Standardized Cloning and Curing of Plasmids

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Abstract

Plasmids are highly useful tools for studying living cells and for heterologous expression of genes and pathways in cell factories. Standardized tools and operating procedures for handling such DNA vectors are core principles in synthetic biology. Here, we describe protocols for molecular cloning and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate the application of this system to cure SEVA constructs (all vectors are available at SEVA/Addgene).
Original languageEnglish
Title of host publicationSynthetic Biology. Methods in Molecular Biology
EditorsJ. Braman
Volume1772
Place of PublicationNew York
PublisherHumana Press
Publication date2018
Pages469-476
Chapter28
ISBN (Print)978-1-4939-7794-9
ISBN (Electronic)978-1-4939-7795-6
DOIs
Publication statusPublished - 2018
SeriesMethods in Molecular Biology
ISSN1064-3745

Keywords

  • SEVA
  • Plasmid
  • DNA assembly
  • Plasmid curing
  • pFREE
  • Vector backbone exchange
  • Cell factory design

Cite this

Lauritsen, I., Kim, S. H., Porse, A., & Nørholm, M. H. H. (2018). Standardized Cloning and Curing of Plasmids. In J. Braman (Ed.), Synthetic Biology. Methods in Molecular Biology (Vol. 1772, pp. 469-476). Humana Press. Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-7795-6_28