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

Abstract

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.

Protocol

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 v....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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