GENE DISRUPTION IN ESCHERICHIA-COLI - TCR AND KM(R) CASSETTES WITH THE OPTION OF FLP-CATALYZED EXCISION OF THE ANTIBIOTIC-RESISTANCE DETERMINANT
GENE DISRUPTION IN ESCHERICHIA-COLI - TCR AND KM(R) CASSETTES WITH THE OPTION OF FLP-CATALYZED EXCISION OF THE ANTIBIOTIC-RESISTANCE DETERMINANT
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DOI：
10.1016/0378-1119(95)00193-a
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发表时间：
1995-05-26
期刊：
影响因子：
3.5
通讯作者：
WACKERNAGEL, W
中科院分区：
文献类型：
--
作者：
CHEREPANOV, PP;WACKERNAGEL, W
开发了两个具有四环素抗性(TC-R)和卡那霉素抗性(Km(R))决定簇的盒，用于构建大肠杆菌中克隆基因的插入和缺失突变体。在两个盒式磁带中，都有抵抗。决定簇的两侧是由酵母FLP重组酶介导的位点特异性重组所需的短直接重复序列(FRT位点)。此外，还构建了一种可在大肠杆菌中瞬时生产FLP酶的温度敏感型复制载体。在体外通过将一个盒插入到给定的基因中而构建基因中断或缺失突变后，突变的基因通过同源重组和对盒提供的抗生素耐药性的选择而转移到大肠杆菌染色体上。如果需要，抗性决定因素随后可以通过FLP作用从体内的染色体上移除，留下一个带有一个FRT位点的短核苷酸序列，并且对下游基因没有极性影响。将该体系应用于大肠杆菌A端缺失突变的构建，该突变可由P1以TC-R为标记转导至感兴趣的遗传背景。然后，如果需要，可以将换能器从TC-R中去除。
Two cassettes with tetracycline-resistance (Tc-R) and kanamycin-resistance (Km(R)) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance. determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzyme in E. coli has been constructed. After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E. coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette. If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes. This system was applied in the construction of an E. coli end A deletion mutation which can be transduced by P1 to the genetic background of interest using Tc-R as a marker. The transductant can then be freed of the Tc-R if required.