NEW METHOD FOR GENERATING DELETIONS AND GENE REPLACEMENTS IN ESCHERICHIA-COLI
NEW METHOD FOR GENERATING DELETIONS AND GENE REPLACEMENTS IN ESCHERICHIA-COLI
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DOI：
10.1128/jb.171.9.4617-4622.1989
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发表时间：
1989-09-01
影响因子：
3.2
通讯作者：
KUSHNER, SR
中科院分区：
文献类型：
--
作者：
HAMILTON, CM;ALDEA, M;KUSHNER, SR
我们描述了一种在大肠杆菌中产生基因替换和缺失的方法。这项技术简单而快速，可以应用于大多数基因，甚至是那些必不可少的基因。使这种方法独特和特别有效的是使用温度敏感的pSC101复制子来促进基因替换。该方法通过染色体上的基因与携带在对DNA复制温度敏感的质粒上的同源序列之间的同源重组进行。因此，在质粒转化到合适的宿主后，可以选择44度的质粒与染色体整合。C导致第二次重组事件，导致它们的分解。根据第二次重组发生的位置，染色体要么经历了基因替换，要么保留了基因的原始副本。该程序也可用于将等位基因从质粒转移到染色体上，或将染色体等位基因拯救到质粒上。由于被分解的质粒可以通过选择维持，因此该技术可用于产生必要基因的缺失。
We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44.degree. C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the origianl copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.