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Open Access Highly Accessed Methodology article

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

Ruth Serra-Moreno1, Sandra Acosta1, Jean Pierre Hernalsteens2, Juan Jofre1 and Maite Muniesa1*

Author Affiliations

1 Department of Microbiology. Faculty of Biology. University of Barcelona. Diagonal 645. E-08028 Barcelona. Spain

2 Viral Genetics Laboratory, Faculty of Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium

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BMC Molecular Biology 2006, 7:31  doi:10.1186/1471-2199-7-31

Published: 19 September 2006

Abstract

Background

The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene.

Results

Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol.

Conclusion

This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.