A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria.

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.