Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers

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

Abstract

Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can be fused at the 3'-end with a translational coupling element and an antibiotic resistance gene. Highly expressed target genes can then be selected using a fast and simple whole cell survival assay in the presence of high antibiotic concentrations. Herein we show that the system can be used to select highly expressing clones from libraries sampling translation initiation sites.
Original languageEnglish
Title of host publicationSynthetic Metabolic Pathways
Volume1671
Publication date2018
Pages259-268
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

  • Antibiotic resistance
  • Gene expression
  • Library screening
  • Protein production optimization
  • Selection
  • Translational coupling

Cite this

Rennig, Maja ; Daley, Daniel O. ; Nørholm, Morten H. H. / Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers. Synthetic Metabolic Pathways. Vol. 1671 2018. pp. 259-268 (Methods in Molecular Biology).
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Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers. / Rennig, Maja; Daley, Daniel O.; Nørholm, Morten H. H.

Synthetic Metabolic Pathways. Vol. 1671 2018. p. 259-268 (Methods in Molecular Biology).

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

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T1 - Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers

AU - Rennig, Maja

AU - Daley, Daniel O.

AU - Nørholm, Morten H. H.

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AB - Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can be fused at the 3'-end with a translational coupling element and an antibiotic resistance gene. Highly expressed target genes can then be selected using a fast and simple whole cell survival assay in the presence of high antibiotic concentrations. Herein we show that the system can be used to select highly expressing clones from libraries sampling translation initiation sites.

KW - Antibiotic resistance

KW - Gene expression

KW - Library screening

KW - Protein production optimization

KW - Selection

KW - Translational coupling

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