Design of M13 phage systems dual-displaying two functional proteins for developing a rapid biosensing probe

Area of Science:

• Biotechnology
• Biosensor Development
• Molecular Biology

Background:

• M13 phage display systems are promising for rapid biosensor creation.
• Co-presentation of targeting and reporter elements on M13 phage is key.
• Characterizing different display-site combinations is crucial for optimizing performance.

Purpose of the Study:

• To systematically evaluate M13 phage dual-display capacity.
• To compare co-display strategies using a Vibrio parahaemolyticus-specific nanobody (Nb20) and NanoLuc luciferase (Nluc).
• To identify optimal coat protein sites and strategies for enhanced biosensing.

Main Methods:

• Co-display of Nb20 on pIII and Nluc on pVI, pVII, pVIII, or pIX.
• Evaluation under monovalent and multivalent display strategies.
• Assessment of display efficiency, luminescent activity, and detection capabilities.

Main Results:

• Display efficiency varied significantly based on coat protein site and helper phage infection time.
• The pVIII-based monovalent display yielded the highest specific luminescent activity.
• A functional co-display proportion of up to 2.20% was achieved with the pVIII system.
• The optimized probe detected Vibrio parahaemolyticus with high specificity and sensitivity (10^2 cfu/mL).

Conclusions:

• The pVIII protein site is optimal for M13 phage dual-display biosensors.
• Monovalent display on pVIII offers a practical framework for high-performance, one-step biosensing.
• This study provides fundamental insights for constructing advanced phage-based diagnostic tools.