Nature Biotechnology
20, 1018 - 1023 (2002)
Published online: 16 September 2002; | doi:10.1038/nbt740
Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision systemByung Jo Yu1, 4, Bong Hyun Sung1, 4, Michael D. Koob2, Choong Hoon Lee1, Jun Hyoung Lee1, Won Sik Lee1, Mi Sun Kim3
& Sun Chang Kim11
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, Korea. 2
Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA. 3
Biomass Team, Korea Institute of Energy Research, Taejon 305-343, Korea. 4
These two authors contributed equally to this work.
Correspondence should be addressed to Sun Chang Kim sckim@mail.kaist.ac.krAn 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 loxP 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.
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