Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes

Leonard Schärfen, Miloš Tišma, Michael Schlierf

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Fluorescence microscopy has become a powerful tool in molecular cell biology. Visualizing specific proteins in bacterial cells requires labeling with fluorescent or fluorogenic tags, preferentially at the native chromosomal locus to preserve expression dynamics associated with the genomic environment. Exploring protein function calls for targeted mutagenesis and observation of differential phenotypes. In the model bacterium Escherichia coli, protocols for tagging genes and performing targeted mutagenesis currently involve multiple steps. Here, we present an approach capable of simultaneous tagging and mutagenesis of essential and nonessential genes in a single step. We require only the insertion of a stretch of the target gene into an auxiliary plasmid together with the tag. Recombineering-based exchange with the native locus is then carried out, where the desired mutation is introduced during amplification with homology-bearing primers. Using this approach, multiple tagged mutants per gene can be derived quickly.

Original languageEnglish
Pages (from-to)2203-2207
JournalACS Synthetic Biology
Volume9
Issue number8
DOIs
Publication statusPublished - 2020

Keywords

  • fluorescence microscopy
  • genome engineering
  • point mutagenesis
  • protein tagging
  • recombineering

Fingerprint Dive into the research topics of 'Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes'. Together they form a unique fingerprint.

Cite this