Beyond directed evolution: Darwinian selection as a tool for synthetic biology

Abstract

Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed.

Keywords: Darwinian selection; Directed evolution; Synthetic biology.

Fig. 1

Strategies based on the proposed Darwinian selection approach aimed at identifying biological components suitable for SB, such as strong promoters (a), protein coding sequences such as cellulases (b) and sequences coding for transcription regulators: activators (c) and inhibitors (d). Natural (i.e. meta-genomic) DNA variants are cloned as the components subjected to selection (white arrows). Dark and light gray arrows correspond to other components. Suitable media allowing selection of the fittest components are indicated. Notice than solid, rather than liquid media should be used, since B-lactamase, as well as some cellulases are secreted and bystander cells might survive if liquid media was used

Fig. 2

Strategy for directing the evolution of an oscillatory circuit though a well calibrated master circuit. An oscillatory circuit A (white line) is subjected to fluctuating selection by a master B oscillator (thick grey line) through the action of a coupler circuit (shown below). P1 and P2, promoter sequences. The behavior of circuit A must be within certain limits marked by the output of B by transcriptional activation/inhibition of a death gene on the coupling circuit. When the output of master circuit B decreases, so too should the output of circuit A, in order to keep the death gene under the control of P1 inhibited. When the output of master circuit B rises, high inhibitory levels of A must balance activation of P2 by B