Generation of Single-Stranded DNA with Phagemid Vectors

Introduction

The vectors derived from filamentous phages containing a plasmid’s origin of replication are called phagemids (Qi et al., 2012). Phagemids comprise typical high-copy-number plasmids equipped with a major intergenic region (508bp in length) of a filamentous phage. This region does not encode any proteins; however, it comprises all the cis-acting sequences required to initiate and terminate viral DNA synthesis. These sequences are also involved in the structural development of bacteriophages. 

Foreign DNA segments can be inserted into these phagemids and then proliferate like plasmids. However, when a filamentous phage infects a male E. coli strain possessing a phagemid, the replication of phagemid alters because of the viral gene products. The gene II  product (gene II protein) of the helper virus generates a site-specific nick in the plasmid’s intergenic region, which results in the initiation of rolling circle replication. Consequently, the clones of the plasmid’s one strand are produced. These single-stranded replicas of plasmid DNA are packaged into the bacteriophage coats, and the progeny viral particles are released into the surrounding medium. We can use polyethylene glycol (PEG) precipitation to recover these progeny particles and phenol extraction for purifying the ssDNA.     

Improvements in Helper Viruses and Phagemids

The pEMBL vectors were the first generation of vectors and were not very successful. They generated poor yields of single-stranded DNA after superinfection with helper phages because of multiple factors, including:

• Multiplicity of infection
• Interval of time after infection
• Culture density at the time of infection 

Even under optimal conditions, the progeny particles predominantly comprised the helper phages rather than single-stranded phagemid DNA. To eliminate these problems, a modified version of helper virus-containing mutated gene II favorably activates the replication’s phagemid origin. Moreover, E. coli strains like DH11S, MV118, and TG2 have been synthesized. These strains are efficient enough to get easily transformed by plasmids, easily infected by helper bacteriophages, and produce contamination-free preparations of single-stranded DNA.

The multiple cloning site (MCS) orientation of the phagemid vector and the orientation of bacteriophage origin of replication determine the particular strand of foreign DNA that has to be packaged into phage particles. Therefore, most commercially available phagemids have four possible chilarities. The orientation of MCS is opposite in one pair of vectors and the orientation of the intergenic region in the other pair. The positive and negative orientations of the intergenic region facilitate the helper bacteriophages to rescue single-stranded sense and anti-sense DNAs. For the researchers working with the phagemids for the first time, it is better to use a reliable helper phage (e.g., M13K07 used in the given experiment) and a dependable phagemid in a suitable E. coli strain such as DH11S in this case. 

Helper Bacteriophages

Various filamentous phages have been engineered genetically to increase the yield of single-stranded phagemid DNA packaged into the viral particles. The phagemid to helper genome ratio after superinfection should be 20:1. A 1.5ml culture should be enough for four to eight sequencing reactions. 

M13K07

This helper phage is an M13 phage derivative with plasmid origin of replication, transposon Tn903 gene for kanamycin resistance, and a mutated gene II product (a guanine residue at 6125 replaced by thymine). After infection, the bacterial enzymes convert single-stranded helper phage DNA into double-stranded DNA. This dsDNA then uses p15 plasmid origin to replicate. The viral gene products do not govern the accumulation of double-stranded DNA. Thus, there is a very small chance that resident phagemids interrupt the helper phage genome replication. Over time, all genomes of the M13K07 phages are expressed as required for the production of progeny particles. However, due to the insertion of LacZ sequences, the mutated gene II product interacts less efficiently with the phage origin of replication on its genome.

Consequently, more positive strands are produced from phagemids compared to helper viruses to ensure that the progeny viral particles contain single-stranded DNA from phagemid in bulk. When M13K07 is grown in the absence of a phagemid vector, the mutated gene II interacts with the disrupted origin of replication to produce sufficient M13 phages. 

Other Helper Viruses 

Another widely used helper phage is R408. Apart from M13K07 and R408, many other phages are used. However, the M13K07 phages give the best yield of single-stranded phagemid DNA.

Materials

Buffers, Solutions, and Gels

• 2% w/v SDS  Solution
• Sucrose gel-loading buffer
• 10mg/ml Kanamycin
• 0.7% agarose gel, cast in 0.5x TBE, having 0.5ꭒg/ml EtBr

Media

• 2x YT medium 
• YT agar plates with 60ꭒg/ml ampicillin 
• YT agar plates with 25ꭒg/ml kanamycin
• 2x YT agar plates with 60ꭒg/ml ampicillin
  • Add 5mM Mg+2 to the medium when working with M13 strains with a low multiplicity of infection.

• Agar plates supplemented with minimal M9 medium. Minimal M9 medium is used when working with E. coli strains in which the proline biosynthetic operon is deleted and compliments the pro-AB genes on F’ plasmid.

Bacterial and Viral Strains

• Helper M13K07 Bacteriophage 
• E. coli strain with F’ plasmid
• E. coli strain DH11S
• E. coli strain DH11S transformed with helper M13 phage

Other Equipment

• Water bath (preset to 65oC)
• Shaker 
• Petri plates 

Methods

Preparation of high-titer helper bacteriophage stock

1. Pick a single colony of E. coli DH11S strain from supplemented minimal agar plates and prepare its culture in 20ml of 2x YT medium. Incubate this culture at 37oC with moderate shaking until the optical density at 600nm reaches 0.8. 
2. Prepare a series of ten-fold serial dilutions of M13K07 bacteriophage in a 2x YT medium. Plate the aliquots of the M13K07 strain to get well-isolated plaques on a lawn of DH11S strain by plating bacteriophage.
3. Pick a single well-isolated bacteriophage plaque and inoculate it into 2-3ml of 2x YT medium (having 25ꭒg/ml of kanamycin) in a 15ml tube. Place the infected cultures on a shaker at 37oC with moderate agitation for 12-16 hours (250 cycles/minute).
4. Take 1.5ml sterile centrifuge tubes and shift the infected culture t these tubes. Centrifuge at 4oC for 2 minutes at maximum speed, transfer the supernatants to fresh tubes, and store them at 4oC. 
5. Plate the contents of each tube on a separate plate having a suitable E. coli strain carrying F’ plasmid (that supports the growth of bacteriophage M13). Measure the titer of each stock by the plaque it forms on the plate. 

Recombinant Phagemid growth with Helper Virus

1. Prepare two YT agar plates supplemented with 60ꭒg/ml ampicillin. Streak DH11S cells were transformed with a recombinant phagemid vector and an empty phagemid vector. Incubate them at 37oC for 16 hours.
2. Pick numerous recombinant phagemid transformed colonies and one or two-parent vector transformed colonies. Shift them to sterile 15ml culture tubes having 2-3ml of 2x YT medium (having 60ꭒg/ml ampicillin). The single-stranded DNA yield is affected by multiple factors. So, picking many recombinant colonies increases the chances of success. 
3. To each aliquot, add M13K07 bacteriophage to a final concentration of 2×107pfu/ml. Incubate the infected cultures at 37oC for about one and a half hours with vigorous agitation (300 cycles/ minute). The culture should be slightly turbid after this period of incubation. If the turbidity is too high, add 2x YT medium into it and keep diluting until the turbidity is just slightly visible. 
4. Now, add kanamycin to achieve a final concentration of 25ꭒg/ml and incubate at 37oC for another 14-18 hours. The reason behind adding kanamycin is that the bacteriophage strain used here contains a kanamycin-resistant gene, so only the desired infected cells will survive in the medium. Other helper phages do not carry “antibiotic resistance marker.” Be frugal enough to check the genotype of the helper phage before adding kanamycin.
5. Now transfer the obtained cell suspensions to a fresh microfuge tube and centrifuge at maximum speed for 5 minutes at room temperature. Pour the supernatants into fresh tubes and store them at 4oC.

Electrophoretic Estimation of the Yield of Single-stranded Phagemid DNA 

1. In 0.5ml microfuge tubes, add 2ꭒl of 2% SDS along with 40ꭒl of supernatant and mix the contents of the tubes by gently tapping on their sides. Incubate the tubes at 65oC for 5 minutes. 
2. Add 5ꭒl of sucrose gel loading buffer to each sample, mix and then load into separate wells of a 0.7% agarose gel.
3. Run the gel at 6V/cm and wait until bromophenol blue has migrated half the length of the gel. Examine the gel under ultraviolet light and photograph it. The yield of the DNA is generally about 1ꭒg/ml. However, it more precisely depends upon the size and nature of the foreign insert.
4. Isolate ssDNA phagemid DNA from the aliquot of supernatant having the largest amount of the DNA. Scale up the volume of the DNA up to two to three-fold. Depending on the size and nature of the foreign DNA, one can achieve the yield of single-stranded phagemid DNA up to five to ten folds. The larger size of the foreign insert results in poor yield. The foreign DNAs of equivalent sizes also suppress the yield of ssDNA. For instance, the segments of yeast DNA easily propagate in phagemids, whereas the human DNA inserts result in poor yields. There are other reasons as well that are still unknown. The orientation of the insert DNA and the bacteriophage origin of replication also affect the yield of the DNA. Therefore, to resolve the low yield issue, sometimes reclone the insert in the opposite orientation or the bacteriophage origin of replication in the opposite orientation.

Applications

• The single-stranded DNA obtained from phagemids, just like recombinant phage-derived DNA, is used for Sanger sequencing, site-directed mutagenesis, and the synthesis of radiolabeled probes. Furthermore, the ssDNA obtained from phagemids can be propagated by inappropriate E. coli strains to prepare ssDNA containing uracil instead of a proportion of thymine residues. These DNAs substituted with uracil residues serve as an exceptional substrate for various types of oligonucleotide-directed mutagenesis. 
• Phagemids possess small genomes and can accommodate relatively larger foreign DNA sequences, due to which they are the most common phage display vectors. 
• Extremely diverse phage display libraries are prepared from phagemids because of their greater transformation efficiency (Qi et al., 2012). 
• Phagemid genomes carry a wide range of restriction endonuclease recognition sites that facilitate DNA recombination and gene manipulation. 
• The expression level of fusion proteins formed can be easily modulated.
• In multiple propagations, the phagemids are more stable than recombinant phages.  
• Phagemids possess a positive selectable positive marker to select bacteria for transformation.
• Phagemids generate higher amounts of double-stranded DNA.
• The use of phagemids saves one from the time-consuming process of sub-cloning plasmid DNA fragments into filamentous phage vectors. 
• The use of phagemids reduces the chances of deletions and rearrangements in ssDNA.
• The fragments of several kilobases’ lengths can be isolated in single-stranded form by using phagemids. 

One can create a complete expression cassette in a phagemid—for instance, a gene promoter, a transcription terminator, or a gene of interest. The ssDNA obtained this way can be used for site-directed mutagenesis and then transformed into a suitable expression system such as E. coli or yeast.

Summary

• Phagemids combine the features of plasmids with filamentous bacteriophages. Plasmids with high copy numbers are equipped with the intergenic region of a filamentous bacteriophage. 

• The yield of single-stranded phagemid DA is affected by various factors, including the multiplicity of infection, the interval of time after infection, and culture density at the time of infection. 

• The packaging of a particular strand of DNA is determined by the orientation of the multiple cloning site (MCS) of the phagemid vector and the orientation of the bacteriophage origin of replication.

• M13K07 and R408 are the most commonly used helper phages. 

• Higher transformation efficiency, smaller genomes, a variety of restriction endonuclease recognition sites, and various other properties enable phagemids to have a wide range of molecular biology and bioengineering applications.

References

1. Qi, H., Lu, H., Qiu, H. J., Petrenko, V., & Liu, A. (2012). Phagemid vectors for phage display: properties, characteristics, and construction. Journal of molecular biology, 417(3), 129-143.
2. Sambrook, J., & Russell, D. W. (2006). Producing Single-stranded DNA with Phagemid Vectors. Cold Spring Harbor Protocols, 2006(1), PDB-prot4019.