Screening and selection of artificial riboswitches

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Synthetic riboswitches offer precise control over gene expression in response to small molecules.
• Developing robust screening tools is crucial for engineering functional synthetic riboswitches.
• Existing methods face challenges in efficiently selecting and optimizing these engineered genetic elements.

Purpose of the Study:

• To develop and validate a novel dual-color reporter system for the selection and optimization of synthetic riboswitches.
• To demonstrate the utility of this reporter system in identifying and refining aptamers for riboswitch applications.
• To enhance the efficiency of engineering synthetic riboswitches responsive to non-endogenous small molecules.

Main Methods:

• Engineered synthetic riboswitches by combining in vitro-selected aptamer libraries with a randomized expression platform.
• Developed a dual-color reporter system utilizing the E. coli fimbriae phase variation system (FimE recombinase and fimS invertible DNA segment).
• Utilized constitutive expression of green fluorescent protein (GFPa1) in the absence of analyte and red fluorescent protein (mKate2) upon analyte-induced inversion.

Main Results:

• The dual-color reporter system successfully distinguished between analyte-present and analyte-absent conditions by switching fluorescence from green to red.
• Incorporating enriched aptamer libraries into the riboswitch architecture increased the proportion of potential ligand binders.
• The system demonstrated efficient quantification of synthetic riboswitch function and facilitated selection of structure-switching aptamers.

Conclusions:

• The developed dual-color reporter system is an effective tool for selecting and optimizing synthetic riboswitches in E. coli.
• This system streamlines the engineering process by providing a quantifiable readout of riboswitch activity.
• The approach enables the creation of synthetic riboswitches with enhanced specificity and responsiveness to target molecules.