Team:MIT phage design
From 2010.igem.org
(Difference between revisions)
(3 intermediate revisions not shown) | |||
Line 28: | Line 28: | ||
<li><a href="https://2010.igem.org/Team:MIT_tmodel">Modelling</a></li> | <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> | ||
- | <li><a href="https://2010.igem.org/Team: | + | <li><a href="https://2010.igem.org/Team:MIT_composite">Characterization</a></li> |
</ul> | </ul> | ||
</dd> | </dd> | ||
Line 71: | Line 71: | ||
<b>OVERVIEW</b> | <b>OVERVIEW</b> | ||
<br> | <br> | ||
- | + | ||
+ | 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. | ||
+ | <br><br> | ||
+ | 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. | ||
+ | <br><br> | ||
+ | <b>HYPERPHAGE</b> | ||
<br> | <br> | ||
+ | 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). | ||
<br> | <br> | ||
- | <b> | + | <div style="text-align:center"> |
+ | <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> | ||
+ | </div> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <b>P8-FUSION DESIGN</b> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2010/f/fa/Phage_design.png" width=630px> | ||
+ | <br><br> | ||
+ | Above is the genetic design of our fusion construct. | ||
+ | <br><br> | ||
+ | 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). | ||
+ | <br><br> | ||
+ | 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. | ||
+ | <br><br> | ||
+ | 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. | ||
+ | <br><br> | ||
+ | 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>. | ||
+ | This construct can be easily assembled with BioBrick assembly into the circuit described below. | ||
+ | <br><br> | ||
+ | <b>PROPOSED CIRCUIT</b> | ||
<br> | <br> | ||
<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> | ||
Line 90: | Line 117: | ||
</ol> | </ol> | ||
<br><br> | <br><br> | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
Latest revision as of 01:20, 28 October 2010
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 ![]()
|