Team:MIT phage background

From 2010.igem.org

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<ul>
<ul>
                         <li><a href="http://2010.igem.org/Team:MIT_toggle">Overview</a></li>
                         <li><a href="http://2010.igem.org/Team:MIT_toggle">Overview</a></li>
 +
                        <li><a href="http://2010.igem.org/Team:MIT_tmodel">Modelling</a></li>
<li><a href="http://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
<li><a href="http://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
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<li><a href="#">Characterization</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_composite">Characterization</a></li>
</ul>
</ul>
</dd>
</dd>
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</dl>
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<dl id ="specialnav">
<dt><b>Phage</b></dt>
<dt><b>Phage</b></dt>
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</ul>
</ul>
</dd>
</dd>
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</dl>
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<dl id ="nav">
<dt><b>Mammalian</b></dt>
<dt><b>Mammalian</b></dt>
<dd>
<dd>
<ul>
<ul>
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                         <li><a href="http://2010.igem.org/Team:MIT_mammalian_Overview">Overview</a></li>
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                         <li><a href="http://2010.igem.org/Team:MIT_mammalian">Overview</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Standard">Standard and Design</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Standard">New Mammalian Standard </a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Experiments">Experiments</a></li>
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                        <li><a href="http://2010.igem.org/Team:MIT_mammalian_Circuit">Circuit Design</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Mechanosensation"> Mechanosensation</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Bone"> Bone Formation</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Switch"> Synthetic Switch</a></li>
</ul>
</ul>
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<table width=650px style="background-color: white; margin-top:5px; padding: 10px;"><tr><td><div class="bodybaby">hairy cells and polymerizing phage - background</div></td>
<table width=650px style="background-color: white; margin-top:5px; padding: 10px;"><tr><td><div class="bodybaby">hairy cells and polymerizing phage - background</div></td>
<tr><td>
<tr><td>
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<div style="font-size:8pt"><i>Arap, 2005</i></div>
<img style="float: left" src="http://2010.igem.org/wiki/images/e/ed/P3_image.jpg" height=400px title="Image from: Arap, Phage Display Technology – Applications and Innovations, 2005">
<img style="float: left" src="http://2010.igem.org/wiki/images/e/ed/P3_image.jpg" height=400px title="Image from: Arap, Phage Display Technology – Applications and Innovations, 2005">
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<br>
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<b>M13 ANATOMY</b>
<b>M13 ANATOMY</b>
<br>
<br>
-
M13 bacteriophage is a long, filamentous virus that infects bacteria via interactions with the F-pilus.  Below is a list of genes:
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M13 bacteriophage is a long (930 nm in length, 6.5 nm in diameter), filamentous virus that infects bacteria via interactions with the F-pilus.  Below is the list of genes that comprise the phage:
<br><br>
<br><br>
<ul><li>
<ul><li>
-
PIII/PVI (pointed end) – mediate termination of assembly and particle release from the membrane.  About five copies.</li>
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pIII/pVI (pointed end) – mediate termination of assembly and particle release from the membrane; is present at around five copies</li>
<li>
<li>
-
PVII/PIX (blunt end) – small coat proteins.  About five copies.</li>
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pVII/pIX (blunt end) – small coat proteins; is present at around five copies</li>
<li>
<li>
-
PVIII – major coat protein.  About 2700 copies.</li>
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pVIII – major coat protein; is present at around 2700 copies</li>
<li>
<li>
-
PII – nicking enzyme, allows for replication to occur</li>
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pII – nicking enzyme, allows for replication to occur</li>
<li>
<li>
-
PX – last third of PII; may inhibit PII in a regulatory manner</li>
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pX – last third of PII; may inhibit PII in a regulatory manner</li>
<li>
<li>
-
PIV – channel for phage exit</li>
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pIV – channel for phage exit</li>
<li>
<li>
-
PI – may hydrolyze ATP to promote assembly</li>
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pI – may hydrolyze ATP to promote assembly</li>
<li>
<li>
-
PXI – last third of PI, may interact with PI to form a channel that works with PIV</li>
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pXI – last third of PI, may interact with PI to form a channel that works with PIV</li>
<li>
<li>
-
PV – dimerizes and binds ssDNA, packaging it into a rod for assembly</li></ul>
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pV – dimerizes and binds ssDNA, packaging it into a rod for assembly</li></ul>
-
<br><br>
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<br><br><br><br>
<b>M13 ASSEMBLY</b>
<b>M13 ASSEMBLY</b>
-
<br>
 
-
<img src="http://2010.igem.org/wiki/images/0/0f/M13_assembly.png" height=400px title="Image from: Barbas III et al. Phage Display: a Laboratory Manual. 2004.">
 
<br>
<br>
Assembly of M13 happens through a three-part process:
Assembly of M13 happens through a three-part process:
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<ul>
<ul>
<li>
<li>
-
Initiation: PVII/PIX and PVII interaction with the DNA packaging signal.  This is mediated by PI.</li>
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Initiation: pVII/pIX and pVII interaction with the DNA packaging signal.  This is mediated by PI.</li>
<li>
<li>
-
Elongation: DNA is extruded with PV being replaced by PVIII.  This is mediated by PI.  The particle elongates through the PIV channel.</li>
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Elongation: DNA is extruded with pV being replaced by pVIII.  This is mediated by pI.  The particle elongates through the pIV channel.</li>
<li>
<li>
-
Termination: PIII/PXI are added to the end of the particle
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Termination: pIII/pXI are added to the end of the particle. When pIII is not present due to a knockout, elongation proceeds through multiple phage genomes, resulting in particles that are tethered to the membrane and up to 20x normal length.
</li></ul>
</li></ul>
 +
<br>
 +
<div style="text-align:center" 2px solid #aaa;>
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<img src="http://2010.igem.org/wiki/images/0/0f/M13_assembly.png" height=300px title="Image from: Barbas III et al. Phage Display: a Laboratory Manual. 2004."><div style="font-size:8pt"><i>Barbas III et al., 2004</i></div></div>
<br>
<br>
<b>PHAGE DISPLAY</b>
<b>PHAGE DISPLAY</b>
<br>
<br>
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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.  
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The goal of phage display is to use M13 to carry a foreign protein or peptide on its surface, and package the DNA corresponding to that foreign peptide. One accomplishes this by genetically fusing a the peptide sequence of interest to one of the coat proteins. This modification can be genomic, in which case all copies of the coat protein will display the peptide, or the fusion can be expressed on a separate plasmid. In the separate plasmid (phagemid) system, the fusion proteins are integrated along with the wild type coat proteins.
 +
  <br><br>
 +
Historically, phage display has been used as a mechanism for screening libraries of proteins or peptides 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.
<br><br>
<br><br>
-
<b>HYPERPHAGE</b>
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We use principles of phage display only for the display property (rather than the packaging property). We also use a phagemid system. See <a href="http://2010.igem.org/Team:MIT_phage_design">Design</a> page for details.
<br>
<br>
-
Hyperphage is a plasmid with the gene for pIII truncated.  The pIII protein is required for phage exit from the host cell membrane; phage without a proper pIII grow into long fibril-like structures called polyphage. The goal is to crosslink these polyphage.
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<img style="float: left; width:130px; padding:20px" src="http://2010.igem.org/wiki/images/b/be/Coiled_coil.png">
<br><br>
<br><br>
 +
<b>COILED COILS</b>
 +
<br>
 +
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. For more information on coiled coils, see Branden and Tooze (1999). <br><br>All these pairs of coils have been successfully displayed on M13, and in the case of Fos/Jun and ACID/BASE cross-linking of phage has been observed (Wang et al. 2010, Sweeney et al. 2007).
 +
<br><br><br><br><br><br><br><br>
<div style="text-align:center">
<div style="text-align:center">
&larr; <a href="http://2010.igem.org/Team:MIT_phage">Introduction</a>
&larr; <a href="http://2010.igem.org/Team:MIT_phage">Introduction</a>

Latest revision as of 00:58, 28 October 2010

Phage
hairy cells and polymerizing phage - background
Arap, 2005
M13 ANATOMY
M13 bacteriophage is a long (930 nm in length, 6.5 nm in diameter), 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. When pIII is not present due to a knockout, elongation proceeds through multiple phage genomes, resulting in particles that are tethered to the membrane and up to 20x normal length.

Barbas III et al., 2004

PHAGE DISPLAY
The goal of phage display is to use M13 to carry a foreign protein or peptide on its surface, and package the DNA corresponding to that foreign peptide. One accomplishes this by genetically fusing a the peptide sequence of interest to one of the coat proteins. This modification can be genomic, in which case all copies of the coat protein will display the peptide, or the fusion can be expressed on a separate plasmid. In the separate plasmid (phagemid) system, the fusion proteins are integrated along with the wild type coat proteins.

Historically, phage display has been used as a mechanism for screening libraries of proteins or peptides 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.

We use principles of phage display only for the display property (rather than the packaging property). We also use a phagemid system. See Design page for details.


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. For more information on coiled coils, see Branden and Tooze (1999).

All these pairs of coils have been successfully displayed on M13, and in the case of Fos/Jun and ACID/BASE cross-linking of phage has been observed (Wang et al. 2010, Sweeney et al. 2007).







Introduction       Design