Precision design of stable genetic circuits carried in highly-insulated E.coli genomic landing pads.

Yongjin Park,Jonghyeon Shin,Amin Espah Borujeni,Thomas E Gorochowski,Christopher A Voigt

doi:10.15252/msb.20209584

Yongjin Park, Jonghyeon Shin + Show 3 more

Open Access

https://doi.org/10.15252/msb.20209584

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Abstract

Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high‐expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA + strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible.

Highlights

Cells use regulatory networks, encoded in their genomes, to determine which genes need to be expressed based on cellular needs or to adapt to the environment (McAdams & Shapiro, 1995)
If allowed to enter the circuit DNA, genomic RNA polymerases (RNAPs) flux can cause the circuit to malfunction (Lee et al, 2016)
The opposite can be problematic, where the RNAP flux from the circuit can exit and cause genes encoded in the genome to be expressed incorrectly

Summary

Introduction

Cells use regulatory networks, encoded in their genomes, to determine which genes need to be expressed based on cellular needs or to adapt to the environment (McAdams & Shapiro, 1995). They can lead to instability, cell-to-cell heterogeneity, and metabolic burden (Summers & Sherratt, 1984; Chiang & Bremer, 1988; Summers, 1991; Stoebel et al, 2008; Kittleson et al, 2011; Gyorgy et al, 2015; Borkowski et al, 2016; Wang et al, 2016b) This can lead to evolutionary forces breaking a circuit through plasmid loss or mutagenesis to the plasmid or genome (to reduce the copy number; Mayo et al, 2006; Stoebel et al, 2008; Klumpp et al, 2009; Sleight et al, 2010; Chen et al, 2013; Sleight & Sauro, 2013; Fernandez-Rodriguez et al, 2015; Gyorgy et al, 2015; Ceroni et al, 2018; Liu et al, 2018; Moser et al, 2018, 2012). It is standard practice in industrial biotechnology to introduce recombinant DNA in the genome, if selective pressure from antibiotics is impossible or costprohibitive (Singh et al, 2011; Isabella et al, 2018)

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