Engineering transmembrane signal transduction in synthetic membranes using two-component systems

Area of Science:

• Synthetic biology
• Biochemistry
• Membrane biophysics

Background:

• Cells utilize transmembrane signal transduction to perceive and react to external stimuli.
• Synthetic systems mimicking cellular signaling offer potential for novel biosensing and therapeutics.
• Bacterial two-component systems (TCSs) are key mediators of cellular signal transduction.

Purpose of the Study:

• To investigate the feasibility of reconstituting bacterial TCSs for transmembrane signaling in synthetic membranes.
• To engineer a nitrate-sensing TCS (NarX-NarL) for cell-free applications.
• To explore the impact of membrane properties and protein engineering on TCS performance.

Main Methods:

• Utilized cell-free protein expression systems to synthesize components of the NarX-NarL TCS.
• Integrated the histidine kinase (NarX) into synthetic lipid bilayers with varying biophysical properties.
• Employed protein engineering to modify the ligand-binding domain of NarX.
• Validated sensing capabilities in relevant environmental samples.

Main Results:

• Successfully reconstituted the NarX-NarL TCS function in a cell-free system with synthetic membranes.
• Demonstrated that membrane composition influences TCS sensitivity and performance.
• Engineered NarX variants capable of detecting diverse ligands.
• Confirmed the system's ability to sense target ligands in complex sample matrices.

Conclusions:

• Transmembrane signaling via bacterial TCSs can be effectively replicated in cell-free systems using synthetic membranes.
• Membrane biophysics and protein engineering are critical for optimizing synthetic signaling systems.
• This work expands the toolkit of cell-free systems for practical biosensing applications.