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Team:UCLA/Project
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| - | + | Others have utilized phage display techniques in order to exploit the Mtd’s natural stability to generate enormously diverse protein libraries. These pursuits have been conducted <i>in-vivo</i>, using the BPP-1 phage’s native diversity generation system (Overstreet). Our team hopes to develop a fully <i>in-vitro</i> 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. | 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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| + | <b>REFERENCES</b> | ||
| + | <p>Overstreet, C M., <i>et al.</i> Self-made phage libraries with heterologous inserts in the Mtd of <i>Bordetella bronchiseptica</i>. <i>Protein Eng. Des. Sel. </i> <b>145</b> (2012)</p> | ||
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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 21:29, 26 September 2013
Overall project
In-Vitro Immune System
Diversity is an important feature of biological systems, and it can be harnessed to serve as a useful tool in synthetic biology.
Many organisms have molecular mechanisms to generate diversity at the DNA level in order to accelerate evolution. This is true in particular with host/pathogen interactions, where the host must mutate to protect itself from the pathogen and the pathogen mutates to evade host defenses. One important example of such diversity generating mechanisms is found in the Bordetella bacteriophage BPP-1. Bordetella is a genus of bacteria that can infect the mammalian respiratory tract, causing diseases such as pertussis (whooping cough). The infectious cycle of the Bordetella bacteria involves a switch between a non-infectious and an infectious stage with different virulence factors and surface receptors expressed. Interactions between bacteria surface receptors and phage tail fiber proteins are extremely specific. 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. In order to successfully infect both phases of the host bacteria, the BPP-1 phage uses a complex genetic mechanism to generate diversity at the host-recognition site of the Mtd protein.
Though the general structure of the Mtd protein is constant and 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.
Others have utilized phage display techniques in order to exploit the Mtd’s natural stability to generate enormously diverse protein libraries. These pursuits have been conducted in-vivo, using the BPP-1 phage’s native diversity generation system (Overstreet). 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.
REFERENCES
Overstreet, C M., et al. Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica. Protein Eng. Des. Sel. 145 (2012)
