Technical Notes

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

Ralf Heermann¹ , Tim Zeppenfeld² and Kirsten Jung¹

¹Ludwig-Maximilians-Universität München, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Str. 1a, D-80638 München, Germany

²Gene Bridges GmbH, Im Neuenheimer Feld 584, 69120 Heidelberg, Germany

author email corresponding author email

Microbial Cell Factories 2008, 7:14doi:10.1186/1475-2859-7-14

Published:	24 April 2008

Abstract

Background

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.

Results

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^®/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.

Conclusion

Chromosomal 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.