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What Are Phagemids and What Are They Used for?
Keywords
Antimicrobial peptides, phagemids, targeted bacterial killing, bacterial toxins, synthetic gene networks, bacteriophage resistance, bacteriophage particle, bacterial host.
Introduction
A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. These vectors carry, in addition to the origin of plasmid replication, an origin of replication derived from bacteriophage. Unlike commonly used plasmids, phagemid vectors differ by having the ability to be packaged into the capsid of a bacteriophage, due to their having a genetic sequence that signals for packaging.
Phagemids are used in a variety of biotechnology applications; for example, they can be used in a molecular biology technique called "Phage Display". The term "phagemid" or "phagemids" was offered to the world science community by a group of Soviet scientists, who discovered them, named them, and published the article in April 1984 in Gene magazine. After that, the world community started using this term.
Single-stranded DNA (ssDNA) is the optimal template for most polymerase-based molecular-biology applications, including DNA sequencing and site-directed mutagenesis. Phagemids are chimeric vectors, derived from the ssDNA bacteriophages M13, fd, or f1, that normally replicate as plasmids in bacterial hosts.
Phagemids typically lack the genes required for packaging into virions and, thus, “helper” bacteriophage is often required to package phagemids as ssDNA into viral particles, which are subsequently released into the culture medium. SsDNA isolated from these virions is suitable for use as a template in a variety of procedures with no further manipulation, as denaturation is not required prior to its use as a template.
Phagemids usually encode no or only one kind of coat protein. In addition, other elements such as molecular tags and selective markers are introduced into the phagemids to facilitate the subsequent operations, such as gene manipulation and protein purification.
Technology
Bacteriophages are bacteria-specific viruses that infect and lyse specific species and strains of bacteria. The technology uses phagemids to specifically target and kill bacteria. Phages were long ago proposed as a potential antibiotic therapy due to their exquisite specificity, however, when phage lyse their host bacteria the bacterial membrane bursts and releases endotoxins that can induce strong inflammatory reactions in patients.
This technology uses a modified phage system, called phagemids, which efficiently kill bacteria without lysis and the resulting toxin release. The phagemid system uses two DNA vectors, one that expresses antimicrobial peptides (AMPs), and a second that specifically packages the AMP vector.
When transformed into a strain of packaging bacteria, these vectors produce large amounts of replication-defective phage that can be purified and used as an antibiotic. This phagemid technology has several important improvements over existing technologies.
Firstly, the phagemids are replication-deficient and do not result in propagation of more phage, which eliminates the potential for unregulated phage evolution. Secondly, the non-lytic nature of the phagemids reduces toxicity due to bacterial bursting and reduces the formation of bacteriophage resistance.
Features of Phagemids
- They contain the origin of replication (ori) for double stranded replication and can also be used in the absence of helper phage, as well as an f1 ori to enable single stranded replication and packaging into phage particles.
- A multiple cloning site (MCS) flanked by T3 and T7 promoters to be read in opposite directions on two strands.
- An inducible lac promoter (lacI), upstream of lacZ region, which is complimentary to E. coli (lacZ-) and provides the facility for selection of chimeric vector DNA using the white colonies.
- A gene having ampicillin resistance for antibiotic selection.
Key Points
- 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 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.
Advantages
- Modular vector system allows rapid customization of phage characteristics and customizable choice of antimicrobial peptides.
- Non-lytic bacterial killing reduces toxicity.
- Non-replicative phage eliminates unregulated evolution.
- Reduces the formation of bacterial resistance.
- Demonstrated in vivo effectiveness in a mouse model of peritonitis.
Conclusion
Single-stranded DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. The yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized.
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