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

Özet

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp. (2018)

This video demonstrates the phage-mediated transfer of donor DNA carrying an excisable antibiotic resistance cassette into Escherichia coli. The integrated cassette replaces the target gene via homologous recombination, resulting in a stable gene deletion.

Protokol

1. Bacteriophage P1 Transduction
   1. Preparing the recipient cells
      1. Grow the recipient strain BL21ΔABCF in LB (Lysogeny Broth) supplemented to an optical density at 600 nm (OD₆₀₀) of ~1.0. Use a spectrophotometer to measure the OD₆₀₀ value.

      2. Calculate the volume of the phage lysate needed to achieve a multiplicity of infection (MOI) value of 0.5. To calculate the MOI, estimate the number of bacteria based on the OD₆₀₀ of the culture. Assume that an OD₆₀₀ value of 1.0 corresponds to ~10⁹ cfu/mL. Calculate the volume required based on the known titer of the lysate.

         Example:                    

         

      3. Add CaCl₂ to the recipient strain culture to 10 mM and mix. Take 1 mL of the culture for transduction.
2. Performing transduction
   1. Add the appropriate volume of phage lysate to 1 mL of recipient culture (including CaCl₂ at 10 mM) as calculated in step 1.1.2 and mix gently.
      NOTE: Be careful to pipette the sample from the top of the lysate to avoid transferring chloroform to the mix.
   2. Statically incubate the mix for 20 min at 37 °C.
   3. Stop the infection by adding sodium citrate, pH 5.5, to 100 mM.
   4. Centrifuge the bacteria (5,000 x g for 2 min) and remove the supernatant, then resuspend them in 1 mL of fresh LB supplemented with 100 mM sodium citrate, pH 5.5.
   5. Wash the cells twice more to ensure the removal of free phages and calcium.
   6. Resuspend the bacteria in 1 mL of fresh LB supplemented with 100 mM sodium citrate, pH 5.5. Incubate the bacteria at 30 °C for 1 h with shaking (> 100 rpm).
   7. Collect the bacteria by centrifugation (5,000 x g for 2 min) and resuspend them in ~100 μL of LB with 100 mM sodium citrate, pH 5.5.
   8. Spread the bacteria on an LB plate supplemented with Kanamycin at 25 µg/mL and 10 mM sodium citrate, pH 5.5, and grow the bacteria at 30 °C until colonies appear (~24 h).
3. Selecting the transductants
   1. Once colonies have grown on the selection plate, restreak them on LB + Kanamycin for single colonies and grow at 30 °C till single colonies appear.

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
Escherichia coli BL21DABCF	Addgene	102270	Derived from BL21(DE3)
P1 virus	NIG	HR16	Generally transducing bacteriophage
pCP20	CGSC	14177	conditionally replicating plasmid with FLP
Acetic acid	ThermoFisher	33209
Agar	BD Bacto	214010
Agarose	Lonza	50004
Anhydrotetracycline	Abcam	ab145350
Calcium chloride	Merck	102382
Kanamycin	Applichem	A1493
Sodium chloride	VWR	27808
Incubating shaker	Infors HT	Minitron
Incubator	VWR	390-0482
Microcentrifuge	Eppendorf	5415D
Tabletop centrifuge	Beckman Coulter	B06322
Vortex mixer	Scientific Industries	SI-0236