Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes

Research output: Contribution to journalResearch articleContributedpeer-review



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
Number of pages5
JournalACS synthetic biology
Issue number8
Publication statusPublished - 21 Aug 2020

External IDs

ORCID /0000-0002-6209-2364/work/142237616
Scopus 85089787046



  • protein tagging, point mutagenesis, recombineering, genome engineering, fluorescence microscopy, ESCHERICHIA-COLI, RECOMBINATION, CLONING