Genetics and Genomics Commons^™

A Mini-Tn5-Derived Transposon With Reportable And Selectable Markers Enables Rapid Generation And Screening Of Insertional Mutants In Gram-Negative Bacteria, Eric S. Nazareno, Bimala Acharya, C. Korsi Dumenyo

Agricultural and Environmental Sciences Faculty Research

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The …