pBRINT-Ts:: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZgene of Escherichia coli
pBRINT-Ts:: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZgene of Escherichia coli
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DOI：
10.1016/s0378-1119(98)00168-1
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发表时间：
1998-11-26
期刊：
影响因子：
3.5
通讯作者：
Gosset, G
中科院分区：
文献类型：
--
作者：
Le Borgne, S;Palmeros, B;Gosset, G
大肠杆菌的pBRINT-T-s家族整合载体的构建是通过使用来自pSC 101的温度敏感复制子、lacZ基因的同源区域和用于选择整合体的各种抗生素抗性标记（卡那霉素、氯霉素和庆大霉素）。可以将待整合的基因或基因组插入多克隆位点，侧翼为抗生素抗性标记和lacZ序列。开发了一种简单而快速的程序，用于选择发生等位基因交换的细胞。用这种方法，用几个E.大肠杆菌菌株。从具有整合的pBRINT-T-s质粒的菌落中，我们检测到平均等位基因交换事件频率为7.5%。作为对该系统的测试，我们整合了编码来自B的α-淀粉酶的amy基因。将嗜热脂肪菌的lacZ基因插入大肠杆菌W3110的lacZ基因中。发现α-淀粉酶的产生与拷贝数成比例;每条染色体多达10个拷贝。(C)1998 Elsevier Science B. V.保留所有权利。
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.