PCR-based tandem epitope tagging system for Escherichia coli genome engineering.

TitlePCR-based tandem epitope tagging system for Escherichia coli genome engineering.
Publication TypeJournal Article
Year of Publication2006
AuthorsCho B-K, Knight EM, Palsson BØ
PubMed Date2006 Jan
KeywordsBlotting, Western, Chromatin Immunoprecipitation, Epitopes, Escherichia coli, Genes, myc, Genomics, Polymerase Chain Reaction, Protein Engineering, Recombinant Fusion Proteins, Tandem Repeat Sequences, Transcription Factors

Biological discovery in the postgenomic era requires a systematic and high-throughput experimental approach. To this end, a versatile PCR-based tandem epitope tagging system is described, which inserts a tandem epitope coding sequence into any desired position of the Escherichia coli chromosome. Template plasmids were constructed that carry tandem copies of the epitope encoding sequence, Flp recombinase target (FRT) sites, and antibiotic resistance genes. The linear DNA fragment, amplified from the template plasmid with extensions homologous to the end of the target gene and part of its downstream region, was transformed into E. coli K-12 MG1655 harboring the bacteriophage gamma Red recombination system. The antibiotic resistance gene was then removed from the inserted heterologous PCR fragment using Flp recombinase. This epitope tagging system was applied to global transcription factors of E. coli to obtain proteins fused with tandem c-myc epitope tags. The tandem myc epitope-fused transcription factors were successfully detected by Western blot analysis and chromatin immunoprecipitation with increased detection sensitivity and higher yield. Higher copy numbers of the epitope molecule allowed the use of more stringent experimental conditions to increase the signal-to-noise ratio in subsequent experimental applications. Furthermore, judging from the measurement of gene expression using reverse transcription PCR (RT-PCR), the epitope-fused transcription factors retained their normal function for gene regulation in vivo.

Alternate JournalBioTechniques
PubMed ID16454042



417 Powell-Focht Bioengineering Hall

9500 Gilman Drive La Jolla, CA 92093-0412

Contact Us

Contact Us

In Silico Lab:  858-822-1144

Wet Lab:  858-246-1625

FAX:   858-822-3120

Website Concerns: sbrgit@ucsd.edu


Visit the Official SBRG YouTube Channel

User Login