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|Blue: Coat Protein pIII; Brown: Coat Proteín pVI; Red: Coat Protein pVII; Limegreen: Coat Protein pVIII; Fuchsia: Coat Proteín pIX; Purple: Single Stranded DNA|
Group II (ssDNA)
M13 is a filamentous bacteriophage composed of circular single-stranded DNA (ssDNA) which is 6407 nucleotides long encapsulated in approximately 2700 copies of the major coat protein P8, and capped with 5 copies of two different minor coat proteins (P9, P6, P3) on the ends. The minor coat protein P3 attaches to the receptor at the tip of the F pilus of the host Escherichia coli. Infection with M13 lysogenic phage. The infection causes turbid plaques in E. coli lawns, of intermediary opacity in comparison to regular lysis plaques. However, a decrease in the rate of cell growth is seen in the infected cells. M13 plasmids are used for many recombinant DNA processes, and the virus has also used for phage display, directed evolution, nanostructures and nanotechnology applications.
The phage coat is primarily assembled from a 50 amino acid protein called pVIII (or p8), which is encoded by gene VIII (or g8) in the phage genome. For a wild type M13 particle, it takes approximately 2700 motion 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. For example, when the phage genome was mutated to reduce its number of DNA bases (from 6.4 kb to 221 bp), then the number of p8 copies was decreased to fewer than 100, causing the p8 coat to shrink in order to fit the reduced genome. The phage appear to be limited at 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.
There are four other proteins on the phage surface, two of which have been extensively studied. At one end of the filament are five copies of the surface exposed pIX (p9) and a more buried companion protein, pVII (p7). If p8 forms the shaft of the phage, p9 and p7 form the "blunt" end that is seen in the 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 pIII (p3) and its less exposed accessory protein, pVI (p6). These form the rounded tip of the phage and are the first proteins to interact with the E. coli host during infection. p3 is also the last point of contact with the host as new phage bud from the bacterial surface.
Replication in E. coli
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
- A phage protein, pII, nicks the (+) strand in the RF
- 3'-hydroxyl acts as a primer in the creation of new viral strand
- pII circulizes 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 pII, pX, and pV, and they are part of the replication process of DNA. The other phage proteins are synthesized and inserted into the cytoplasmic or outer membranes.
- pV dimers bind newly synthesized single-stranded DNA and prevent conversion to RF DNA
- RF DNA synthesis continues and amount of pV reaches critical concentration
- DNA replication switches to synthesis of single-stranded (+) viral DNA
- pV-DNA structures from about 800 nm long and 8 nm in diameter
- pV-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 III whose protein pIII was externally accessible. M13 does not have this unique Bam site in gene III. 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. 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 or to pack carbon nanotubes into straight bundles for use in photovoltaics.
- Opella SJ, Stewart PL, Valentine KG (February 1987). "Protein structure by solid-state NMR spectroscopy". Quarterly Reviews of Biophysics. 19 (1–2): 7–49. doi:10.1017/S0033583500004017. PMID 3306759.
- Khalil AS, Ferrer JM, Brau RR, Kottmann ST, Noren CJ, Lang MJ, Belcher AM (March 2007). "Single M13 bacteriophage tethering and stretching". Proceedings of the National Academy of Sciences of the United States of America. 104 (12): 4892–7. doi:10.1073/pnas.0605727104. PMC 1829235. PMID 17360403.
- Suthiwangcharoen N, Li T, Li K, Thompson P, You S, Wang Q (May 2011). "M13 bacteriophage-polymer nanoassemblies as drug delivery vehicles. Nano Research". 4 (5): 483–93. doi:10.1007/s12274-011-0104-2. Cite journal requires
- Esvelt, Kevin M.; Carlson, Jacob C.; Liu, David R. (2011-04-28). "A System for the Continuous Directed Evolution of Biomolecules". Nature. 472 (7344): 499–503. Bibcode:2011Natur.472..499E. doi:10.1038/nature09929. ISSN 0028-0836. PMC 3084352. PMID 21478873.
- Specthrie L, Bullitt E, Horiuchi K, Model P, Russel M, Makowski L (December 1992). "Construction of a microphage variant of filamentous bacteriophage". Journal of Molecular Biology. 228 (3): 720–4. doi:10.1016/0022-2836(92)90858-h. PMID 1469710.
- Huang Y, Chiang CY, Lee SK, Gao Y, Hu EL, De Yoreo J, Belcher AM (July 2005). "Programmable assembly of nanoarchitectures using genetically engineered viruses". Nano Letters. 5 (7): 1429–34. Bibcode:2005NanoL...5.1429H. doi:10.1021/nl050795d. PMID 16178252.
- Nam KT, Kim DW, Yoo PJ, Chiang CY, Meethong N, Hammond PT, et al. (May 2006). "Virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes". Science. 312 (5775): 885–8. Bibcode:2006Sci...312..885N. doi:10.1126/science.1122716. PMID 16601154.
- Dang X, Yi H, Ham MH, Qi J, Yun DS, Ladewski R, et al. (April 2011). "Virus-templated self-assembled single-walled carbon nanotubes for highly efficient electron collection in photovoltaic devices". Nature Nanotechnology. 6 (6): 377–84. Bibcode:2011NatNa...6..377D. doi:10.1038/nnano.2011.50. PMID 21516089.
- Barbas CF, Burton DR, Silverman GJ (October 2004). Phage Display: A Laboratory Manual (1st ed.). Cold Spring Harbor Laboratory Press. ISBN 978-0-87969-740-2.
- Messing J (1993). "M13 Cloning Vehicles" (PDF). In Griffin H.G., Griffin A.M. (eds.). DNA Sequencing Protocols. Methods in Molecular Biology™. Methods in Molecular Biology. 23. Humana Press. pp. 9–22. doi:10.1385/0-89603-248-5:9. ISBN 0-89603-248-5. PMID 8220775. Archived from the original on 2012-02-19.CS1 maint: unfit url (link)