Recombineering Applications for the Mutational Analysis of Bacterial RNA-Binding Proteins and Their Sites of Action
Genetics remains a powerful tool to study structure–function relationships in proteins and RNA. Structural elements important for the biological activity of these molecules can be dissected through the isolation of mutations and analysis of their effects on the mechanism under study. In suitable model organisms, this approach can greatly benefit from the ability to introduce mutations directly in the chromosomal context in ways that do not perturb neighboring sequences. Methods for performing such “markerless” site-directed chromosomal mutagenesis in bacteria have been developed in recent years. One such technique, used routinely in our laboratory, is described here.
Key wordsRecombineering Lambda red Site-directed mutagenesis Counterselection Markerless Scarless
We would like to thank Kelly Hughes and Fabienne Chevance for initially encouraging us to develop the protocol described here. This work was supported by French National Research Agency (ANR) ANR-13-BSV3-0005-01.