Le Borgne S, Palmeros B, Valle F, Bolivar F, Gosset G
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
Gene. 1998 Nov 26;223(1-2):213-9. doi: 10.1016/s0378-1119(98)00168-1.
A pBRINT-Ts 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-Ts 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 alpha-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.
通过使用源自pSC101的温度敏感复制子、与lacZ基因的同源区域以及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），构建了用于大肠杆菌的pBRINT-Ts整合载体家族。待整合的基因或基因组可插入多克隆位点，两侧为抗生素抗性标记和lacZ序列。开发了一种简单快速的程序来选择发生等位基因交换的细胞。通过该程序，在使用几种大肠杆菌菌株时，观察到整合效率约为10-3。从整合了pBRINT-Ts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，我们将编码嗜热栖热放线菌α-淀粉酶的amy基因整合到大肠杆菌W3110的lacZ基因中。发现α-淀粉酶的产生与拷贝数成正比；每条染色体最多10个拷贝。
