M13 bacteriophage

The phage coat is primarily assembled from a 50 amino acid protein called p8, which is encoded by gene 8 in the phage genome. For a wild type M13 particle, it takes approximately 2700 copies of p8 to make the coat about 900 nm long. The coat's dimensions are flexible though and the number of p8 copies adjusts to accommodate the size of the single stranded genome it packages.[7] The phage appear to be limited to approximately twice the natural DNA content. However, deletion of a phage protein (p3) prevents full escape from the host E. coli, and phage that are 10-20X the normal length with several copies of the phage genome can be seen shedding from the E. coli host.

At one end of the filament are five copies of the surface exposed protein (p9) and a more buried companion protein (p7). If p8 forms the shaft of the phage, p9 and p7 form the "blunt" end that is seen in micrographs. These proteins are very small, containing only 33 and 32 amino acids respectively, though some additional residues can be added to the N-terminal portion of each which are then presented on the outside of the coat. At the other end of the phage particle are five copies of the surface exposed (p3) and its less exposed accessory protein (p6). These form the rounded tip of the phage and are the first proteins to interact with the E. coli host during infection. Protein p3 is also the last point of contact with the host as new phage bud from the bacterial surface.

Below are steps involved with replication of M13 in E. coli.

• Viral (+) strand DNA enters cytoplasm
• Complementary (-) strand is synthesized by bacterial enzymes
• DNA Gyrase, a type II topoisomerase, acts on double-stranded DNA and catalyzes formation of negative supercoils in double-stranded DNA
• Final product is parental replicative form (RF) DNA
• Transcription and translation of the viral genome begins by host resources, including p2.
• A phage protein, p2, nicks the (+) strand in the RF
• 3'-hydroxyl acts as a primer in the creation of new viral strand
• p2 circularizes displaced viral (+) strand DNA
• Pool of progeny double-stranded RF molecules produced
• Negative strand of RF is template of transcription
• mRNAs are translated into the phage proteins

Phage proteins in the cytoplasm are p2, p10 and p5, and they are part of the replication process of DNA. The other phage proteins are synthesized and inserted into the cytoplasmic or outer membranes.

• p5 dimers bind newly synthesized single-stranded DNA and prevent conversion to RF DNA. The timing and attenuation of p5 translation is essential.
• RF DNA synthesis continues and amount of p5 reaches critical concentration
• DNA replication switches to synthesis of single-stranded (+) viral DNA
• p5-DNA structures from about 800 nm long and 8 nm in diameter
• p5-DNA complex is substrate in phage assembly reaction

George Smith, among others, showed that fragments of EcoRI endonuclease could be fused in the unique Bam site of f1 filamentous phage and thereby expressed in gene 3 whose protein p3 was externally accessible. M13 does not have this unique Bam site in gene 3. M13 had to be engineered to have accessible insertion sites, making it limited in its flexibility in handling different sized inserts. Because the M13 phage display system allows great flexibility in the location and number of recombinant proteins on the phage, it is a popular tool to construct or serve as a scaffold for nanostructures.[8] For example, the phage can be engineered to have a different protein on each end and along its length. This can be used to assemble structures like gold or cobalt oxide nano-wires for batteries[9] or to pack carbon nanotubes into straight bundles for use in photovoltaics.[10]

• Phage display
• Phagemid
• Filamentous bacteriophage