Bypassing bacterial infection in phage display by sequencing DNA released from phage particles

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Phage display is a powerful technique for selecting proteins with specific binding properties.
• Traditional phage display requires bacterial infection for phage amplification and clone identification.
• Mutant or modified phage often exhibit reduced bacterial infection rates, hindering selection efficiency.

Purpose of the Study:

• To evaluate the feasibility of directly sequencing DNA from phage particles after panning.
• To determine if this method can accurately represent the phage population without bacterial amplification.
• To establish a protocol for phage display with compromised or non-infective phage particles.

Main Methods:

• Phage panning was performed on target molecules.
• DNA was extracted directly from eluted phage particles.
• High-throughput sequencing was employed to analyze the DNA from phage particles.
• Sequence coverage was compared to traditional methods using amplified DNA from infected cells.

Main Results:

• Essentially all phage clones could be identified by direct DNA sequencing from phage particles.
• Sequence coverage for mutants was comparable to amplified wild-type phage DNA.
• The method effectively bypasses the bacterial infection step.

Conclusions:

• Direct DNA sequencing from phage particles is a viable alternative to bacterial amplification in phage display.
• This approach significantly enhances phage display for modified phage with low infectivity.
• It broadens the applicability of phage display, including for non-infective phage particles.