Team:MIT phage background

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Phage
hairy cells and polymerizing phage - background
Arap, 2005
M13 ANATOMY
M13 bacteriophage is a long, filamentous virus that infects bacteria via interactions with the F-pilus. Below is the list of genes that comprise the phage:

  • PIII/PVI (pointed end) – mediate termination of assembly and particle release from the membrane; is present at around five copies
  • PVII/PIX (blunt end) – small coat proteins; is present at around five copies
  • PVIII – major coat protein; is present at around 2700 copies
  • PII – nicking enzyme, allows for replication to occur
  • PX – last third of PII; may inhibit PII in a regulatory manner
  • PIV – channel for phage exit
  • PI – may hydrolyze ATP to promote assembly
  • PXI – last third of PI, may interact with PI to form a channel that works with PIV
  • PV – dimerizes and binds ssDNA, packaging it into a rod for assembly




M13 ASSEMBLY
Assembly of M13 happens through a three-part process:

  • Initiation: PVII/PIX and PVII interaction with the DNA packaging signal. This is mediated by PI.
  • Elongation: DNA is extruded with PV being replaced by PVIII. This is mediated by PI. The particle elongates through the PIV channel.
  • Termination: PIII/PXI are added to the end of the particle

Barbas III et al., 2004

PHAGE DISPLAY
By genetically modifying the phage, fusion proteins can be created that become integrated into the phage coat in a defined manner. Usually these new proteins are linked to the pIII or pVIII coat proteins (though PVII or PIX can be used). Thus, novel proteins can be displayed on the phage coat. This has historically been used as a mechanism for screening libraries of proteins or peptides (e.g., antibodies) for binding to a specific substrate or ligand of interest. In a process called panning, a population of phage is iteratively enriched for those that bind a substrate. Phage with proteins that do not bind are washed away and the remaining phage can then be amplified through infection.


COILED COILS
Coiled coils are a common structural motif found in proteins. Repetitive amino acid sequences in a pair of alpha-helices provides a mechanism for the helices to interact with each other, coiling around each other. Leucine zippers are of this class--every seventh residue is a leucine. The leucines and other hydrophobic residues form a hydrophobic core, allowing for the helices to "zip" together. In our project we're using two leucine zipper pairs: Fos+Jun and ACID+BASE, and one pair of modified coiled coils: GR1+GR2. Expressing these on the polyphage surfaces should allow for the coiled coils to interact and dimerize, creating linkage. For more information on coiled coils, see Branden and Tooze (1999).







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