Engineering riboswitch‐based whole cell biosensors in Escherichia coli

Abstract

In vitro diagnostics (IVD) have become increasingly popular tool for tackling global health concerns, but conventional IVD tests, such as microscopy and nucleic‐acid amplification methods, are often expensive and complex to use, limiting their use in low‐resource settings. Whole‐cell biosensors in bacterial systems offer an attractive solution as an easy‐to‐use, portable device that non‐specialists could use for clinical purposes outside clinical locations. However, a strategy is needed to engineer ligand specific sensors for new targets. An appealing candidate for a sensing module is the riboswitch. Genetic RNA switches, termed riboswitches, are genetic regulatory elements found in the 5′‐UTR (untranslated region) of some prokaryotic mRNA and contain two functional components: an aptamer binding domain and an expression platform that modulates downstream gene expression. We developed an engineering platform coupling genetic selections and Fluorescence‐Activated Cell Sorting (FACS) screens to identify novel riboswitches from a 108 random‐sequence library in which we replaced the aptamer sequence of the ThiM#2 riboswitch with a degenerate 40‐nucleotide sequence. In a proof‐of‐principle validation of the platform, we identified novel riboswitches for the small molecule theophylline, which are distinct from the existing synthetic theophylline switches. Our best riboswitch hit displays a 2.3‐fold activation in response to theophylline and does not respond to the structurally unrelated molecule thiamine. We performed directed evolution on this riboswitch hit by mutagenizing its sequence and performing additional rounds of selections and screens, resulting in a variant with a 3.6‐fold activation. Current efforts are focused on assessing the efficacy of the selection platform when isolating riboswitches for dopamine. Furthermore, a variant of the engineering platform is being used side‐by‐side to compare if a dopamine riboswitch can be selected from a library composed of an in vitro selected aptamer coupled with a 15‐nucleotide degenerate sequence. We anticipate that these efforts will provide general guidelines toward the development of novel riboswitches for a variety of natural and non‐natural ligands.Support or Funding InformationNSF CBET 1258307Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.