A PBRINT FAMILY OF PLASMIDS FOR INTEGRATION OF CLONED DNA INTO THE ESCHERICHIA-COLI CHROMOSOME

Plasmid pBRINT is an efficient vector for chromosomal integration of c loned DNA into the lacZ gene of Escherichia coli [Balbas et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (Cm-R or Gm(R) or Km(R)) and a larger mu ltiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. T heir functionality as integration vectors has been ascertained by inte grating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobac terium leiognathi lux genes. To evaluate the level of expression obtai ned after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compa red their enzymatic activities with those obtained from a strain carry ing cat on a multicopy plasmid.