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A plasmid system with tunable copy number

Author

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  • Miles V. Rouches

    (Cornell University)

  • Yasu Xu

    (Cornell University)

  • Louis Brian Georges Cortes

    (Cornell University)

  • Guillaume Lambert

    (Cornell University)

Abstract

Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 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 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 systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems.

Suggested Citation

  • Miles V. Rouches & Yasu Xu & Louis Brian Georges Cortes & Guillaume Lambert, 2022. "A plasmid system with tunable copy number," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-31422-0
    DOI: 10.1038/s41467-022-31422-0
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    References listed on IDEAS

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    1. Laurent Potvin-Trottier & Nathan D. Lord & Glenn Vinnicombe & Johan Paulsson, 2016. "Synchronous long-term oscillations in a synthetic gene circuit," Nature, Nature, vol. 538(7626), pages 514-517, October.
    2. Tanita Wein & Nils F. Hülter & Itzhak Mizrahi & Tal Dagan, 2019. "Emergence of plasmid stability under non-selective conditions maintains antibiotic resistance," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
    3. Xinyi Wan & Filipe Pinto & Luyang Yu & Baojun Wang, 2020. "Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    4. Bin Shao & Jayan Rammohan & Daniel A. Anderson & Nina Alperovich & David Ross & Christopher A. Voigt, 2021. "Single-cell measurement of plasmid copy number and promoter activity," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
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    Cited by:

    1. Shivang Hina-Nilesh Joshi & Chentao Yong & Andras Gyorgy, 2022. "Inducible plasmid copy number control for synthetic biology in commonly used E. coli strains," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    2. Kanakov, Oleg & Chen, Shangbin & Zaikin, Alexey, 2024. "Learning by selective plasmid loss for intracellular synthetic classifiers," Chaos, Solitons & Fractals, Elsevier, vol. 179(C).
    3. Yuanli Gao & Lei Wang & Baojun Wang, 2023. "Customizing cellular signal processing by synthetic multi-level regulatory circuits," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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