Generating Gene Deletions in E. coli via Phage-Mediated Transfer of an Excisable Antibiotic Cassette

Take a suspension containing recipient E. coli, calcium chloride, and phage particles.

The phages carry donor DNA with an excisable antibiotic resistance cassette.

During incubation, calcium chloride facilitates phage adsorption to the recipient cells. The phage injects the donor DNA and then detaches.

The donor DNA, carrying an excisable antibiotic resistance cassette, replaces the target gene region, resulting in a gene deletion.

Add a chelator to bind calcium chloride and inhibit further phage adsorption.

Centrifuge to separate the bacteria from the unadsorbed and detached phages. Remove the supernatant.

Resuspend the bacteria in fresh chelator-supplemented media.

Repeat the wash to remove residual contaminants.

Resuspend again in chelator-containing media and incubate with shaking.

Centrifuge and discard the supernatant.

Resuspend the pellet, then spread it onto nutrient-rich agar supplemented with an antibiotic and a chelator, and incubate.

Antibiotic-resistant colony formation indicates successful transduction of the recipient E. coli with gene deletion.

For P1 transduction, grow the recipient strain in LB to an optical density of one at 600 nanometers and add calcium chloride to the rec