PHEIGES: all-cell-free phage synthesis and selection from engineered genomes

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacteriophages are a vast biological resource for medicine and biotechnology.
• Current methods for engineering and selecting phages are slow and inefficient.
• Cell-free transcription-translation (TXTL) systems offer a potential solution for rapid phage engineering.

Purpose of the Study:

• To develop a rapid method for engineering and selecting bacteriophages.
• To demonstrate the utility of cell-free TXTL for phage genome assembly and expression.
• To establish a genotype-phenotype linkage for efficient phage selection.

Main Methods:

• Developed PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage and E. coli TXTL.
• Assembled phage genomes in vitro from PCR fragments and expressed them in batch TXTL reactions.
• Created phage genomes with tail fiber mutant libraries for selection.

Main Results:

• Achieved production of up to 10^11 PFU/ml engineered phages within one day.
• Demonstrated genotype-phenotype linkage in bulk TXTL for rapid selection.
• Successfully selected phages with altered rough lipopolysaccharides specificity.
• Showcased scalability of PHEIGES with fluorescent gene integration and genome reduction.

Conclusions:

• PHEIGES provides a rapid and efficient platform for bacteriophage engineering and selection.
• Cell-free TXTL is a powerful tool for assembling, expressing, and selecting engineered phages.
• This method significantly advances the exploitation of bacteriophages in biotechnology and medicine.