Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red(R)/ET(R) Recombination.

Microbial Cell Factories
Ralf HeermannKirsten Jung

Abstract

Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red(R)/ET(R) Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red(R)/ET(R) Recombination has been applied to several enteric bacteria before.

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Citations

May 27, 2011·Folia Microbiologica·Mahesh MadyagolStanislav Stuchlík
Apr 25, 2009·BMC Molecular Biology·Joanna I KatashkinaSergey V Mashko
Dec 3, 2008·Microbial Cell Factories·Maurilio De FeliceAntonio Villaverde
Nov 5, 2010·Journal of Biomedicine & Biotechnology·Charles WardenHua Zhu
Feb 16, 2016·Biological Procedures Online·Michela CasanovaPaolo Magni
Sep 24, 2013·Journal of Molecular Biology·Martina RauschmeierKirsten Jung
Oct 16, 2015·Scientific Reports·Chris R Reisch, Kristala L J Prather
Oct 2, 2012·Journal of Microbiological Methods·Luitpold FriedKirsten Jung
Nov 28, 2012·Biochemical Society Transactions·Katrin Richter, Johannes Gescher
Mar 25, 2015·Nucleic Acids Research·Varnica KhetrapalSwaine L Chen
Feb 17, 2015·Nature Chemical Biology·Jürgen LassakKirsten Jung
Jun 29, 2018·PloS One·Magdalena Motz, Kirsten Jung
Feb 22, 2012·Applied and Environmental Microbiology·Huruma Nelwike TuntufyeBruno Maria Goddeeris
Jun 12, 2012·Journal of Bacteriology·Tobias KraxenbergerKirsten Jung
Jun 27, 2017·Biotechnology for Biofuels·Jae Hyung Lim, Gyoo Yeol Jung
Nov 2, 2019·Mammalian Genome : Official Journal of the International Mammalian Genome Society·Lukasz PiszczekCornelius Gross
Apr 23, 2020·International Journal of Molecular Sciences·Si Won KimTae Sung Jung
Oct 26, 2018·Frontiers in Microbiology·Xiang AoYingying Guo
Feb 18, 2018·Research in Microbiology·Lacey L WestphalKarin E Kram
May 26, 2016·EcoSal Plus·Kenan C Murphy
Jul 10, 2020·ACS Synthetic Biology·Leonard SchärfenMichael Schlierf

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Methods Mentioned

BETA
PCR

Software Mentioned

Red
Ec2

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