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

被引：17

作者：

Le Borgne, S ^{[1
]}

Palmeros, B ^{[1
]}

Valle, F ^{[1
]}

Bolivar, F ^{[1
]}

Gosset, G ^{[1
]}

机构：

[1] Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Mol Microbiol, Cuernavaca 62191, Morelos, Mexico

来源：

GENE | 1998年 / 223卷 / 1-2期

关键词：

genetic engineering; homologous recombination; heterologous gene expression; gene dosage alpha-amylase; Bacillus stearothermophilus;

D O I：

10.1016/S0378-1119(98)00168-1

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

A pBRINT-T-s family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region 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 multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10(-3) was observed, using several E. coli strains. From 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 integrated the amy gene that codes for the cc-amylase from B. stearothermophilus, into the lacZ gene of E, coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome. (C) 1998 Elsevier Science B.V. All rights reserved.

引用

页码：213 / 219

页数：7

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