An efficient recombination system for chromosome engineering in Escherichia coli

Daiguan Yu, Hilary M. Ellis, E. Chiang Lee, Nancy A. Jenkins, Neal G. Copeland, Donald L. Court

Research output: Contribution to journalArticle

1280 Scopus citations

Abstract

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective λ prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective λ prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

Original languageEnglish (US)
Pages (from-to)5978-5983
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number11
DOIs
StatePublished - May 23 2000

ASJC Scopus subject areas

  • General

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