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

Nature Biotechnology volume 20, pages 1018–1023 (2002)Cite this article

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 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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Figure 1: Tn5-mediated insertions of the Cre/loxP excision system and deletion of the target regions.
Figure 2: Physical maps of the Cmr and Kmr inserts.
Figure 3: Deletion of genomic segments and confirmation by PCR.

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Acknowledgements

This work was supported in part by grants from the National Research Laboratory Program (2000-N-NL-01-C-160) and the Molecular Medicine Research Program (00-J-MM-01-B-01) from the Korean Ministry of Science and Technology.

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Author notes

  1. Byung Jo Yu and Bong Hyun Sung: These two authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, Korea

    Byung Jo Yu, Bong Hyun Sung, Choong Hoon Lee, Jun Hyoung Lee, Won Sik Lee & Sun Chang Kim

  2. Institute of Human Genetics, University of Minnesota, Minneapolis, 55455, MN, USA

    Michael D. Koob

  3. Biomass Team, Korea Institute of Energy Research, Taejon, 305-343, Korea

    Mi Sun Kim

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  1. Byung Jo Yu
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Correspondence to Sun Chang Kim.

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Yu, B., Sung, B., Koob, M. et al. Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol 20, 1018–1023 (2002). https://doi.org/10.1038/nbt740

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