Directed evolution of transcriptional switches using dual-selector systems

Yuki Kimura, Daisuke Umeno*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter


Genetic switches provide core components of gene expression induction systems, genetic circuits, and metabolite sensors, including those for high-throughput screening and selection of enzyme functions. However, it is rare that natural transcription factors meet the required specifications for each application. Fortunately, the directed evolution of transcription switches is a straightforward process, given that the two states of genetic switches (on and off) are both selectable, using a wide range of positive and negative selection tools developed in the field of molecular genetics. On/off-state selections based on bactericidal mechanism allow greatly accelerate the entire process. The key to success is finding the selection conditions that minimize false-positive and false-negative clones as with any directed evolution experiment. We introduce a reliable and automatable directed evolution platform to rapidly evolve genetic switches with desired specifications in this chapter. Highlighting the importance and ease of screening for selection conditions, we demonstrate how selection conditions influence the resultant population of the selected pools.

Original languageEnglish
Title of host publicationEnzyme Engineering and Evolution
Subtitle of host publicationSpecific Enzyme Applications
EditorsDan S. Tawfik
PublisherAcademic Press Inc.
Number of pages17
ISBN (Print)9780128244319
Publication statusPublished - 2020
Externally publishedYes

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Aminoglycoside phosphotransferase
  • Genetic selection
  • Genetic switch
  • Thymidine kinase
  • Transcription factor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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