Bacteriophage Synthesis Using a Cell-Free Transcription-Translation System

Take a crude bacterial cell extract containing ribosomes, RNA polymerase, and a DNA-degrading enzyme.

Add an energy buffer mix containing nucleotides, tRNAs, and high-energy phosphate donors.

Introduce an amino acid mix and an inhibitor that blocks the DNA-degrading enzyme to prevent DNA degradation.

Add bacteriophage DNA, and mix the solution.

Finally, add a molecular crowding agent to mimic the crowded intracellular environment.

Vortex the solution to mix well, then incubate.

This solution now contains the essential molecular machinery for a cell-free transcription-translation or TXTL system.

The phage DNA is transcribed by RNA polymerase into messenger RNA or mRNA.

The mRNA then recruits the ribosome to initiate translation, where tRNA delivers amino acids to synthesize viral proteins using energy from high-energy phosphate donors.

Phage DNA and viral proteins self-assemble to form a new phage particle.

The bacteriophages synthesized by the cell-free TXTL system are ready for further analysis.

To perform the cell-free reaction, aliquot the indicated volume of crude extract, 33% of the final reaction volume, into a microcentrifuge tube. Prepare the master mix as listed in the text protocol. Add the appropriate volume of each component to the crude extract.

After adding the last component, homogenize the reaction by vortexing. Then split the master mix into n microcentrifuge tubes. Next, add the indicated volumes of the variable components to the master mix array.

Add water to each reaction to reach the desired final reaction volume before vortexing. Then incubate the microcentrifuge tubes at 29 degrees Celsius for at least eight hours or overnight.