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A Modified PCR-Directed Gene Replacements Method Using $lambda$-Red Recombination Functions in Escherichia coli  

KIM SANG-YOON (Department of Bioindustry and Technology, Sangji University)
CHO JAE-YONG (Department of Bioindustry and Technology, Sangji University)
Publication Information
Journal of Microbiology and Biotechnology / v.15, no.6, 2005 , pp. 1346-1352 More about this Journal
Abstract
We have developed a modified gene replacement method using PCR products containing short homologous sequences of 40- to 50-nt. The method required $\lambda$-Red recombination functions provided under the control of a temperature-sensitive CI857 repressor expressed from the $P_{lac}$ promoter in the presence of IPTG on an easily curable helper plasmid. The method promoted the targeted gene replacements in the Escherichia coli chromosome after shifting cultures of the recombinogenic host, which carries the helper plasmid, to $42^{\circ}C$ for 15 min. Since this method employs $\lambda$-Red recombination functions expressed from the easily curable helper plasmid, multiple rounds of gene replacements in the E. coli chromosome would be possible. The procedures described herein are expected to be widely used for metabolic engineering of E. coli and other bacteria.
Keywords
Gene replacement; cI857; helper plasmid; Escherichia coli;
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Times Cited By Web Of Science : 9  (Related Records In Web of Science)
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