Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

Ida Lauritsen, Virginia Martinez, Carlotta Ronda, Alex Toftgaard Nielsen, Morten H. H. Nørholm

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest. The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool.
Original languageEnglish
Title of host publicationSynthetic Metabolic Pathways
Volume1671
Publication date2018
Pages27-37
ISBN (Print)978-1-4939-7294-4
ISBN (Electronic)978-1-4939-7295-1
DOIs
Publication statusPublished - 2018
SeriesMethods in Molecular Biology
ISSN1064-3745

Keywords

  • CRISPR interference
  • CRISPR-Cas9
  • CRMAGE
  • CRiPi
  • Essential genes
  • Genome editing
  • N-Degron
  • N-End rule pathway
  • PROTi
  • Protein stability

Cite this

Lauritsen, I., Martinez, V., Ronda, C., Nielsen, A. T., & Nørholm, M. H. H. (2018). Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability. In Synthetic Metabolic Pathways (Vol. 1671, pp. 27-37). Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-7295-1_3
Lauritsen, Ida ; Martinez, Virginia ; Ronda, Carlotta ; Nielsen, Alex Toftgaard ; Nørholm, Morten H. H. / Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability. Synthetic Metabolic Pathways. Vol. 1671 2018. pp. 27-37 (Methods in Molecular Biology).
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Lauritsen, I, Martinez, V, Ronda, C, Nielsen, AT & Nørholm, MHH 2018, Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability. in Synthetic Metabolic Pathways. vol. 1671, Methods in Molecular Biology, pp. 27-37. https://doi.org/10.1007/978-1-4939-7295-1_3

Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability. / Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta; Nielsen, Alex Toftgaard; Nørholm, Morten H. H.

Synthetic Metabolic Pathways. Vol. 1671 2018. p. 27-37 (Methods in Molecular Biology).

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

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AU - Martinez, Virginia

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AU - Nielsen, Alex Toftgaard

AU - Nørholm, Morten H. H.

PY - 2018

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N2 - In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest. The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool.

AB - In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest. The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool.

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Lauritsen I, Martinez V, Ronda C, Nielsen AT, Nørholm MHH. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability. In Synthetic Metabolic Pathways. Vol. 1671. 2018. p. 27-37. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7295-1_3