Prolonging genetic circuit stability through adaptive evolution of overlapping genes

Author:

Chlebek Jennifer L1ORCID,Leonard Sean P1,Kang-Yun Christina1,Yung Mimi C1,Ricci Dante P1,Jiao Yongqin1,Park Dan M1ORCID

Affiliation:

1. Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory , Livermore , CA  94550, USA

Abstract

Abstract The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or ‘entangled’ entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more ‘benign’ mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

Funder

U.S. Department of Energy

Lawrence Livermore National Laboratory

Publisher

Oxford University Press (OUP)

Subject

Genetics

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