Minimization of the Escherichia coli genome using a Tn5-targeted Cre/IoxP excision system

Byung Jo Yu, Bong Hyun Sung, Michael D. Koob, Choong Hoon Lee, Jun Hyoung Lee, Won Sik Lee, Mi Sun Kim, Sun Chang Kim

Research output: Contribution to journalArticlepeer-review

122 Scopus citations

Abstract

An increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. To facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. We made two large pools of independent transposon mutants in Escherichia coli using modified Tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated IoxP recombination, we obtained E. Coli strains in which large genomic fragments (59-117 kilobases) were deleted. Some of these individual deletions were then combined into a single "cumulative deletion strain" that lacked 287 open reading frames (313.1 kilobases) but that nevertheless exhibited normal growth under standard laboratory conditions.

Original languageEnglish (US)
Pages (from-to)1018-1023
Number of pages6
JournalNature biotechnology
Volume20
Issue number10
DOIs
StatePublished - Oct 2002

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