Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

This method is useful in the Microbiology field, such as finding out the functions of specific genes. The main advantage of this method is that it is a fast and reliable way of generating knockout mutants of E.Coli. Demonstrating this procedure will be Thomas Trunk, A grad student from my laboratory.

To infect a donor strain containing a gene deletion based on an excisable antibiotic cassette, first, grow the bacteria in five milliliters of lysogeny broth, or LB, with ten millimolar calcium chloride, and optionally, with 25 micrograms per milliliter of kanamycin, to an optical density at 600 nanometers of about 1.0. Make a dilution series of an existing P1 phage stock in the LB medium, and and mix 200 microliters of the bacterial suspension with 100 microliters each phage dilution in individual 15 milliliter centrifuge tubes. Incubate the tubes statically for 20 minutes at 37 degrees Celsius.

At the end of the incubation, add approximately three milliliters of molten top agar, supplemented with ten millimolar calcium chloride to the tubes. Mix the contents by vortexing, and pour the mixtures onto pre-warmed LB plates to make even layers. Then, incubate the plates overnight at 37 degrees Celsius.

The next morning, select a plate with a semi-confluent growth of phage plaques and use an inoculation loop to scrape the top agar layer into a centrifuge tube. Under fume hood, add one to two milliliters of LB and a drop of chloroform to the tube, and vortex the tube vigorously for one minute. Pellet the agar and bacterial cells by centrifugation and transfer the supernatant to a microcentrifuge tube, avoiding debris from the pellet.

Then, add two drops of chloroform and store the lysate at four to ten degrees Celsius. 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 ten millimolar with mixing. Next, add the appropriate volume of phage lysate from the top of the sample to avoid transferring the chloroform to one 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 bacteria phages. Resuspend the pellet in one 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 one milliliter of fresh LB, supplemented with 100 millimolar sodium citrate. for a one hour incubation at 30 degrees Celsius at 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 ten millimolar sodium citrate, and grow the bacteria at 30 degrees Celsius until colonies appear. To select the transductants, restreak the colonies onto new LB plus kanamycin plates for single colony growth at 30 degrees Celsius until single colonies appear. To excise the kanamycin cassette, make the kanamycin-resistant recipient strain electrocompetent.

Add one picogram of the recombinase-encoding pCP20 plasma DNA to the cell suspension. Mix the suspension gently, and transfer it to a cooled, one millimeter electroporation cuvette. Set the electroporator to 1.8 kilovolts for electroporation of the cells and rescue the transformed cells with one milliliter of SOC medium for one hour at 30 degrees Celsius.

Then, plate 100 microliters of the cells on LB plus ampicillin plates for overnight culture at 30 degrees Celsius. The next morning, pick single colonies for inoculation in fresh LB without antibiotics and grow the cells overnight at the non-permissive temperature of 43 degrees Celsius to induce the expression of flippase recombinase. Then, plate 50 microliter serial dilutions on non-selective plates for overnight culture at 30 degrees Celsius, and screen for clones that have lost their antibiotic resistance cassette.

After P1 transduction and kanamycin cassette excision as just demonstrated, several clones sensitive to both ampicillin and kanamycin can be obtained, and the successful deletion of the tamA gene within the clones can be verified by colony PCR. After inducing protein production, the outer membrane fractions of the expression cultures can be isolated and analyzed by SDS-PAGE. No major differences are observed between the expression levels in the parent and tamA knockout recipient strains, although YadA appears to be produced at somewhat lower levels in the knockout strain.

The opposite appears to be true for EibD. To examine whether the lack of tamA might influence the correct folding or transport of the proteins, the ability of the proteins to bind to ligans can be tested by a collagen far-western blot. In this representative experiment, YadA-bound collagen was detected at a similar level in both the parent and knockout strains, demonstrating that the protein is correctly-folded and functional.

Similarly, the IgG-binding activities of EibD in the two strains correlated with the expression of the protein, demonstrating that the deletion of tamA does not have a significant effect on trimeric autotransporter adhesin biogenesis. When attempting this procedure, it is important to remember to use good aseptic technique and to remember to grow pCP20-containing bacteria at 30 degrees or lower. Don't forget that working with chloroform can be extremely hazardous, and that precautions, such as working in a fume hood and ensuring personal protection by wearing chemical-resistant gloves and a lab coat, should always be taken while performing this procedure.