Rapid engineering of bacterial reporter gene fusions by using Red recombination.
Reporter gene fusions are essential tools for the investigation of gene regulation. Such fusions are traditionally generated by transposon mutagenesis and identified by a suitable selection procedure. Alternatively, specific reporter fusions can be generated by cloning of DNA fragments containing promoters or other regulatory elements in reporter plasmids. Here, we describe a novel approach for the rapid generation of reporter gene fusions in single copies at defined positions in bacterial genomes. This technique utilizes the Red recombinase for the homologous recombination of PCR-generated cassettes containing various currently used reporter genes, such as those for beta-galactosidase, luciferase, and green fluorescent protein. The approach allows the generation of transcriptional or translational reporter fusions in a single step without the requirement for recombinant DNA constructs and is applicable to various enterobacterial species. Generation of reporter fusions by Red recombination is rapid, overcomes the current limitations of transposon mutagenesis or reporter plasmids, and offers new options for the study of bacterial gene regulation.
Gerlach
Roman G
RG
Mikrobiologisches Institut, Universitätsklinikum Erlangen, Wasserturmstr 3-5, Erlangen, Germany.
Hölzer
Stefanie U
SU
Jäckel
Daniela
D
Hensel
Michael
M
D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D045522:Recombinases; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics; D012763:Shigella flexneri / Q000235:genetics
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D045522:Recombinases; D049452:Green Fluorescent Proteins; D008156:Luciferases