Replacing the Kanamycin Resistance Cassette with a Chloramphenicol Resistance Cassette in Plasmids for Transformation into Kanamycin Resistant Bacterial Strains Including the Keio Collection Using a Modified Gibson Assembly

Abstract

The kanamycin resistance (Kan^R) cassette used in the Escherichia coli Keio collection prevents the introduction of Kan^R plasmids into these strains, limiting their compatibility with common genetic tools. To overcome this barrier, we developed a protocol to replace the Kan^R cassette in plasmids with a chloramphenicol resistance (Cam^R) cassette using a modified Gibson Assembly strategy. This method involves linearization of the plasmid at a blunt-cutting site within the Kan^R gene, PCR amplification of the Cam^R cassette with homologous flanking regions, and assembly using the GeneArt™ Gibson Assembly® EX kit. The resulting Cam^R plasmid is suitable for transformation into Kan^R E. coli strains, including Keio knockouts, enabling co-selection and plasmid maintenance.