An Integrated System for Precise Genome Modification in Escherichia coli

PloS One
Huseyin TasThomas E Kuhlman

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.

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Citations

Apr 14, 2016·Journal of Microbiological Methods·Shalini S DebArvind M Lali
Apr 14, 2016·ACS Synthetic Biology·Marcelo C BassaloRyan T Gill
Jun 15, 2016·Proceedings of the National Academy of Sciences of the United States of America·Neil H KimThomas E Kuhlman
Jan 12, 2017·ACS Synthetic Biology·Zihe LiuHuimin Zhao
Jun 7, 2020·G3 : Genes - Genomes - Genetics·Nicholas A Sherer, Thomas E Kuhlman
Jun 14, 2016·Biopolymers·Tyler M EarnestZaida Luthey-Schulten
Nov 30, 2018·The Journal of General and Applied Microbiology·Gumpanat MahipantAlisa S Vangnai
Nov 21, 2018·Proceedings of the National Academy of Sciences of the United States of America·Gloria LeeThomas E Kuhlman
Oct 6, 2020·Frontiers in Microbiology·Ursula FelsPetra Van Damme
Jun 13, 2019·Cell Reports·Andrew F SchoberKimberly A Reynolds
May 1, 2021·Microorganisms·Ruben D Arroyo-OlarteEdgar Morales-Ríos
Sep 4, 2021·Nucleic Acids Research·Kok Zhi LeeKevin V Solomon

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Datasets Mentioned

BETA
KR071147

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

MATLAB
ALFIRE
MAGIC
PLRS
KIKO
FRUIT

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