Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Riboswitches are genetic devices that control gene expression.
• Cell-free protein synthesis (CFPS) systems offer a versatile platform for biological research and synthetic biology applications.
• Orthogonal control of gene expression is crucial for complex synthetic systems.

Purpose of the Study:

• To design and characterize novel synthetic riboswitches.
• To demonstrate the function of these riboswitches in a reconstituted cell-free protein synthesis (CFPS) system.
• To achieve switchable and orthogonal control over gene expression using external stimuli.

Main Methods:

• Design and synthesis of novel riboswitches targeting ASP2905 and theophylline.
• Reconstitution of a cell-free protein synthesis (CFPS) system.
• Encapsulation of the CFPS system and DNA-templated reporter genes within liposomes.
• Stimulation with cognate ligands to assess riboswitch activity and orthogonality.

Main Results:

• Two novel synthetic riboswitches responsive to ASP2905 and theophylline were successfully created.
• These riboswitches demonstrated functionality within a reconstituted cell-free protein synthesis (CFPS) system.
• Liposome encapsulation enabled switchable and orthogonal control of gene expression via external ligand stimulation.

Conclusions:

• Novel synthetic riboswitches can effectively regulate gene expression in cell-free systems.
• Liposome-encapsulated CFPS systems provide a platform for precise, ligand-inducible genetic control.
• The developed riboswitches offer orthogonal control, paving the way for complex synthetic gene circuits.