), and Designer then generates complete reaction networks that represent transcription, translation, regulation, and degradation of those parts. A Systems Biology Markup Language (SBML) or NetCDF file is generated with the model. We have also adapted SynBioSS Designer to automatically generate a kinetic model from a construct composed entirely of BioBricks. This was the work of the iGEM 2008 Minnesota team. With Designer, a user can quickly build a network of reactions to represent any synthetic gene regulatory network. As a demonstration, we present the steps in Designer for building the comparator, a six-gene synthetic construct we are building in 2008 iGEM. The comparator is a genetic device that receives two inputs (in our case, isopropyl-β-D-1-thiolgalactopyranoside [IPTG] and anhydrous tetracycline [aTc]) and has two potential outputs (in our case, a green fluorescence protein [GFP] and a red fluorescence protein [RFP]). The comparator compares the relative amounts of the inputs, turns on GFP if aTc>IPTG or turns on RFP if IPTG>aTc (for more information, see the Minnesota iGEM Wiki: http://2008.igem.org/Team:Minnesota/HomeComparator). Here is the set of simple steps followed for any synthetic gene network: 1.	Go to Designer’s home at http://neptune.cems.umn.edu/designer/interface1.php 2.	Enter parts in order (i.e., Promoter→RBS→DNA→Terminator). From a drop-down menu, characterize each of these parts, one at a time. 3.	Provide the name of the protein for each coding DNA region (C0051 is repressor CI from lambda phase, C0072 is repressor P22mnt, E0040 is GFP, and J06504 is RFP) and characterize the protein (Activator; Repressor [CI and P22mnt]; Reporter [GFP and RFP]; Enzyme; Other). 4.	Specify promoters as constitutively ON or OFF (all ON in this example). 5.	Add operators to promoters and specify their relative position. This is an important step for defining regulatory relationships. R0040 has tetO, which is the operator bound by TetR; R0010 has lacO, which is the operator bound by LacI (lactose repressor); K101000 is a promoter we built. It is dually repressed by TetR and P22mnt, so we add two operator sites, tetO and p22O, for the transcription factors to bind; K101001 is the second promoter we built. It is dually repressed by LacI and CI, so we add two operator sites, lacO and CIO, for the transcription factors to bind. 6.	Enter any proteins constitutively expressed. In the comparator example, these are TetR and LacI, since we cloned the comparator in DH5aPro E. coli cells that constitutively express these two repressors. 7.	Specify protein oligomeric structure (monomer [GFP, RFP], dimer [TetR2, CI2, P22mnt2], tetramer [LacI4]). 8.	Specify where transcription factors bind (TetR2-tetO; CI2-ciO; P22mnt2-p22O; LacI4-lacO). 9.	Enter any relevant effector molecules (e.g., inducers) present in the system (aTc and IPTG). 10.	Specify how many times each effector binds to a protein (two aTc can bind to TetR2; four IPTG to LacI4). Finally, the user can click on a button to generate a reaction network with all the interactions and save the reaction network in SBML or NetCDF file format. Designer generates the reactions with default kinetic constants. These are taken from known biomolecular interactions and applied to the various interaction types (e.g., RNAp binding on promoters, ribosome binding on RBS, protein dimerization, protein-operator, protein-effector). For the comparator, a set of 98 reactions is generated. The files are ready to upload on SynBioSS Desktop (DS) to run numerical simulations. The user can also upload the file on SynBioSS Wiki and carefully check all the reactions and parameters, searching in the Wiki database for available information on any interaction. If there is no available information, the user can choose to retain the default value entered (default values are taken from known interactions, as discussed in detail later in the proposal) or conduct simulations over a range of parameter values for a sensitivity analysis. Uploading the SBML or NetCDF file with the reaction network in SynBioSS DS (see next section for instructions to upload a model in SynBioSS DS and conduct simulations) allows the user to visually and carefully check the reactions, the kinetic constants, and the initial conditions and run numerical simulations. For the comparator, we have conducted a series of numerical simulations at various aTc and IPTG concentrations. Results and a discussion are available in the iGEM 2008 Minnesota Wiki site (http://2008.igem.org/Team:Minnesota). To generate reactions, Designer processes the user-supplied information with biological first principles. For example, in Designer the basal transcription process with an arbitrary number of activating or repressing transcription factors starts with the formation of the holoenzyme complex on the promoter, called RNAP, which binds and unbinds to the promoter DNA in the reaction, RNAP + Promoter = RNAP:P. A series of standard reactions are generated that model transcription initiation, elongation and the production of mRNA. More information in tutorials in http://neptune.cems.umn.edu/designer/interface1.php Although generally true that there is dearth of quantitative information on biomolecular interactions, this is not the case for a number of widely used BioBricks, such as components of the tetracycline operon, the lactose operon and the arabinose operon. For these system, there is a wealth of kinetic and equilibrium constant information, and the challenge becomes finding this information. Thus we are building SynBioSS Wiki, a searchable repository of quantitative information, and have already conducted the tedious search of the available information for the tetracycline, lactose, and arabinose operons, populating the Wiki database with more than 100 distinct molecular species and 130 reactions. We also provide literature links and information on the experimental context used to determine the constants. While we believe this is a good start, we recognize that only a community-wide effort could pay significant dividends for quantitative biology and we believe that a wiki format, which has been enormously successful in other applications (e.g., Wikipedia or OpenWetWare), can facilitate this effort. Simulating gene regulatory networks is simple. With the third component of SynBioSS, the Desktop Simulator, a user can run sophisticated numerical simulations of complex reaction networks quickly and seamlessly on a PC. SynBioSS Desktop Simulator can be downloaded as an installation executable for Windows (we have a beta MacOS version). The steps are: 1.	Go to synbioss.sourceforge.net 2.	Click on “Simulator” on the upper left corner. This will take you to http://synbioss.sourceforge.net/simulator/ 3.	Click on “Download” in the middle of the webpage. This will take you to the sourceforge file directory. 4.	Click on SynBioSSDSInstaller-1.0.1.exe. This downloads the installation executable on your computer. 5.	Run the executable. This will install the current version of SynBioSS on your computer. 6.	Click on the Start Menu to find and click the SynBioSS icon. This will launch SynBioSS. 7.	The user can build a new model or upload one generated in Designer and Wiki. We have done this for bistable switches, oscillators, logic gates and the comparator.