An Integrated System for Precise Genome Modification in Escherichia coli

• Published in: PloS one Vol. 10; no. 9; p. e0136963
• Main Authors: Tas, Huseyin, Nguyen, Cac T, Patel, Ravish, Kim, Neil H, Kuhlman, Thomas E
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02.09.2015; Public Library of Science (PLoS)
• Subjects: Animal behavior; Antibiotics; Antiporters - genetics; Bacteria; Bacterial Proteins - genetics; Base Sequence; Biophysics; CRISPR; Deoxyribonucleic acid; DNA; E coli; Endonuclease; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - growth & development; Fluorescence; Galactose; Galactose - genetics; Gene Deletion; Gene expression; Gene Fusion; Gene Targeting; Genes; Genetic aspects; Genetic engineering; Genetic Engineering - methods; Genome, Bacterial; Genomes; Genomics; Homing; Homing endonuclease; Homologous Recombination; Homology; Integration; Lactose operon; Landing; Molecular biology; Molecular Sequence Data; Mutation; Nickel - metabolism; Nucleotide sequence; Oligonucleotides; Operons; Physics; Plasmids; Plasmids - genetics; Sugar; Transformation, Genetic; United States; United States > US; Illinois
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0136963

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.