Team:MIT phage design

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                         <li><a href="https://2010.igem.org/Team:MIT_toggle">Overview</a></li>
                         <li><a href="https://2010.igem.org/Team:MIT_toggle">Overview</a></li>
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                        <li><a href="https://2010.igem.org/Team:MIT_tmodel">Modelling</a></li>
<li><a href="https://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
<li><a href="https://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="https://2010.igem.org/Team:MIT_composite">Characterization</a></li>
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<b>OVERVIEW</b>
<b>OVERVIEW</b>
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When designing the system we had to consider two critical aspects: linkage creation and circuitry. We decided to use an M13 bacteriophage-based system.  By knocking out pIII production we could get the infected cells to create polyphage "hairs".  To make this easy, instead of using wild-type M13, we used a product called Hyperphage, which comes with a truncated gene for pIII.  For linkage, we decided to use phage-display of coiled coils, fusing them to the pVIII coat protein. The circuitry used is similar to the circuitry for the RFP output: our fusion protein is controlled by the toggle, allowing UV input to begin fusion protein production and hopefully polymerization (amongst a heterogeneous population with matching linkers).  Additionally, we wanted to have the exterior of the UV pattern have no polymerization, so we designed an inverter to be ON where UV is OFF.  See below for further details.
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We envision a phage material composed of polyphage strands, produced by cells that carry hyperphage. In addition to producing all the proteins to form the polyphage, each cell will produce a p8-fusion from a separate plasmid to be displayed on the polyphage coat. The polyphage strands cross-link with one another via the coiled-coil interactions of proteins displayed on the phage coat.  
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<img src="https://static.igem.org/mediawiki/2010/f/f9/Polymer_block.jpg" style="float: left; height: 200px; padding:5px">
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We designed the phage material formation module to be integrated with the UV controller module. Our fusion protein is controlled by the toggle, allowing linkage to occur only where UV light has set the state of the toggle to low CI/ high LacI. p3 is under control of an inverter such that where UV light has set the state of the toggle to low CI/ high LacI, there is no p3 production, and thus polyphage formation.  
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<b>HYPERPHAGE</b>
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Our goal is to obtain something like that pictured to the left: a solid polymer. But this polymer was obtained through means of external cross-linking; we would like ours to be patterned by UV and controlled completely by genetic programming--no external linkers required.
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Hyperphage is a commercially available version of M13 with the gene for pIII truncated. Recall that pIII is required for the termination of assembly and exit from the host cell membrane, and thus hyperphage can be used as a polyphage "generator." Hyperphage can be obtained from <a href="http://www.progen.de/hyperphage-small.html">Progen Biotechnik</a> in Germany.  Below you can see a hyperphage plasmid map next to a M13 KO7 plasmid map.  Notice the size difference in the green highlighted gIII gene (gIII produces pIII).
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<div style="font-size:8px"><i>Willis, et al. 2007 </i></div>
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<a href="https://static.igem.org/mediawiki/2010/5/54/Hyp_v_ko7.jpg" class="thickbox" ><img src="https://static.igem.org/mediawiki/2010/5/54/Hyp_v_ko7.jpg" width=630px></a>
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<b>P8-FUSION DESIGN</b>
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<img src="https://static.igem.org/mediawiki/2010/f/fa/Phage_design.png" width=630px>
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Above is the genetic design of our fusion construct.
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p8 was chosen to "carry" our linker coil. Since repeating copies of p8 makes up the vast majority of the surface area of the phage, more fusion proteins are likely to be displayed (recall that in single phage, p8 is present in 2700 copies, compared to p9's 5 copies. The proportion is even more drastic in polyphage). The specific protein used, "opti-p8" is a mutational analysis-derived version that has been shown to incorporate fusion proteins into the phage coat at a higher efficiency (Weiss et al. 2000).
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The p8 leader sequence is responsible for localization of p8 to the membrane, where it is cleaved and the protein is allowed to be incorporated into the phage. We took the first 27 amino acids from M13 strain M13KE.
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In between the coil and the p8 protein are the "tag" and the "linker" sequence. The tag was introduced to facilitate western blotting experiments, and is either an HA tag or a Myc tag, depending on the coil. Like the choice of "opti-p8," the "linker" sequence was chosen because of its property of higher fusion incorporation efficiency.
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For additional information about the fusion design, please see <a href="https://static.igem.org/mediawiki/2010/c/c3/Fusion_design.pdf">this PDF</a>.
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This construct can be easily assembled with BioBrick assembly into the circuit described below.
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<b>PHAGE-RELEVANT CIRCUITRY DESIGN</b>
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<b>PROPOSED CIRCUIT</b>
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<img src="https://static.igem.org/mediawiki/2010/f/f4/Igem_circuit_phage.png" width=630px>
<img src="https://static.igem.org/mediawiki/2010/f/f4/Igem_circuit_phage.png" width=630px>
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<b>HYPERPHAGE</b>
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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.  Hyperphage can be obtained from <a href="http://www.progen.de/hyperphage-small.html">Progen</a> in Germany.
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<b>P8-FUSION DESIGN</b>
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<img src="https://static.igem.org/mediawiki/2010/f/fa/Phage_design.png" width=630px>
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The above shows the RBS + p8-fusion genetic design.  We chose p8 because of the large number of copies, increasing the potential for incorporation, and hopefully allowing for large-scale polymerization instead of small-scale clumping which might happen with p9 (due to the small number of p9 produced).  This genetic fusion is designed to allow the expression of a leucine zipper (coil in the diagram) on the p8 phage coat protein.  There are six total p8 fusions for the six different zippers.  To assist in proper cellular trafficking to the membrane, a leader sequence was introduced.  Additionally, HA and Myc tags were introduced (one for each half of a zipper pair), which were used in western blotting experiments to test for the expression of the p8-fusion genes.  A special version of p8 has been used, called opti-p8, which displays proteins better (see <i>Weiss et al. Mutational analysis of the major coat protein of M13 identifies residues that control protein display. 2000.</i>)  For additional information about the fusion designs, please see <a href="https://static.igem.org/mediawiki/2010/c/c3/Fusion_design.pdf">this PDF</a>.
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Latest revision as of 01:20, 28 October 2010

Phage
hairy cells and polymerizing phage - design

OVERVIEW
We envision a phage material composed of polyphage strands, produced by cells that carry hyperphage. In addition to producing all the proteins to form the polyphage, each cell will produce a p8-fusion from a separate plasmid to be displayed on the polyphage coat. The polyphage strands cross-link with one another via the coiled-coil interactions of proteins displayed on the phage coat.

We designed the phage material formation module to be integrated with the UV controller module. Our fusion protein is controlled by the toggle, allowing linkage to occur only where UV light has set the state of the toggle to low CI/ high LacI. p3 is under control of an inverter such that where UV light has set the state of the toggle to low CI/ high LacI, there is no p3 production, and thus polyphage formation.

HYPERPHAGE
Hyperphage is a commercially available version of M13 with the gene for pIII truncated. Recall that pIII is required for the termination of assembly and exit from the host cell membrane, and thus hyperphage can be used as a polyphage "generator." Hyperphage can be obtained from Progen Biotechnik in Germany. Below you can see a hyperphage plasmid map next to a M13 KO7 plasmid map. Notice the size difference in the green highlighted gIII gene (gIII produces pIII).



P8-FUSION DESIGN


Above is the genetic design of our fusion construct.

p8 was chosen to "carry" our linker coil. Since repeating copies of p8 makes up the vast majority of the surface area of the phage, more fusion proteins are likely to be displayed (recall that in single phage, p8 is present in 2700 copies, compared to p9's 5 copies. The proportion is even more drastic in polyphage). The specific protein used, "opti-p8" is a mutational analysis-derived version that has been shown to incorporate fusion proteins into the phage coat at a higher efficiency (Weiss et al. 2000).

The p8 leader sequence is responsible for localization of p8 to the membrane, where it is cleaved and the protein is allowed to be incorporated into the phage. We took the first 27 amino acids from M13 strain M13KE.

In between the coil and the p8 protein are the "tag" and the "linker" sequence. The tag was introduced to facilitate western blotting experiments, and is either an HA tag or a Myc tag, depending on the coil. Like the choice of "opti-p8," the "linker" sequence was chosen because of its property of higher fusion incorporation efficiency.

For additional information about the fusion design, please see this PDF. This construct can be easily assembled with BioBrick assembly into the circuit described below.

PROPOSED CIRCUIT


  1. Toggle - The toggle-switch works by mutual repression. Through addition of IPTG, it can be set to “off”, and UV, “on”. The “on” state lets RecA cleave cI, allowing for leaky transcription from the hybrid luxR/cI-regulated promoters. For full transcription, the LuxR-AHL complex must bind. AHL can thus be added to induce transcription while LuxR is produced constitutively. This toggle was donated from the Collins lab. It has been biobricked as K415300 (a low-power version is K415301).

  2. & 3. Pattern Formation - Constitutive LuxR allows for expression of mCherry and pVIII fusion proteins where there is no cI (i.e., where there is UV induction). Two distinct pVIII phage proteins are expressed in two populations of cells which then should bind together via leucine zipper interactions to allow for polymerization in the UV-induced area. Part (2) is K415010, Part (3) combined with Part (2) creates our K415147-152 parts.

  3. Inverter - In order to inhibit phage polymerization outside of the UV-induced region, an inverter allows for transcription only in areas where UV has not been introduced. pIII production then prohibits polyphage from forming which precludes leucine zipper interactions and polymerization. This part is currently in the construction phase and has not been incorporated into existing circuitry. (There's also another inverter being developed using the CymR system.)


← Background       Construction →

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