Molecular Biology. Practical Guide - Velikov V.A. 2013

Isolation of Plasmid and Phage DNA
Isolation of the M13 Phage Replicative Form (RF)

Bacteriophages (phages) are bacterial Viruses that replicate inside Cells, typically resulting in Cell lysis. Chemically, they are nucleoprotein complexes consisting of a protein coat (capsid) enclosing a single-stranded or, less commonly, double-stranded DNA or RNA molecule.

Closely related filamentous phages such as M13, f1, and fd, which contain single-stranded DNA virions, are classified as temperate phages—they do not lyse the host cells in which they propagate (parasitize). Vectors based on filamentous phages are well-suited for sequencing because single-stranded DNA of the +/- strands can be readily isolated from phage particles. Interest in these phages is also driven by the fact that they form The basis of phage display, a premier affinity technique in Protein Engineering used to study Protein-Structure/156.html">Protein Interactions alongside Yeast two-hybrid systems and protein microarrays. Gene cloning is performed within the double-stranded DNA of a filamentous phage, which exists inside the infected bacterial population as a plasmid. If a gene of interest is cloned in-frame with phage gene III (or VIII) encoding the capsid protein gp3 (gp8), the resulting Translation product is displayed On the surface of the mature virion as a fusion construct. Such phages, as well as gene libraries constructed in M13 vectors, are referred to as "phage display" systems, as they display Peptides on The surface of phage particles (McCafferty et al., 1990).

The isolation of the double-stranded circular replicative form (RF) of M13 phage from E. coli cells can be carried out using any standard plasmid purification method. M13 phage infects exclusively F+ strains of Escherichia coli by adsorbing to F-pili.

This section outlines the Procedure for infecting (transfecting) E. coli cells with M13 phage, followed by Propagation of the RF form to yield double-stranded DNA.

Materials and Equipment

F+ E. coli strain XL1-Blue (TG-1, JS5, or alternative), M13 K07 phage in TE buffer at a titer of 10 particles/mL, centrifuge, shaker.

Solutions

- Solution I (Procedure 4.1)

- Solution II (Procedure 4.1)

- 10 M Ammonium acetate. Dissolve 770 g in 800 mL of Water and adjust to 1 L.

- Kanamycin. 100 mg/mL solution in water.

Procedure

1. Grow an overnight culture of the F+ E. coli strain in 5 mL of liquid 2YT medium. Cell concentrations can reach up to 10¹⁰ cells/mL.

2. Infect the culture with the phage at a multiplicity of infection (MOI) of 10:1 (approximately 10 phage particles per bacterial cell). To do this, add 10 µL of the phage particle suspension to The Cell tube.

3. Incubate without shaking for 1 h at 37°C. During this time, the M13 K07 phage adsorbs to the F-pili, penetrates the cell, and initiates RF synthesis. The transfection efficiency of E. coli exceeds that of transformation (Topic 8).

4. Add the appropriate antibiotic ^a (500 µL of 100 mg/mL kanamycin solution) to 500 mL of fresh 2YT medium, and transfer the phage-infected E. coli cells into it.

5. Cultivate the culture with aeration overnight at 37°C.

6. Pellet the cells by centrifugation at 4000 rpm for 10–15 min. Decant the supernatant containing the culture medium into a clean flask ^b.

7. Isolate the double-stranded circular replicative-form DNA of the phage from the cell pellet using a miniprep protocol (Procedure 4.1).

Notes

^a The M13 K07 phage (Viera, Messing, 1987) is recombinant and carries a kanamycin-resistance gene (see Appendix 1). Wild-type M13 phage is grown without Antibiotics.

^b The culture fluid contains phage particles released from the cells. These can be readily isolated, as described in Procedure 4.4, by proportionally scaling up the volumes. The titer of phage particles is determined by transfecting E. coli cells and subsequently plating them onto an Agar growth medium containing the antibiotic.