A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.
Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor.
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering Institute, Seoul National University, Seoul, Republic of Korea.
Ji-Nu
JN
Kim
Jeong Sang
JS
Yi
Bo-Rahm
BR
Lee
Eun-Jung
EJ
Kim
Min Woo
MW
Kim
Yoseb
Y
Song
Byung-Kwan
BK
Cho
Byung-Gee
BG
Kim
D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D005818:Genetic Engineering; D016680:Genome, Bacterial / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000276:immunology*
D001426:Bacterial Proteins; D000939:Epitopes