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Team:UCLA/Project
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We plan on first generating a diverse DNA library of the Mtd gene, then using mRNA-display to detect and screen for the variants of the protein that can bind to a selected target. In our case, the target will be the surface of E. Coli bacterial cells. | We plan on first generating a diverse DNA library of the Mtd gene, then using mRNA-display to detect and screen for the variants of the protein that can bind to a selected target. In our case, the target will be the surface of E. Coli bacterial cells. | ||
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A major concern is that the mtd protein may not behave the same way in vitro as it does in the natural bordatella system. Although we are theoretically able to generate more variants of mtd than in the natural phage system, it is possible that by isolating it from the phage could seriously affect its binding affinity. ‘For example, the native phage has several tail fibers, each with a copy of the mtd that could potentially bind a target. It may be that one isolated mtd has a very low binding affinity and that it is necessary to have several in order to create a strong enough binding interaction with the target. | A major concern is that the mtd protein may not behave the same way in vitro as it does in the natural bordatella system. Although we are theoretically able to generate more variants of mtd than in the natural phage system, it is possible that by isolating it from the phage could seriously affect its binding affinity. ‘For example, the native phage has several tail fibers, each with a copy of the mtd that could potentially bind a target. It may be that one isolated mtd has a very low binding affinity and that it is necessary to have several in order to create a strong enough binding interaction with the target. | ||
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Revision as of 22:07, 25 September 2013
Overall project
In-Vitro Immune System
Diversity is an intrinsic characteristic of nature, and it can be harnessed to serve as a useful tool in synthetic biology.
In particular, this diversity is exemplified in a species of bacteriophage known as the Bordetella BPP-1 phage. The Bordetella bacteria, a genus of small Gram-negative bacteria, frequently changes its surface topography over the course of its life cycle. To successfully infect its constantly-changing host, the BPP-1 phage uses diversity as its weapon of choice. The tip of each virus tail-fiber is a protein called the major tropism-determinant protein (mtd), which binds to the hosts’ surface and allows the phage to infect it.
Though the general structure of the mtd protein is constant and is very stable, its active ends are extremely variable. Each phage produces a slightly different structural variant of the protein, with the hope that at least one phage can successfully bind to the host and infect it- akin to the way our immune system produces numerous variants of antibodies so that at least one can bind to the antigen of interest.
The mtd gene, extracted from the BPP-1 phage genome, has the capability of generating a diverse library of stable proteins. Our team hopes to develop a fully in-vitro system to express and select for mtd variants of interest.
We plan on first generating a diverse DNA library of the Mtd gene, then using mRNA-display to detect and screen for the variants of the protein that can bind to a selected target. In our case, the target will be the surface of E. Coli bacterial cells.
