Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes |
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Authors: | Ruth Serra-Moreno Sandra Acosta Jean Pierre Hernalsteens Juan Jofre Maite Muniesa |
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Institution: | (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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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. |
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