Hamilton C M, Aldea M, Washburn B K, Babitzke P, Kushner S R
Department of Genetics, University of Georgia, Athens 30602.
J Bacteriol. 1989 Sep;171(9):4617-22. doi: 10.1128/jb.171.9.4617-4622.1989.
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 degrees C. Subsequent growth of these cointegrates at 30 degrees 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 original 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.
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。该技术简单快速，可应用于大多数基因，甚至是那些必需基因。使这种方法独特且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，将质粒转化到合适的宿主中后，有可能在44℃下选择质粒整合到染色体中。这些共整合体随后在30℃下生长会导致第二次重组事件，从而使其解离。根据第二次重组事件发生的位置，染色体要么经历了基因替换，要么保留基因的原始拷贝。该程序还可用于实现等位基因从质粒到染色体的转移，或将染色体等位基因拯救到质粒上。由于解离后的质粒可通过选择进行维持，因此该技术可用于产生必需基因的缺失。
