Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

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

doi:10.1007/978-1-4939-7295-1_3

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 proceeding › Book chapter › Research › peer-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 language	English
Title of host publication	Synthetic Metabolic Pathways
Volume	1671
Publication date	2018
Pages	27-37
ISBN (Print)	978-1-4939-7294-4
ISBN (Electronic)	978-1-4939-7295-1
DOIs	https://doi.org/10.1007/978-1-4939-7295-1_3
Publication status	Published - 2018

Series	Methods in Molecular Biology
ISSN	1064-3745

Keywords

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

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10.1007/978-1-4939-7295-1_3

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title = "Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability",

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.",

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language = "English",

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T1 - Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

AU - Lauritsen, Ida

AU - Martinez, Virginia

AU - Ronda, Carlotta

AU - Nielsen, Alex Toftgaard

AU - Nørholm, Morten H. H.

PY - 2018

Y1 - 2018

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.

KW - CRISPR interference

KW - CRISPR-Cas9

KW - CRMAGE

KW - CRiPi

KW - Essential genes

KW - Genome editing

KW - N-Degron

KW - N-End rule pathway

KW - PROTi

KW - Protein stability

U2 - 10.1007/978-1-4939-7295-1_3

DO - 10.1007/978-1-4939-7295-1_3

M3 - Book chapter

C2 - 29170951

SN - 978-1-4939-7294-4

VL - 1671

T3 - Methods in Molecular Biology

SP - 27

EP - 37

BT - Synthetic Metabolic Pathways

ER -

Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

Abstract

Keywords

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