Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination

Ralf Heermann,Kirsten Jung,Tim Zeppenfeld

doi:10.1186/1475-2859-7-14

Abstract

BackgroundIntroducing 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.ResultsHere 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®/ET® 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.ConclusionChromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.

Highlights

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
Chromosomal modifications performed by rpsL counter-selection may be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before
A powerful counter-selection system based on the rpsL gene and streptomycin selection for the introduction of point mutations into Bacterial Artificial Chromosomes (BACs) has been described [4], and this method was further useful for the recombination of large DNA-fragments into BACs [5]

Summary

Introduction

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. A powerful counter-selection system based on the rpsL gene (rpsL-neo) and streptomycin selection for the introduction of point mutations into Bacterial Artificial Chromosomes (BACs) has been described [4], and this method was further useful for the recombination of large DNA-fragments into BACs [5]. This counter-selection system is based on the rpsL gene encoding the S12 ribosomal protein, which is the target of streptomycin. Many E. coli strains commonly used for protein overproduction and/or metabolic engineering, including MC4100 [7], JM110 [8], Rosetta DE3 (Novagen, Darmstadt), HB101 (ATCC 33694), and (page number not for citation purposes)

Objectives

Methods

Results

Discussion

Conclusion