Design

Overall Mechanism

We are so accustomed to designing synthetic circuits in a traditional way (i.e., to build a circuit by concatenating different biobricks together) that we even believe this is all about synthetic biology. However, with the advent of the second wave of synthetic biology^[1], larger circuits, systems and even circuit networks are being designed. Traditional methods are increasingly incompatible with trends in synthetic biology.

So FLAME might change the way we view softwares for synthetic biology. Instead of choosing each biobrick we need in order to construct a circuit, FLAME utilizes frameworks as design prototype. Users choose the inputs (e.g., IPTG, aTc, etc.), outputs (e.g., GFP) and the topologies of circuits, and FLAME can automatically finish the construction. Then users are able to tune the details of the circuits (for example, change a RBS for higher expression efficiency).

How can FLAME achieve all these? It adopts the following three mechanisms.

1. Input-Receptor-Promoter Relationships

At the beginning, users should choose the inputs, outputs and the topology for his/her circuits. Thus far, synthetic biologists have constructed most circuits from a limited number of commonly used parts (e.g., LacI, TetR and lambda repressor protein)^[2], which makes possible the automatic design of genetic circuits. Through the choice of inputs, FLAME can determine what parts (for example, promoters) should be used.

For instance, when users choose IPTG as the input of the circuits, FLAME will then select LacI as the receptor protein of IPTG. In the absence of IPTG, LacI will repress the expression from lac promoter (P_lac), and the binding of IPTG with LacI abolishes the repression imposed by LacI. Then an “IPTG-LacI-P_lac” relationship is determined for further use.

Figure 1 LacI represses the expression from P_lac, and binding of IPTG with LacI abolishes this repression.

2. Framework-Based Design

Next, frameworks help the automatic design. What are the functions of frameworks?

Supposing that a synthetic circuit was reported in literature (like toggle switch, see Figure 2), and we want to improve its performance or design a better one with desired performance, the easiest way may be that we would refer to this reported circuits and try to change its components or its topologies.

So, the reported circuits are there to provide the necessary information about how to arrange the parts in place to construct a functional circuit. For example, there must be at least one promoter, one RBS, one protein coding sequence and one terminator in a circuit, and the order of all these parts is: promoter-RBS-protein coding sequence-terminator. The primary function of frameworks is to make sure that the constructed circuit contains the necessary parts in a correct, functional order.

But that is not enough. Frameworks can help users with automatic design of circuits, system and ever larger networks. With the help of frameworks, users can think of design as putting circuits in place to construct a system or network, rather than a bottom-up method starting from every biobrick.

Figure 2 Frameworks can help users with easy designs. For instance, Gardner et al.^[3] reported a toggle switch design in 2000. This design can be used as a “frame”, or a guide to users for designing toggle switches using different biological parts (Adapted from Gardner et al., 2000).

3. Biobricks Still Matter

It is of note that FLAME is still based on biobricks. Biobricks are “bricks” for construction of large “buildings”--circuits, systems or even networks in synthetic biology. As mentioned above, we must place the necessary parts in a “promoter-RBS-protein coding sequence-terminator” order to construct a functional circuit. The utilization of characterized and standardized biobricks guarantees this framework-based automatic method adopted by FLAME.

WORK FLOW

After introduction to the mechanisms of FLAME, we will briefly introduce the work flow of our software. There are four modules: Design, Display, Simulation and Experiment.

1. DESIGN

This is the first module of FLAME, and also the beginning of circuit designs. This module is aimed at getting FLAME ready for automatic design. FLAME offers users with two methods of starting a circuit design.

1) Users choose the input, output and then set the logic between them (that is, the topologies of the circuits). After that, FLAME goes on with the obtained information.

2) Users can also design a circuit with the help of a truth table if they are not clear what design frameworks they should choose. At first, users are required to select inputs and outputs. Then they can fill in the truth table. Users are not required to finish the entire table, which means they just need to complete a few lines. Then FLAME will recommend the topologies of circuits that are in accordance with the truth table.

2. DISPLAY

Users can examine and tune their designed circuits at a variety of levels in the Display module. According to synthetic biologists and iGEMers’ usual practice, there are four sub-modules in Display module: Device, Parts, DNA and Vector. They are in the order of their scales.

Device

Detailed information (e.g., which Biobricks have been automatically selected for this circuit) will be displayed in this page. You can change the performance of the circuit via switching a promoter, altering an RBS, etc. In this way, you can make subtle adjustments so that our recommendation will fit your desires.

Parts

This sub-module provides users with another dimension of the circuits, that is, a view of Biobricks. If you want to know the specific length of any Biobrick or of the material connecting them, just visit this page. In this page you can also make small changes to your circuit like that in Device page.

Vector

Plasmid is the vector of parts. In this page you can get an overall view of the vector. Furthermore, Vector sub-module will also show you the restriction sites on plasmids.

DNA

Small changes in DNA sequence can be made in this DNA page. In addition to the DNA sequence of every parts, you can also know about the position of restriction sites in the sequence. In short, the DNA sub-module offers you with information on the DNA level.

3. SIMULATION

After a long work of design, users may want to know how it works in reality. In the Simulation module, FLAME will not only provide you two types of curve graphs, Static Performance and Dynamic Performance, to inform you with the possible performance of the circuit, but also help you find the perfect RBS that best function in the circuit.

4. EXPERIMENT

For researchers aiming to bring their design into reality(e.g., go into a lab and turn their design into a vector), FLAME can offer you a general procedure of your wetlab experiments.

[1] Purnick, P.E.M. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Bio 10, 410-422 (2009).

[2] Lu, T.K., Khalil, A.S. & Collins, J.J. Next-generation synthetic gene networks. Nat Biotechnol 27, 1139-1150 (2009).

[3] Gardner, T.S., Cantor, C.R. & Collins, J.J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339-342 (2000)