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Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E. coli

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    Rights statement: ACS AuthorChoice - This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Copyright © 2017 American Chemical Society

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http://pubs.acs.org/doi/10.1021/acssynbio.7b00328
Original languageEnglish
Pages (from-to)553-564
Number of pages12
JournalACS Synthetic Biology
Volume7
Issue number2
Early online date5 Jan 2018
DOIs
Publication statusPublished - 16 Feb 2018

Abstract

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behavior of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guidance for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host.

    Research areas

  • gene circuit, host circuit interaction, orthogonality, metabolic burden, RNA-Seq, synthetic biology

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