The field of synthetic biology has produced genetic circuits capable of emulating functional paradigms commonly seen in digital electronic circuits such as bistable switches, oscillators, and logic gates. In this work we combine synthetic bioengineering experiments with multiscale models to design, build, and characterize a high-fidelity logical AND gate in bacteria.

Experimentally, we constructed an in vivo synthetic hybrid system consisting of multiple operators within a single promoter. The operator sequences employed are derived from three unrelated natural regulatory elements: the tetracycline (tet), lactose (lac) and λ-phage operons arranged logically within a single transcriptional unit. Specifically, we built six single-promoter regulatory motifs by shuffling tet and lac operator sites (T and L, respectively) in and around the PL (λ-phage): LLT, LTL, TLL, TLT, TTL and LTT . The promoters drive expression of green fluorescent protein (GFP). The regulatory architecture is designed such that each operator position efficiently interferes with RNA polymerase (RNAp) promoter binding while causing the least perturbance to promoter function. To study the fidelity of logical gate behavior among the designed variants, the synthetic promoter sequences were incorporated in the reporter vector pGlow to direct the transcription of GFP. We engineer the system in a Escherichia Coli strain that constitutively expresses lactose repressor (LacI) and tetracycline repressor (TetR) proteins.

What is novel in the approach is the combination of experiments with sophisticated models to guide the design of synthetic constructs. A reductionist modeling approach is pursued. That is, instead of developing the simplest possible model that can capture the behavior of the biological system, we include all the biomolecular interactions involved in transcription, translation, regulation and induction in order to explicitly relate each model parameter to a real in vivo reaction event. Our models are multiscale because they are detailed and they are detailed so they are general. Synthetic biology efforts would benefit from a design tool that allows one to use the input-output characteristics of simple parts and then design a more complicated system from them.

We will present the Synthetic Biology Software Suite (synbioss.sourceforge.net), a suite of tools that facilitate synthetic biology [1]. There are three parts to the suite:

1) Designer. The user enters gene network components, such as BioBricks. These are becoming a widely used standard, and our codes can make use of them. The Designer uses general principles of bacterial expression to generate reaction networks. Networks of dozens of reactions modeling transcription, translation, regulation and induction can be built in minutes and stored in SBML 2) Wiki. Recognizing the difficulty of finding kinetic parameters, we introduce a database with a wiki front. First, for a lot of the widely used components (components of the tetracycline, lactose, arabinose operons, green fluorescence proteins, etc.) the information is scattered around in the literature and difficult to find and put to use. We have populated the database with dozens of known interactions of the tetracycline, lactose and arabinose operons.Second, the wiki can be used as a portal for the community to enter new quantitative information and a repository for the community to search and find this information. The Wiki stores reaction kinetic data in a formatted and searchable scheme with references to the relevant literature. Therefore, we provide a platform for facilitating inclusion of quantitative information, a necessary step to bridge gaps between models and experiments.

3) Desktop simulator. Besides Open License files for UNIX/Linux, a single Windows executable can be downloaded from synbioss.sourceforge.net (we are working on MacOS). It will install SynBioSS DS on any PC (a little icon appears in the Start Menu). When executed, SynBioSS launches a GUI for the user to build a new reaction network or read any SBML file with an existing reaction network, e.g. one generated with Designer. The GUI can be used to add, edit and delete reactions. It can be used for specifying parameters, such as the volume of a cell, the time of cell duplication, initial concentrations of chemical species, etc. The GUI can be used to specify simulation parameters, such as simulated time, time steps for integrating stochastic differential equations and others. Default values are already entered. The GUI can be used to launch a simulation of multiple stochastic trajectories and then save Excel files with histograms of concentrations of molecules, such as GFP.

With SynBioSS, models acquire a predictive character that renders them well suited for engineering purposes. Because they are stochastic, the models generate probability distributions of phenotypes that are directly comparable to experimentally observed variation. Because they are detailed, the models provide the opportunity to gain molecular level insight and quantify previously undetermined biomolecular interactions.

We will present the wet-lab techniques, the software package that rationalizes our efforts and the results of simulations and experiments to construct high-fidelity logic-AND gates in bacteria. The simulation results, coupled with in vivo data, not only identify important design degrees of freedom, but provide parameters that can be used to guide future synthetic designs using these common regulatory elements.

[1] SynBioSS: The Synthetic Biology Modeling Suite

A.D. Hill, J.R. Tomshine, E.M.B. Weeding, V. Sotiropoulos and Y.N. Kaznessis, Bioinformatics, 2008

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