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A Plasmid System with Tunable Copy Number

View ORCID ProfileMiles V. Rouches, View ORCID ProfileYasu Xu, View ORCID ProfileLouis Cortes, View ORCID ProfileGuillaume Lambert
doi: https://doi.org/10.1101/2021.07.13.451660
Miles V. Rouches
1Field of Biophysics, Cornell University, Ithaca, NY 14853, USA
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Yasu Xu
1Field of Biophysics, Cornell University, Ithaca, NY 14853, USA
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Louis Cortes
2School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA
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Guillaume Lambert
2School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA
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  • For correspondence: lambert@cornell.edu
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Abstract

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted July 13, 2021.
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A Plasmid System with Tunable Copy Number
Miles V. Rouches, Yasu Xu, Louis Cortes, Guillaume Lambert
bioRxiv 2021.07.13.451660; doi: https://doi.org/10.1101/2021.07.13.451660
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A Plasmid System with Tunable Copy Number
Miles V. Rouches, Yasu Xu, Louis Cortes, Guillaume Lambert
bioRxiv 2021.07.13.451660; doi: https://doi.org/10.1101/2021.07.13.451660

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