pBRINT-T-s: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZgene of Escherichia coli

A pBRINT-T-s family of integrative vectors for Escherichia coli was constru cted by using a temperature-sensitive replicon derived from pSC101, a regio n of homology to the lacZ gene, and various antibiotic resistance markers ( kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multipl e cloning site, flanked by an antibiotic resistance marker and lacZ sequenc es. A simple and rapid procedure was developed for the selection of cells w here allelic exchange had occurred. With this procedure, the efficiency of integration of around 10(-3) was observed, using several E. coli strains. F rom colonies with an integrated pBRINT-T-s plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we int egrated the amy gene that codes for the cc-amylase from B. stearothermophil us, into the lacZ gene of E, coli W3110. Production of alpha-amylase was fo und to be proportional to copy number; at up to 10 copies per chromosome. ( C) 1998 Elsevier Science B.V. All rights reserved.