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We constructed a transposon ( t ransposon  a ssisted  g ene  i nsertion  t echnology, or TAGIT) that allows the random insertion of  gfp  (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan R  to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the  kan  and  lacZ  genes  in vivo . The resulting  gfp  insertions maintain target gene reading frame (to the 5′ and 3′ of  gfp ) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the  Escherichia coli  lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the  lacZ  operon. Several of these latter GFP insertions localize to  lacO  arrays integrated in the  E. coli  chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins