质粒
生物
复制子
遗传学
枯草芽孢杆菌
基因座(遗传学)
大肠杆菌
细菌圆形染色体
染色体
可选择标记
DNA
基因
分子生物学
细菌
作者
Ian C. Blomfield,V Vaughn,R F Rest,B I Eisenstein
标识
DOI:10.1111/j.1365-2958.1991.tb00791.x
摘要
Summary To facilitate efficient allelic exchange of genetic information into a wild‐type strain background, we improved upon and merged approaches using a temperature‐sensitive plasmid and a counter‐selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild‐type chromosomal sequences, at either the fimB–A or lacZ–A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn 5 , but, unlike another similar cassette, it lacks IS 1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature‐sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non‐selective conditions at 42°C, and then in the presence of sucrose at 30°C, allowing positive selection for both plasmid excision and curing. Despite marked locus‐specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non‐essential parts of the E. coli chromosome.
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