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Team:MIT phage design
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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. | 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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<div style="font-size:8px"><i>Willis, et al. 2007 </i></div> | <div style="font-size:8px"><i>Willis, et al. 2007 </i></div> | ||
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Revision as of 20:52, 27 October 2010
iGEM 2010
hairy cells and polymerizing phage - design |
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OVERVIEW 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. 
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.
Willis, et al. 2007
PHAGE-RELEVANT CIRCUITRY DESIGN 
HYPERPHAGE 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 cross-link these polyphage. Hyperphage can be obtained from Progen in Germany. P8-FUSION DESIGN 
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 Weiss et al. Mutational analysis of the major coat protein of M13 identifies residues that control protein display. 2000.) For additional information about the fusion designs, please see this PDF. |
