Phage plasmids skillfully combine the characteristics of plasmids and filamentous phages. In addition to the basic features, these plasmids are usually high copy number and carry a modified filamentous phage with a major intergenic region. This region ( 508 bp in the wild type) does not express proteins but contains all of the cis-acting sequences that are essential for the initiation and termination of viral DNA synthesis and the morphogenesis of phage particles. The source of this experiment is "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Preparation of single-stranded DNA from phage vectors
Principle
Phage plasmids skillfully combine the characteristics of plasmids and filamentous phages. In addition to the basic features, these plasmids are usually high copy number and carry a modified filamentous phage with a major intergenic region. This region (508 bp in the wild type) does not express protein but contains all of the cis-acting sequences that are essential for the initiation and termination of viral DNA synthesis and for the morphogenesis of phage particles.
Materials and Instruments
E. coli F' strain E. coli DH11S Move I. Materials For more product details, please visit Aladdin Scientific website.
Kanamycin SDS solution Sucrose gel spiking buffer
Agarose gel M9 basic medium plate YT agar plate Water bath
1. Buffers and solutions
Kanamycin (10 mg/ml)
SDS solution (2%, m/V)
Sucrose gel spiking buffer
2. Gel
Agarose gel (0.7%) suspended in 0.5 X TBE, containing 0.5 μg/ml ethidium bromide
3. medium
Gaffer's M9 Basic Medium Plate
YT agar plates containing 60 μg/ml ampicillin.
2 X YT medium
2 X YT medium with 60 μg/ml Ampicillin
2 X YT medium with 25 μg/ml kanamycin
4. Specialized equipment
Water bath, set to 65°C
5. Carriers and strains
Phage M13K07 ( auxiliary)
E. coli F' strain
E. coli DH11S
E. coli DH11S, transformed with phage vector of M13 phage
E. coli DH11S, transformed with a recombinant phage vector of M13 phage with exogenous DNA
II.
Preparation of high titer auxotrophic phage prototypes
The key to the successful use of phages is the preparation of auxotrophic progeny with known precise titers.
1. Pick a fresh single colony of E. coli DH11S from a Garfield basic agar plate, inoculate with 20 ml of 2 X YT medium, and incubate at 37°C with gentle motions until an OD600 of 0.8 is reached.
2. Prepare a series of 10-fold dilutions of M13K07 phage in 2 X YT medium and spread the diluted phage on plates to obtain well-separated phage spots in the DH11S cell layer.
3. Pick the well-isolated single phage spots and inoculate them into 15 ml culture tubes containing 2~3 ml of 2 X YT medium containing 25 μg/ml kanamycin. Incubate at 37℃ for 12-16 h with gentle shaking (250 r/min).
Important: The following steps use M13K07 prototypes obtained from a single freshly picked phage spot.
4. Transfer the infected supernatant to a 1.5 ml sterile microcentrifuge tube and centrifuge on a microcentrifuge at maximum speed at 4°C for 2 min. Transfer the supernatant to a new tube and store at 4°C.
5. Determine the titer of each phage progeny by forming a phage spot in an E. coli F' strain (TGI, DH11S, NM522, or XLl-Bkie) that supports the growth of the M13 phage.
The titer of the infectious phage particles of the proto-species should be 1010 pfu/ml. discard all proto-species with low titers.
Proliferation of recombinant phage particles with helper phage
6. On two YT agar plates containing 60 μg/ml ampicillin, streak the recombinant phage-transformed and null phage-transformed DH11S bacteria and incubate for 16 h at 37℃.
7. Pick several recombinant phage-transformed clones and 1~2 clones transformed by their parental vectors, and inoculate them into 15 ml culture tubes containing 2~3 ml of 2 X YT medium containing 60 μg/ml ampicillin.
Due to the difficulty in determining the factors affecting the yield of single-stranded DNA, multiple clones should be picked from each phage recombinant, which is difficult to improve the success rate.
8. Add M13K07 helper phage to each tube to a concentration of 2 X 107 pfu/ml. incubate for 1.0~1.5 h at 37℃ with vigorous shaking (300 r/min).
After this short period of incubation, the bacterial culture should be only slightly turbid. If growth is excessive, dilute the culture with pre-warmed 2 X YT medium until turbidity is just visible. 
9. Add kanamycin to the culture to a final concentration of 25 μg/ml and incubate for 14-18 h at 37°C.
Since phage M13K07 contains a kanamycin resistance gene, only those infected bacteria will survive the addition of antibiotic at this step.
Other helper phages (e.g. R408 ) do not carry the antibiotic resistance marker. The genotype of the helper phage should be checked before adding antibiotics.
10. Transfer the suspension to a microcentrifuge tube and centrifuge at maximum speed in a microcentrifuge for 5 min at room temperature to separate the bacterial body from the culture medium, and transfer the supernatant to a new tube and store it at 4℃.
Estimate the rate of phage single-stranded DNA by gel electrophoresis.
11. Add 40 μl of supernatant and 2 μl of 2% SDS into a 0.5 ml microcentrifuge tube, tap the wall of the tube to mix the contents, and incubate at 65℃ for 5 min.
Estimate the yield of virus particles containing single-stranded copies of phage. The supernatant was first diluted and infected with E. coli DH11S, and then spread on YT agar plates containing ampicillin (60 μg/ml). The number of virus particles containing phage single-stranded DNA in the supernatant was calculated according to the number of ampicillin-resistant clones produced after 24 h incubation at 37 ℃. 2 X 1011~5 X 1011 pfu/ml of supernatant could achieve a satisfactory yield of purified phage single-stranded DNA.
12. Add 5 μl of Sampling Buffer to each phage DNA sample, mix it and add it to the sampling well of 0.7% agarose gel.
13. Electrophoresis at 6 V/cm for several hours until the bromophenol blue has migrated to approximately half of the gel. Check and photograph under UV light.
The yield varies with the size and persistence of the exogenous DNA on the phage. However, it is usually about 1 μg/ml of culture.
14. Isolate phage single-stranded DNA from supernatant containing large single-stranded DNA. follow the procedure in protocol "M13 Preparation of phage single-stranded DNA", and amplify the volume by 2-3 times.
As with the M13 phage vector, the yield of phage single-stranded DNA can vary from five to ten times depending on the size and characteristics of the exogenous DNA. Usually, the larger the fragment, the lower the yield. Moreover, even if the exogenous DNA is of the same size, the yield of single-stranded DNA can vary considerably, e.g., most yeast DNA fragments seem to have no problem with phage proliferation, whereas the same size of human genomic DNA may result in disappointing single-stranded DNA yields. The direction of insertion of exogenous DNA on the phage and the orientation of the phage DNA replication start point can also have an important effect on the yield. Therefore, the problem of low yield can sometimes be solved by recloning the fragment in the opposite direction or by switching to vectors with phage DNA replication starting points in the opposite direction.