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 recipient strain culture to 10 millimolar with mixing. Next, add the appropriate volume of phage lysate from the top of the sample to avoid transferring the chloroform to 1 milliliter of recipient culture to achieve a multiplicity of infection of 0.5, and statically incubate the mix for 20 minutes at 37 degrees Celsius.

At the end of incubation, stop the infection by adding sodium citrate to 100 millimoles per liter and centrifuge the transduced bacteria.

Adding sodium citrate after the transduction is an essential step as it prevents further infection by wild-type bacteriophages.

Resuspend the pellet in 1 milliliter of fresh LB supplemented with 100 millimolar sodium citrate and wash the cells two more times to ensure the removal of free phages and calcium.

After the last wash, resuspend the bacteria in 1 milliliter of fresh LB, supplemented with 100 millimolar sodium citrate for a one-hour incubation at 30 degrees Celsius and 100 RPM. At the end of the incubation, collect the bacteria by centrifugation and resuspend the pellet in about 100 microliters of LB supplemented with 100 millimolar sodium citrate.

Then, spread the bacteria on an LB plate supplemented with 25 micrograms per milliliter of kanamycin and 10 millimolar sodium citrate, and grow the bacteria at 30 degrees Celsius until colonies appear.