T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection

Area of Science:

• Synthetic biology
• Molecular biology
• RNA biology

Background:

• Riboswitches are non-coding RNA sensors that regulate gene expression.
• Selecting and evolving functional riboswitches is challenging.
• Existing methods lack efficiency in enriching these genetic sensors.

Purpose of the Study:

• To develop a novel phage-based system for the evolution of theophylline riboswitches.
• To create a dual-selection method for enriching functional riboswitches in both ON and OFF states.
• To demonstrate the efficacy of the T7AE system in evolving a library of riboswitch variants.

Main Methods:

• Engineered bacteriophage T7 by replacing DNA polymerase with a theophylline riboswitch-controlled transcription factor.
• Created two host environments with specific genes (cmk or pifA) for dual selection.
• Applied the T7AE system to a library of 65,536 randomized riboswitch variants.

Main Results:

• Successfully enriched functional theophylline riboswitches from a large library.
• Demonstrated selection in both riboswitch ON (cmk host) and OFF (pifA host) states.
• Observed enrichment of phages encoding riboswitches conferring a fitness advantage.

Conclusions:

• The T7AE system provides a powerful new tool for evolving gene switches, including non-coding RNA-based sensors.
• This technique advances synthetic biology by enabling the development of novel RNA-based regulatory elements.
• The dual-selection mechanism offers a robust approach for selecting functional riboswitches.