Overview

Abstract Currently genetic circuits in synthetic biology are complicated thus are difficult for biologists to design. In our project, we developed the Framework-based Layout And Metacircuit design Engine (FLAME), to simplify the whole design procedure by characterizing numerous published genetic circuits and abstract these circuits into 3 parts: inputs, outputs and logic relationships. A novel simulation module using ordinary differential equations is integrated into our software. Our simulation results were precisely validated by results from published literature and our self-designed wetlab experiments. In conclusion, the combination of a simplified design procedure, a new and effective simulation module and wetlab validation makes complex biological circuits more accessible to synthetic biologists.

One method frequently adopted by synthetic biologists is the reconstructive approach^[1]; that is, by designing and building genetic circuits with similar functions, synthetic biologists can gain insight into the underlying mechanisms of the naturally occuring circuits. Software aiming at facilitating the in silico design of genetic circuits has been developed.

Thus far, a lack of methods for efficient and proof-of-principle design may limit its wide acceptance in synthetic biology. On one hand, for a beginner, a dearth of frameworks for the reliable construction of complex, higher-order systems^[3] makes arrangement of parts into circuit topologies somewhat annoying and frustrating, let alone construction of a FUNCTIONAL circuits only by linking the scattered biobricks together. On the other hand, the performance of a system cannot be deduced through individual parts. An expert synthetic biologist may want not only to build a functional genetic circuit, but also to characterize its performance.

So software endowed with the capacity of more efficient and reliable design is needed. Based on the idea of framework, our latest software FLAME (Framework-based Layout And Metacircuit Design Engine) is a tentative exploration of solutions to the problems mentioned above. Features of FLAME are as follows.

Framework-based Circuit Design -- Core of FLAME

Framework-based method is the core of FLAME to ensure the EFFICIENT construction of a FUNCTIONAL circuit. After users select an input, output and the topologies (provided as “frameworks”) of the system, several solutions are provided when you choose your ideal framework, each of which differs in mechanisms and efficiency (for example, genetic AND GATES can be achieved by protein-protein interactions or substrate-receptor interactions; they are different in many aspects). According to the performance of each solution (shown in the form of rader charts), users can select one and fine tune the details of the circuits. The fact that most synthetic circuits are still made up of a limited but sufficient number of commonly used components (such as LacI, TetR and lambda repressor proteins and regulated promoters)^[2] makes possible the framework-based construction.

Characterization and Standardization of a Circuit or System -- Our Goal

So far, a considerable number of synthetic biological parts and devices have been characterized and standardized^[3], but not circuits nor systems. Although we could theoretically ensure a functional system based on frameworks, we could not determine the actual performance of a system through the parameters of its parts. Furthermore, standardization is useful in creating circuits or systems that can be used in a plug-and-play fashion to construct larger networks^[3], but there still not enough standardized circuits or systems. Cameron et al.^[4] pointed out that in the near future, workflow for a biological circuit engineer will be limited by their capability of analyzing circuit behaviour and incorporating the data into the next design cycle.

To tackle these problems, we can learn from the mature methods with which synthetic biologists and iGEMers characterize and standardize a biobrick. We propose that we could characterize and standardize a circuit or a system in a similar way, though with more inputs and outputs. In the Simulation module, FLAME provides simulations on Dynamic Performance, Static Performance and Expression Efficiency, all of them being important clues on the performance of the designed circuit or system. And the Vector sub-module in FLAME may be helpful in standardization of a circuit.

New Simulation Module -- Improvements as well as Innovations

We develop a new Simulation module for FLAME. We rewrite our algorithms to enhance the simulation efficiency. As mentioned above, the Simulation module is also designed for characterization and standardization of synthetic circuits or systems. This module can be divided into three sections: Dynamic Performance, the response of outputs to an increase in input level at 0 min^[3]; Static Performance, the steady-state relationship between the inputs and outputs^[3]; Expression Efficiency, a section concerning the effects of RBS on gene expression efficiency.

We also developed an online blast tool, Biobrick Blast Online (or BBO for short), as our Human Practice project, to aid in recognition of a possible biobrick from a DNA sequence. We believe that with FLAME and BBO, users can be more confident in their efficient constructions of reliable genetic circuits!

[1] Sprinzak, D. & Elowitz, M.B. Reconstruction of genetic circuits. Nature 438, 443-448 (2005).

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

[3] Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26, 787-793 (2008).

[4] Cameron, D.E., Bashor, C.J. & Collins, J.J. A brief history of synthetic biology. Nature reviews. Microbiology 12, 381-390 (2014).