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| + | <li><a href="/Team:UCLA/Parts">PARTS</a></li> | ||
| + | <li><a href="/Team:UCLA/Modeling">MODELING</a></li> | ||
| + | <li><a href="/Team:UCLA/HumanPractices">HUMAN PRACTICES</a></li> | ||
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| + | <li style="margin-left: 12px;" id = "current"><a href = "https://2013.igem.org/Team:UCLA/Project">Overview</a></li> | ||
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| + | <li><a href="/Team:UCLA/Project/mrnaDisplay">mRNA Display</a></li> | ||
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| - | + | == '''Overall project''' == | |
| - | < | + | <h4><i>In Vitro</i> Immune System</h4> |
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| + | Diversity is an important feature of biological systems, and it can be harnessed to serve as a useful tool in synthetic biology. | ||
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| + | 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 <i>Bordetella</i> bacteriophage BPP-1. <i>Bordetella</i> is a genus of bacteria that can infect the mammalian respiratory tract, causing diseases such as pertussis (whooping cough). The infectious cycle of the <i>Bordetella</i> 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 ([[Team:UCLA/Modeling|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. | ||
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| - | </ | + | 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. |
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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 using synthetic diversity generation techniques to express and select for Mtd variants of interest. | ||
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| + | We plan on first generating a diverse DNA library of the ''mtd'' gene. Next, we plan on using mRNA-display as a means 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 <i>E. coli</i> bacterial cells. | ||
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| + | <b>REFERENCES</b> | ||
| + | <p>Overstreet, C.M., et al. <a href="http://www.ncbi.nlm.nih.gov/pubmed/22286238">"Self-made phage libraries with heterologous inserts in the Mtd of <i>Bordetella bronchiseptica."</i></a> <i>Protein Eng. Des. Sel. </i> 25.4 (2012):145-51. Print.</p> | ||
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| - | == | + | <!-- |
| + | ==Background Information== | ||
| - | + | <h4>The Natural Host</h4> | |
| + | <i>Bordetella</i> is the natural host of the BPP-1 bacteriophage. It expresses a protein on its surface, pertactin, that BPP-1 binds to in the first step of the infection process. However, <i>Bordetella</i> does not always express pertactin. It cycles between two phases: Bvg<sup>+</sup> and Bvg<sup>-</sup>. Pertactin is only expressed in the Bvg<sup>+</sup> phase. In the Bvg<sup>-</sup> phase, pertactin expression is inhibited. Infection by BPP-1 during this phase is much less common, but it still occurs. This indicates that the BPP-1 virus has a mechanism for changing its host specificity, and thus, the binding properties of the proteins on its tail fibers. | ||
| + | <h4>The Virus</h4> | ||
| + | The BPP-1 virus is a bacteriophage belonging to the <i>Podoviridae</i> family. It has an icosahedral head with T=7 symmetry, and a short, noncontractile tail with six tail “spikes” attached to tail fibers. At the end of these tail fibers are major tropism determinant (Mtd) proteins, which bind to the pertactin proteins expressed on the surface of <i>Bordetella</i>. The Mtd protein does not display high affinity for pertactin, but the multiple Mtd proteins possessed by each phage particle coupled with the flexibility of the tail fibers allow BPP-1 as a whole to have high avidity for its host. Following infection, the virus is lytic, and destroys the bacterial host to release more copies of itself. | ||
| + | ==mRNA Display== | ||
| + | <h4>Historical Background</h4> | ||
| + | Richard Roberts and Jack Szostak from Harvard developed this technique in 1997. They were able to generate and identify a myc epitope starting from a random mrna library using this technique. | ||
| + | <h4>What is it?</h4> | ||
| + | mRNA display is a technique used to generate and select for proteins that bind specific targets. MRNA display allows for the identification of these selected proteins because they are covalently attached to the DNA that codes for them. | ||
| + | <h4>How does it work?</h4> | ||
| + | <ul> | ||
| + | <li>First generate a library of DNA using pcr or some other method.</li> | ||
| + | <li>Next DNA library is transcribed <i>in vitro</i> to an RNA library.</li> | ||
| + | <li>With the help of a DNA splint, this RNA strand is ligated to a single strained DNA that contains a puromycin protein at the 3 ' end</li> | ||
| + | <li>This RNA-puromycin fusion is then translated <i>in vitro</i>.</li> | ||
| + | <li>Puromycin mimics a tRNA and is incorporated into the A site of the ribosome. However the puromycin has a non hydrolizable amide bond, therefore terminating translation and releasing the polypeptide, which is still bound to the RNA template through the puromycin.</li> | ||
| + | <li>You now have a library of proteins covalently attached to the RNA that coded for them.</li> | ||
| + | <li>In order to create a more stable structure, the RNA is converted to a DNA-RNA duplex through <i>in vitro</i> reverse transcription</li> | ||
| + | <li>Now out you can screen for the protein out of your library that best binds your target molecule.</li> | ||
| + | <li>Once you have your protein, it can be easily identified because it is still bound to the DNA that coded for it.</li> | ||
| + | <li>Simple pcr of the DNA followed by sequencing will give you the identity of your protein, (ie the sequence that codes for it.)</li> | ||
| + | </ul> | ||
| + | <h4>What does the MTD have to do with mRNA display?</h4> | ||
| + | The MTD protein will be our scaffold for our MRNA display experiment. Most proteins are not capable of undergoing the mutation necessary to create a large library while still maintaining their structure and function. Therefore we needed a protein whose structure allows it to be mutagenized in certain sites and still remain stable. The MTD has evolved in the natural system of bordatella bacteriophage to have precisely this property. Therefore it acts as an effective scaffold because the majority of the protein is able to remain constant and stable while a small localized region of each protein gets mutagenized. | ||
| + | <h4>Other approaches for directed protein targeting?</h4> | ||
| + | -Phage display: | ||
| + | An <i>in vivo</i> technique similar in principle to mrna display in which bacteriophage are allowed replicate to create a diverse library with different binding affinities. The DNA from the bacteriophage that bind the target can be isolated and then sequenced. | ||
| + | |||
| + | -Ribosome display: | ||
| + | Another <i>in vitro</i> technique that fuses the rna transcript to the protein it codes for. Instead of a puromycin linker between the transcript and the protein, it is the ribosome that links the protein and the template that codes for it. | ||
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| + | <h4>What are the advantages of this route / why did we choose this route for our project?</h4> | ||
| + | One of the major advantages of the mRNA display approach is that it takes place completely <i>in vitro</i>. This eliminates the limitations and complications that come with working <i>in vivo</i> and gives us more control over the entire process. Also, using an <i>in vitro</i> method allows us to create a more diverse library. Generating the library <i>in vitro</i> can potentially yield several orders of magnitude more protein variants than <i>in vivo</i> systems such as phage display. | ||
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| + | With respect to ribosome display, which is also an <i>in vitro</i> method, mRNA has an advantage when it comes to screening for protein binding. The disadvantage of ribosome display is that the linker between the protein and the template DNA is the actual ribosome. This is an issue because the ribosome is a very large protein and it has the potential to interact with the target protein, thereby throwing off the selection process. In contrast, the puromycin link in mRNA display is much smaller and is not likely to interfere in any way with the target protein. | ||
| + | |||
| + | <h4>Issues With using this route?</h4> | ||
| + | What we are essentially doing with this project is taking the MTD protein which comes from an <i>in vivo</i> system (bordatella phage) and using it as the scaffold in our <i>in vitro</i> protein selection system. We know that in that native system of bordatella phage, a large library of phage are generated each with unique variants of the mtd protein on its tail fibers. Out of this large library, some phage are able to effectively bind to a certain target using the mtd protein. | ||
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| + | A major concern is that the mtd protein may not behave the same way <i>in vitro</i> 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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== Project Details== | == Project Details== | ||
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== Results == | == Results == | ||
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Latest revision as of 01:52, 28 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 using synthetic diversity generation techniques to express and select for Mtd variants of interest.
We plan on first generating a diverse DNA library of the mtd gene. Next, we plan on using mRNA-display as a means 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. 25.4 (2012):145-51. Print.
