An Integrated System for Precise Genome Modification in Escherichia coli.
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.
Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Huseyin
H
Tas
Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Cac T
CT
Nguyen
Department of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Ravish
R
Patel
Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Neil H
NH
Kim
Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Thomas E
TE
Kuhlman
D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005690:Galactose / Q000235:genetics; D017353:Gene Deletion; D050939:Gene Fusion; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
D017920:Antiporters; D001426:Bacterial Proteins; C079051:tetA protein, Bacteria; C022838:nickel chloride; D009532:Nickel; D005690:Galactose