Figure 1.

Comparative illustration of RIVET (left) and TRIVET (right) fused to a hypothetical geneX (dark grey) in the ON/OFF scenario of its promotor. Additional chromosomal sequences are highlighted in light gray, the res cassette parts in yellow, the integrated suicide vectors (pIVET and pTRIVET) harbouring tnpR in red, and tetR-phoA-cat (tpc) cassette in green flanked by IS10 sites (black). In case of RIVET, the pIVET suicide vector is integrated into V. cholerae hypothetical geneX via homologous recombination resulting in a merodiploid in which geneX and tnpR (resolvase) are transcriptionally fused and controlled by the chromosomal promotor of geneX. TnpR expression via activation of the geneX promotor results in an irreversible excision of the res cassette marked by a change in the resistance profile of the cell [kanamycin resistant (Kn^R) and sucrose sensitive (Suc^S) to kanamycin sensitive (Kn^S) and sucrose resistant (Suc^R)]. Thus, resolved strains (loss of the res cassette) can be selected by their ability to grow in sucrose.