Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes

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.

Direct visualization of four diffusive LexA states controlling SOS response strength during antibiotic treatment

“…Therefore, we expressed LexA E45K and LexA 3x from Plac at the lacZ locus in addition to the untagged chromosomal lexA. The S119A mutation was inserted at the native locus on the chromosome (30) to serve as a negative control for SOS induction. We imaged all mutants under the same conditions as LexA wt in unstressed cells, obtained localizations and tracked single molecules.…”

“…A targeting cassette including ~50 bp homology to the target region on either end was amplified by PCR. We described the construction of the genomic LexA S119A mutation previously (30). Recombineering (63) was carried out using a protocol adapted from (64).…”