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 CAnb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm-R, Km(R) and Gm(R)), wh ich could easily be inserted into the genome and subsequently removed by us ing the Cre/loxP site-specific recombination system of bacteriophage pi. In this manner the same marker could be used more than once in the same backg round, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of t he 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 ori gamma/pir-dependent suicide p lasmid, which contained a dominant Sm-R gene. The cassettes were crossed in to the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Posfai et al. [Nucl. Acids Res. 22 (1 994) 2392-2398], selecting for the Cm-R, Km(R), or Gm(R), for the LacZ(-) o r GalE(-) and for the Sm-S phenotypes (the latter to assure allelic exchang e rather than insertion of the entire plasmid). When required, after select ing the strain with the desired modification, the CmR, KmR, or GmR marker w as excised by supplying the Cre function. Cre was provided by the thermosen sitive plasmid pJW168, which was transformed into the Anb(R) host at 30 deg rees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) ma rker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site. (C) 2000 Elsevier Science B.V. All rights reserved.