![]() ![]() Received: DecemAccepted: AugPublished: September 2, 2015Ĭopyright: © 2015 Tas et al. PLoS ONE 10(9):Įditor: Christophe Herman, Baylor College of Medicine, UNITED STATES 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.Ĭitation: Tas H, Nguyen CT, Patel R, Kim NH, Kuhlman TE (2015) An Integrated System for Precise Genome Modification in Escherichia coli. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl 2 for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. 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. 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). We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome.
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