Protocol

Artificial Riboswitches

Volume 1111 of the series Methods in Molecular Biology pp 57-75

Date:

Artificial Riboswitch Selection: A FACS-Based Approach

  • Zohaib GhaziAffiliated withDepartment of Biochemistry and Biomedical Sciences, McMaster UniversityDepartment of Chemistry and Chemical Biology, McMaster UniversityMichael G. DeGroote Institute for Infectious Disease Research, McMaster University
  • , Casey C. FowlerAffiliated withDepartment of Biochemistry and Biomedical Sciences, McMaster UniversityDepartment of Chemistry and Chemical Biology, McMaster UniversityMichael G. DeGroote Institute for Infectious Disease Research, McMaster University
  • , Yingfu LiAffiliated withDepartment of Biochemistry and Biomedical Sciences, McMaster UniversityDepartment of Chemistry and Chemical Biology, McMaster UniversityMichael G. DeGroote Institute for Infectious Disease Research, McMaster University

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Abstract

Riboswitches have a number of characteristics that make them ideal regulatory elements for a wide range of synthetic biology applications. To maximize their utility, methods are required to create custom riboswitches de novo or to modify existing riboswitches to suit specific experimental needs. This chapter describes such a method, which exploits fluorescence-activated cell sorting (FACS) to quickly and efficiently sort through large libraries of riboswitch-like sequences to identify those with the desired activity. Suggestions for the experimental setup are provided, along with detailed protocols for testing and optimizing FACS conditions FACS selection steps, and follow-up assays to identify and characterize individual riboswitches.

Key words

Artificial riboswitch Aptamer Biosensor Fluorescence-activated cell sorting (FACS) High-throughput Enrichment