An integrated system for precise genome modification in Escherichia coli

Huseyin Tas, Cac T. Nguyen, Ravish Patel, Neil H. Kim, Thomas E. Kuhlman

Research output: Contribution to journalArticle

Abstract

We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl2 for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. We demonstrate the power of this method to make a variety of genome modifications: the exact integration, without any extraneous sequence, of the lac operon (~6.5 kbp) to any desired location in the genome and without the integration of antibiotic markers; the scarless deletion of ribosomal rrn operons (~6 kbp) through either intrachromosomal or oligonucleotide recombination; and the in situ fusion of native genes to fluorescent reporter genes without additional perturbation.

Original languageEnglish (US)
Article numbere0136963
JournalPloS one
Volume10
Issue number9
DOIs
StatePublished - Sep 2 2015

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Tas, H., Nguyen, C. T., Patel, R., Kim, N. H., & Kuhlman, T. E. (2015). An integrated system for precise genome modification in Escherichia coli. PloS one, 10(9), [e0136963]. https://doi.org/10.1371/journal.pone.0136963