Synthetic gene circuit evolution: Insights and opportunities at the mid-scale

Abstract

Directed evolution focuses on optimizing single genetic components for predefined engineering goals by artificial mutagenesis and selection. In contrast, experimental evolution studies the adaptation of entire genomes in serially propagated cell populations, to provide an experimental basis for evolutionary theory. There is a relatively unexplored gap at the middle ground between these two techniques, to evolve in vivo entire synthetic gene circuits with nontrivial dynamic function instead of single parts or whole genomes. We discuss the requirements for such mid-scale evolution, with hypothetical examples for evolving synthetic gene circuits by appropriate selection and targeted shuffling of a seed set of genetic components in vivo. Implementing similar methods should aid the rapid generation, functionalization, and optimization of synthetic gene circuits in various organisms and environments, accelerating both the development of biomedical and technological applications and the understanding of principles guiding regulatory network evolution.

Keywords: DNA shuffling; directed evolution; experimental evolution; selection; synthetic gene circuit.

Figure 1.. Mid-scale evolution lies between directed and experimental evolution.

There is a gap between directed evolution and experimental evolution, which suggests that mid-scale evolution of whole synthetic gene circuits inside living cells would be both useful and informative. See also Table 1.

Figure 2.. Examples of mid-scale evolution for various known gene circuits. a. Oscillator.

Selection should consist of recurring conditions ensuring that some component is periodically beneficial over time, but costly otherwise. Such periodic selection could involve a costly gene within the circuit, which is growth-promoting in some periodically returning environmental factor. b. Switch. Selection should consist of a transient inducer pulse dropping to an intermediate level, which activates a stress resistance gene, followed by persistent stress to select for persistent activation. Evolving toggle switches would require two similar stimuli and two corresponding stresses. c. Pulse generator. Selection should consist of a pulse of stress concurrent with an increase in inducer that subsequently stays on. Highly costly stress-response gene expression will give incentive for subsequently diminished expression. The stress pulse will last a limited time, while the inducer will persist to select against sense-response or switch circuit behaviors, where the costly product would be continuously high, compromising cellular fitness. Therefore, the cells will be pressured to produce a short pulse of the costly product to mitigate the stress in the environment, but not maintain its expression when the stress is gone. d. Sense-response system. Selection should consist of concurrent increase of both inducer and stress, followed by concurrent decrease of both after some plateau. These conditions should be repeated nonperiodically, with various stress levels versus inducer levels corresponding to the desired sense-response function (e.g., gradual or stepwise). e. Noise generator. Selection should consist of a concave fitness landscape to diminish gene expression noise, and convex fitness landscape to amplify noise. Alternating selection for high and low expression could substitute for convex landscapes.

Figure 3.. A recombination-based system for shuffling genetic components.

a. Nonfunctional, scrambled arrangement of seed components with functional elements separated by recombination sites. b.,c. The seed of components can rearrange itself into functional gene circuits by random recombination events of inversions (1), insertions (2), cassette exchanges (3), and excisions (4) as recombinases rearrange and insert/excise the integrated functional elements switching them with extrachromosomal circular DNA fragments.