Synthetic in vitro circuits

Abstract

Inspired by advances in the ability to construct programmable circuits in living organisms, in vitro circuits are emerging as a viable platform for designing, understanding, and exploiting dynamic biochemical circuitry. In vitro systems allow researchers to directly access and manipulate biomolecular parts without the unwieldy complexity and intertwined dependencies that often exist in vivo. Experimental and computational foundations in DNA, DNA/RNA, and DNA/RNA/protein based circuitry have given rise to systems with more than 100 programmed molecular constituents. Functionally, they have diverse capabilities including: complex mathematical calculations, associative memory tasks, and sensing of small molecules. Progress in this field is showing that cell-free synthetic biology is a versatile testing ground for understanding native biological circuits and engineering novel functionality.

Figure 1

The reactions and molecular composition of different synthetic in vitro circuits. Black arrows represent routes of information flow while red arrows represent interactions capable of controlling or modulating this flow. Nucleic acid based systems contain relatively few types of interactions, resulting in more predictable behavior. Hybrid systems, which are capable of producing and degrading nucleic acids, are intermediate on this scale with several new points of control and information flow. Complete systems can replicate the entire `central dogma', but their increased complexity of interactions makes them less predictable. Note: small-molecule inputs and outputs via enzymes, ribozymes, and deoxyribozymes can, in principle, occur in all systems.

Figure 2

Toehold exchange and seesaw gate function and implementation [13]. (A) A ssDNA input binds to a complementary toehold (red) on a seesaw gate and – through strand migration – replaces an output strand of the dsDNA that is then capable of the reverse reaction via a separate toehold (green). (B) A functional AND gate that takes input molecules A and B and outputs G. The left side presents a simplified overview while the right includes molecular detail. At seesaw gate 1, inputs (A and B) displace a strand of DNA (C) that then encounters threshold gate 1(E–F). This gate quickly and irreversibly turns C into waste by having a larger complementary overhang than that of molecule G–H. Only after threshold molecules have been depleted will C bind to G–H and produce the output strand G.

Figure 3

Sample AND gate implementation in hybrid and complete systems. (A) Hybrid systems may use DNA, RNA, or small molecule inputs and outputs. Here, an incomplete promoter (dashed box) is bound by two ssDNA inputs that complete the promoter region. The polymerase enzyme (brown) then binds and transcribes an mRNA output. (B) Complete systems have a diverse array of possible inputs and outputs. Here, two DNA input signals with intact promoters (solid box) are transcribed into mRNA and translated into functional protein products ntrC (red) and σ⁵⁴ (blue). These protein products then bind to a polymerase and allow it to transcribe a gated promoter (dashed box), leading to production of a protein output [55].