Gutterson N I, Koshland D E
Proc Natl Acad Sci U S A. 1983 Aug;80(16):4894-8. doi: 10.1073/pnas.80.16.4894.
An efficient method for the replacement of chromosomal DNA by segments altered in vitro has been developed for bacteria. The method requires (i) a recombinant plasmid with a ColE1-like replicon and (ii) a strain defective in DNA polymerase I (polA), which is unable to replicate the plasmid extrachromosomally. This method is of general use since there are a number of suitable vectors and polA strains are available in both Escherichia coli and Salmonella typhimurium, the two most widely studied bacterial species. Using the method, we have constructed two chromosomal deletions in the chemotaxis gene region of S. typhimurium. In addition, plasmid sequences integrated into the chromosome have been amplified up to 30-fold by varying the concentration of ampicillin or tetracycline in the growth medium.
已为细菌开发出一种通过体外改变的片段替换染色体DNA的有效方法。该方法需要（i）带有ColE1样复制子的重组质粒，以及（ii）DNA聚合酶I（polA）缺陷的菌株，该菌株无法在染色体外复制质粒。由于有许多合适的载体，并且在两种研究最广泛的细菌物种大肠杆菌和鼠伤寒沙门氏菌中都有polA菌株，所以该方法具有普遍用途。使用该方法，我们在鼠伤寒沙门氏菌的趋化性基因区域构建了两个染色体缺失。此外，通过改变生长培养基中氨苄青霉素或四环素的浓度，整合到染色体中的质粒序列已扩增至30倍。
