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Fig. 2 | Microbial Cell Factories

Fig. 2

From: Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome

Fig. 2

Strategy of the high molecular weight DNA integration into the E. coli chromosome. a Figure depicts main steps of the integration of the high molecular weight DNA into the E. coli chromosome. DNA fragments to be integrated into the E. coli chromosome are first tagged with the selectable markers by cloning into plasmids p14kan and pJScav. p14kan and pJScav encode the kanamycin cassette (kan) and chloramphenicol-yellow fluorescent protein cassette (cat-yfp), respectively. DNA fragments attached to kan or cat-yfp cassettes are transformed into the electro-competent E. coli cells harbouring plasmid pKM208. pKM208- encoded lambda Red recombination system integrates DNA fragments into the chromosome by homologous recombination. Successful transformants from the chromosomal integration of the first DNA fragment attached to kan cassette are selected on medium with kanamycin. Second fragment attached to cat-yfp cassette integrates into the chromosome next to the first fragment, thus replacing the kan resistance marker. Alternative use of kan and cat-yfp cassettes allows elongation of the integrated DNA sequence. b Growth and antibiotic selection of E. coli harbouring integrated high molecular weight DNA tagged with kan or cat-yfp cassette. Strains with kan and cat-yfp cassettes grow only on kanamycin and chloramphenicol plates, respectively. Furthermore, cells with cat-yfp cassette produce fluorescent light

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