Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions.
We have developed a general method for the introduction of any cloned sequence into the chromosome of Escherichia coli. This method employs an Hfr strain which carries a fragment of bla (the pBR322 gene imparting ampicillin resistance) between lacI and lacZ. Plasmid-borne inserts which are flanked by sequences from bla and lacZ can be introduced at this locus by homologous recombination. The isolation of recombinants is enhanced by selection for transfer of an integrated copy of the plasmid during conjugation. Once introduced into the chromosome, the inserted sequences can be transferred to other strains by conventional methods such as P1 transduction or conjugation. This method is suitable for the transfer of any cloned sequence to the chromosome and is particularly well suited to the construction of chromosomal gene and operon fusions with lacZ.
Resnik
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Department of Biochemistry, University of Minnesota, Minneapolis 55455.
LaPorte
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D000668:Ampicillin Resistance / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D007522:Isocitrate Lyase / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D001616:beta-Galactosidase / Q000235:genetics
D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D007522:Isocitrate Lyase