Abstract
A synthetic approach to biology is a promising technique for various applications. Recent advancements have demonstrated the feasibility of constructing synthetic two-input logic gates in Escherichia coli cells with long-term memory based on DNA inversion induced by recombinases. Moreover, recent evidences indicate that DNA inversion mediated by genome editing tools is possible. Powerful genome editing technologies, such as CRISPR-Cas9 systems, have great potential to be exploited to implement large-scale recombinase-based circuits. What remains unclear is how to construct arbitrary Boolean functions based on these emerging technologies. In this paper, we lay the theoretical foundation formalizing the connection between recombinase-based genetic circuits and Boolean functions. It enables systematic construction of any given Boolean function using recombinase-based logic gates. We further develop a methodology leveraging existing electronic design automation (EDA) tools to automate the synthesis of complex recombinase-based genetic circuits with respect to area and delay optimization. In silico experimental results demonstrate the applicability of our proposed methods as a useful tool for recombinase-based genetic circuit synthesis and optimization.
Highlights
The development of synthetic biology shows the feasibility to implement computing devices with DNA genetic circuits in living cells
We show a design flow for arbitrary Boolean function construction with cascaded recombinase-based logic gates. This automated design methodology is demonstrated by leveraging synthesis tool ABC14, an electronic design automation (EDA) tool developed at UC Berkeley, to synthesize cascaded multi-level recombinase-based circuits
To have green fluorescent protein (GFP) highly expressed all Ri’s must exist, and this circuit implements a 3-input AND gate. Note that this 3-input AND gate, where the promoter and the reporter gene GFP can be flipped by recombinases, is designed in a different fashion from the 2-input AND gate in Fig. 1(b), where only transcription terminators are inverted by recombinases
Summary
The development of synthetic biology shows the feasibility to implement computing devices with DNA genetic circuits in living cells. Let molecules AHL and aTc be the stimulus inputs to a cell and act as inducers activating the expressions of recombinases Bxb[1] and phiC31, respectively These recombinases when activated will irreversibly invert (flip) the DNA sequences flanked by their recognition sites (denoted by the colored triangle pairs). Motivated by the viability and applicability of recombinase-based circuits, in this paper we formalize the construction of a general multi-input logic gate with its DNA sequence composed of series of promoters and transcription terminators targeted by multiple recombinases. This automated design methodology is demonstrated by leveraging synthesis tool ABC14, an electronic design automation (EDA) tool developed at UC Berkeley, to synthesize cascaded multi-level recombinase-based circuits
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