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

  • Maja Rennig
  • Daniel O. Daley
  • Morten H. H. Nørholm
Part of the Methods in Molecular Biology book series (MIMB, volume 1671)

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Maja Rennig
    • 1
  • Daniel O. Daley
    • 2
  • Morten H. H. Nørholm
    • 1
  1. 1.Novo Nordisk Foundation Center for BiosustainabilityTechnical University of Denmark2800 Kgs. LyngbyDenmark
  2. 2.Department of Biochemistry and Biophysics, Center for Biomembrane ResearchStockholm UniversityStockholmSweden

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